The next frontier in genetic risk estimat - Elsevier

381REFLECTIONS IN MUTATION RESEARCH: 1999 – 2019380 ELSEVIER

Mutation Research 764 (2015) 1–15

Genome-based, mechanism-driven computational modeling of risks of ionizing radiation: The next frontier in genetic risk estimation? ☆, ☆ ☆K. Sankaranarayanan, H. Nikjoo *

Radiation Biophysics Group, Department of Oncology-Pathology, Karolinska Institutet, Box 260, P9-02, Stockholm SE 17176, Sweden

Reflections Article

Genome-based, mechanism-driven computational modeling of risksof ionizing radiation: The next frontier in genetic risk estimation?§,§§

K. Sankaranarayanan, H. Nikjoo *

Radiation Biophysics Group, Department of Oncology-Pathology, Karolinska Institutet, Box 260, P9-02, Stockholm SE 17176, Sweden

1. Historical background

The estimation of genetic risks of exposure of humanpopulations to ionizing radiation has been a major area ofradiobiology since the early 1950s. Genetic risk estimates togetherwith those on cancers provide the scientific basis for radiologicalprotection recommendations [2,3]. From the very beginning ofthese efforts, the paucity of directly usable human data on adversegenetic effects in the progeny of those exposed to radiationnecessitated the indirect estimation of risks using mouse germ celldata on radiation-induced mutations. This is in contrast to cancersfor which risk estimates have always been made from human

epidemiological data, initially on mortality and later on incidence;

see [2]. The notion that radiation-induced mutations would cause

genetic diseases similar to those that occur naturally as a result of

spontaneous single-gene mutations in germ cells dominated the

thinking of scientific committees involved in risk estimation from

the mid-1950s onwards. Consequently, efforts at risk estimation

were focused on finding a suitable method that would allow the

prediction of risks in terms of the number of additional ‘‘cases’’ of

genetic diseases in the progeny of those exposed, over and above

their baseline frequencies in the population. The method chosen,

namely, the ‘doubling dose method’, enabled the conversion of

mutation rate estimates derived from mouse data into estimates of

the ‘risk of genetic disease’ in humans, albeit with a number of

assumptions (reviewed in [4]).While scientific committees pursued the mouse-data-based

approach for estimating genetic risks, genetic epidemiologicalstudies initiated in Japan in the late 1940s in the aftermath of theA-bombings were focused on ascertaining directly whether anyadverse genetic effects could be demonstrated in the children of A-bomb survivors using indicators that were practicable at the timethese studies were initiated. The indicators were: untoward

A R T I C L E I N F O

Article history:

Accepted 18 December 2014

Available online 31 December 2014

Keywords:

Radiation risk

DNA damage

DNA repair

Biophysical models

A B S T R A C T

Research activity in the field of estimation of genetic risks of ionizing radiation to human populations

started in the late 1940s and now appears to be passing through a plateau phase. This paper provides a

background to the concepts, findings and methods of risk estimation that guided the field through the

period of its growth to the beginning of the 21st century. It draws attention to several key facts: (a) thus

far, genetic risk estimates have been made indirectly using mutation data collected in mouse radiation

studies; (b) important uncertainties and unsolved problems remain, one notable example being that we

still do not know the sensitivity of human female germ cells to radiation-induced mutations; and (c) the

concept that dominated the field thus far, namely, that radiation exposures to germ cells can result in

single gene diseases in the descendants of those exposed has been replaced by the concept that radiation

exposure can cause DNA deletions, often involving more than one gene. Genetic risk estimation now

encompasses work devoted to studies on DNA deletions induced in human germ cells, their expected

frequencies, and phenotypes and associated clinical consequences in the progeny. We argue that the

time is ripe to embark on a human genome-based, mechanism-driven, computational modeling of

genetic risks of ionizing radiation, and we present a provisional framework for catalyzing research in the

field in the 21st century.

§ This article is part of the Reflections in Mutation Research series. To suggest

topics and authors for Reflections, readers should contact the series editor, G.R.

Hoffmann (ghoffmann@holycross.edu).§§ This article is also paper XVIII in a series entitled ‘Ionizing radiation and genetic

risks’ authored by K. Sankaranarayanan and colleagues between 1991 and 2013 and

published in Mutation Research (except paper XVI which was published in Int. J.

Radiat. Biol. in 2011 [1]).

* Corresponding author. Tel.: +46 8 517 724 90; mobile: +46 7 2300 8252.

E-mail address: hooshang.nikjoo@ki.se (H. Nikjoo).

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pregnancy outcomes (including stillbirths, neonatal deaths andcongenital malformations in live births in phase I (1948–1954);sex-ratio shifts and survival of live-born infants in phase II (1955–1968); and cancers in F1 children, sex-chromosomal aneuploids,balanced structural rearrangements and mutations affectingprotein charge or function in phase III (1969–1990) (reviewedin [5]). Of note is that these studies were not aimed at detectingpossible increases in the frequencies of children affected byradiation-induced genetic diseases. Reports of progress in thesestudies published from time to time until 1998 [6] provided noevidence of any detectable increase in adverse effects (asmeasured by the indicators mentioned above) in childrenattributable to radiation exposure of the parents. In retrospect,two factors relegated the Japanese data to the sidelines of geneticrisk estimation efforts until the 1990s: (1) most of theseindicators were not sensitive enough to induced mutations atthe low average doses sustained by the survivors, and (2) theadverse effects measured could not be readily compared with orfitted into the framework of the ‘risk of genetic diseases’envisioned by the scientific committees. The principal messagefrom the Japanese studies, namely, low genetic risk at the lowaverage doses sustained by the survivors, however, was finallyreconciled with the risk estimates arrived at by the committees[7,9].

The ‘current’ genetic risk estimates were first published morethan a decade ago [8], and they are summarized in the reports ofthe BEIR VII Committee of the U.S. National Academy of Sciences[7] and the United Nations Scientific Committee on the Effects ofAtomic Radiation [9]. These are presented in Table 1. The estimatesshow that the total risk to the first post-radiation generation is ofthe order of 3000–4700 cases per 106 progeny per Gy, whichrepresents 0.41–0.64% of the baseline risk. With the exception ofcongenital abnormalities, the risk estimates for genetic diseasehave been obtained using the doubling dose method. The risk ofcongenital abnormalities has been estimated using mouse data oninduced developmental abnormalities (i.e., congenital anomaliesascertained in utero, skeletal abnormalities and cataracts) withoutrecourse to the doubling dose method. This is an important pointand will be returned to later.

2. Uncertainties and unsolved problems

Although the estimates presented in Table 1 reflect the stateof the art in the field at the end of the 20th century, severaluncertainties and unsolved problems remain. These have beendiscussed in detail elsewhere [7–9]. In what follows, we brieflyaddress three of the most obvious ones, namely, (a) thedoubling dose method of risk estimation itself; (b) inability todefine the genetic radiosensitivity of human females and (c)lack of evidence for radiation-induced genetic disease inhumans. Radiation risk assessment, genomics and DNA repairall involve specialized terminology. Table 2 explains theacronyms, abbreviations and technical terms that are used inthis paper.

2.1. The doubling dose (DD) method of risk estimation

The conceptual foundations for the doubling dose (DD) and themethod that bears its name were laid by Muller in the 1950s [10–12]. The DD method permits the use of mutation data from mouseradiation studies for estimating the risk of genetic disease inhumans. The DD is the amount of radiation required to produce asmany mutations as those occurring spontaneously in a generation.Ideally, it is calculated by dividing the average spontaneousmutation rate of a set of genes by the average induced rate for thesame set of genes, although this has not always been possible.1 Thequantity [1/DD] is called the relative mutation risk (RMR) per unitdose. The DD estimate in current use for low dose, chronic, low LETirradiation (the radiation conditions used for risk estimation) is1 Gy.

The DD method is based on the theory of equilibrium betweenmutation and natural selection, which population geneticists useto describe the dynamics of mutant genes in populations. Itassumes that the stability of mutant gene frequencies (and henceof disease frequencies) in a population is a reflection of theexistence of a balance or equilibrium between spontaneousmutations that enter its gene pool at a finite rate every generationand natural selection that eliminates these same mutationsthrough failure of survival or reproduction. This assumptionimplies that the baseline frequencies of genetic diseases onemeasures in a population represent those of a population in‘mutation-selection equilibrium’. When such a population sustainsradiation exposure in every generation, induced mutations enterits gene pool and are also subject to natural selection. Eventually,the population reaches a new equilibrium between mutation andselection at a higher mutant frequency, and thus of diseasefrequency.

In the early years of genetic risk estimation, the focus wason ascertaining the ‘total added risk of genetic disease’ at thenew equilibrium under conditions when the populationsustains radiation exposure at a finite rate in every generation.The DD method allows one to estimate this total risk as aproduct of two quantities, namely, the baseline frequencies [P]and [1/DD]:

Total risk per unit dose ¼ P � 1

� �(1)

Estimates of risk for the first, second, and later post-radiationgenerations were derived from the total risk at the new equilibriummaking assumptions about the magnitude of selection. Two new

Table 1Estimates of genetic risks from exposure to low LET, low dose chronic irradiation

(based on [7–9]) and an assumed doubling dose of 1 Gy.

Disease class Baseline

frequency

(per 106

live births)

Risk per Gy

per 106

first-generation

progeny

Mendelian

Autosomal

dominant

16,500 �750–1500

X-linked

Autosomal

recessive

7500 0

Chromosomal 4000 a

Multifactorial

Chronic 650,000b �250–1200

Congenital

abnormalities

60,000 �2000c

Total 738,000 �3000–4700

Total expressed as

percentage of baseline

�0.41–0.64

a Assumed to be subsumed in part under the risk of autosomal dominant and X-

linked diseases and in part under that of congenital abnormalities.b Frequency in the population.c Estimate obtained using mouse data on developmental abnormalities, not with

the doubling dose method. This estimate overlaps with that shown as risk under the

heading of ‘autosomal dominant and X-linked diseases’; see text for details.

1 The DD method has evolved over the years along with revisions of the data used

for estimating DD [4] and of the data on baseline frequencies of genetic diseases

[3]. In the ‘recent’ UNSCEAR [9] (2001) and BEIR VII [7] (2006) reports, the DD was

calculated using a spontaneous mutation rate of human genes (n = 135) and an

induced mutation rate of mouse genes (n = 34).

K. Sankaranarayanan, H. Nikjoo / Mutation Research 764 (2015) 1–152

quantities, MC and PRCF were introduced in the risk equation in theyear 2000 [8] and the risk (now for any post-radiation generation ofinterest) became a product of four quantities:

Risk per unit dose ¼ P � 1

� �� MC � PRCF (2)

where MC is mutation component,2 and PRCF is potentialrecoverability correction factor.3

Two points deserve emphasis here: first, the estimates of thebaseline frequencies of genetic diseases shown in Table 1 areupdates from earlier compilations and derive from differentstudies of Western European and Western-European-derivedpopulations. Consequently, they do not reflect the profile or theaggregate burden of such diseases in any specific population.Further, it is well-known that the spectrum and frequency ofgenetic diseases can vary among different parts of the world andare rapidly changing as a result of the increased mobility andpopulation admixtures that we are witnessing today.

Second, since the DD is a ratio, it is subject to variation in thenumerator (average spontaneous rate) or the denominator(average induced rate) or both. Further, both rates depend onwhich genes are included and on the range of mutation rates ofindividual genes, especially when the rates are not based on thesame set of genes, as is the case with the currently used DD (seefootnote 1). Further, it assumes proportionality between sponta-neous and induced rates of mutations (i.e., genes with highspontaneous rates of mutations will also be more sensitive toradiation-induced mutations), and this is not always the case. BothMuller and the scientific committees that used the DD methodwere certainly cognizant of its shortcomings. However, as Neelobserved somewhat philosophically some sixteen years ago:

‘‘. . . the doubling dose is a convenient concept, but the manyassumptions and practical difficulties in actually deriving adoubling dose were well-enumerated by Muller [12]. Thesituation has not changed materially in the ensuing 39 years . . .

nevertheless, in an imperfect world, the doubling dose conceptsupplies a perspective, if blurred, difficult to obtain by any othermethod . . .’’ [6].

2.2. Inability to define the genetic radiosensitivity of human females

When the mouse was chosen to serve as a model for assessingmutational radiosensitivity of human genes in the late 1940s, theassumption was that the radiosensitivity of the two sexes in themouse would correspond to that of the two sexes in humans and,considerable amounts of radiation data were collected using bothmale and female mice (reviewed in [15–18]). However, severalobservations cast doubt on this assumption: (i) mature andmaturing mouse oocytes responded very differently from imma-ture mouse oocytes to the induction of mutations; and (ii)responses of immature mouse oocytes to cell killing werestrikingly different from those of the immature oocytes of rhesus

Table 2Glossary of acronyms, abbreviations and technical terms.

Alt-NHEJ, Alternate Non-Homologous End-Joining pathway of DNA DSB repair

Artemis, the endonuclease required for the slow repair process of DSB

BEIR, Biological Effects of Ionizing Radiation; a scientific committee of the US

National Academy of Sciences

CNV, Copy Number Variation(s), a structural genomic variant that results in

copy number changes in a specific chromosomal region; if its population

allele frequency is less than 1%, it is referred to as variant

DD, doubling dose in risk estimation

DNA-ligase III, One of the enzymes involved in the repair of DNA single strand

breaks

DNA-PK, DNA-dependent Protein Kinase, a type of enzyme that modifies other

proteins to cause some particular cell function

DSB, Double strand breaks in DNA. Radiation-induced DSBs have been classified

as simple and complex types. The simple type is a clean kind of break; a

complex DSB is accompanied by one or more SSB in the same turn of DNA

double helix

GWAS, Genome-Wide Association Studies

HR, HRR, Homologous Recombination Repair

ICRP, International Commission on Radiological Protection (www.icrp.org)

IHGSC, International Human Genome Sequencing Consortium (www.genome.

INDEL, Small insertion(s) and deletion(s) present in the genome

KURBUC, A Monte Carlo track structure computer code for generation of

radiation tracks at molecular level

LCR, Low Copy Repeat sequences in the genome; also referred to as segmental

duplications (SD)

LET, Linear Energy Transfer in keV/mm

MC, Mutation component in calculation of risk per unit dose. It specifies the

responsiveness of a given disease to induced mutation

Meiosis, a type of cell division that reduces the chromosome number by half.

Meiosis in humans and other animals is limited to germ cells

Mitotic, process of cell division reproducing somatic cells

MMBIR, Microhomology-Mediated Break-Induced Replication

MMEJ, Microhomology-Mediated End-Joining (also known as Ku-independent

NAHR, Non-Allelic Homologous Recombination

NCBI, National Center for Biotechnology Information; US government-funded

national resource for molecular biology information providing access to many

public databases and other references (www.ncbi.nlm.nih.gov)

NRC, National Research Council of the US (www.nationalacademies.org)

Paired-end sequencing, a large-scale genome sequencing method to identify

structural variants �3 kb or larger; in this approach, anchor points derived

from sequences at the ends of clones from a genomic library of a selected

genome are aligned to the reference assembly, and the distance between

them is compared with the expected size of the clone; any discrepancy

highlights potential insertion or deletion variants

PARP-1, Poly[ADP-ribose] polymerase enzyme encoded by the PARP-1 gene; has

a role in the repair of single-strand DNA breaks

PARTRAC, A Monte Carlo track structure computer code for generation of

radiation tracks at molecular level

PRCF, Potential Recoverability Correction Factor, enables one to bridge the gap

between rates of induced mutations at specified loci in mice and the risk of

genetic disease in humans

RAD52 group genes, a group of genes involved in the homologous

recombination DNA repair pathway that operates on DNA double-strand

breaks to promote error-free repair. Most of the RAD52 group genes were

identified by their requirement for the repair of ionizing-radiation-induced

DNA damage in Saccharomyces cerevisiae. Sensitivity to ionizing radiation is a

universal feature of the RAD52 group mutants

RMR, Relative Mutation Risk

SD, Segmental Duplication(s), also known as low copy repeats

SNP, Single Nucleotide Polymorphism(s)

SSA, Single strand annealing, one of the DNA double-strand repair processes

SSB, Single strand break in DNA

SV, Structural Variation

Sv, Sievert, SI unit of dose equivalent. 1 Sv = 1 J/kg = 100 rem

UNSCEAR, United Nations Scientific Committee on the Effects of Atomic

Radiation

XLF (also known as Cernunnos), a core factor for NHEJ repair of DSB, playing a

unique role in bridging DNA-damage sensing and DSB-rejoining steps

XRCC1, X-ray Repair Cross-Complementing protein 1, a DNA repair protein that

interacts with PARP-1 and DNA-ligase III in single-strand break repair

XRCC4, X-ray Repair Cross-Complementing protein 4, a DNA repair protein

involved in the NHEJ pathway of DSB repair

2 The mutation component (MC) specifies the responsiveness of a given disease

to induced mutation. Formally defined, It is the relative increase in disease

incidence (relative to the baseline) per unit relative increase in mutation rate

(relative to the spontaneous rate) and varies depending on the relationship between

mutation and disease for the different classes of genetic disease. This relationship is

straightforward for autosomal dominant and X-linked diseases, slightly compli-

cated for autosomal recessive diseases (an induced recessive mutation does not

result in disease in the following generation) and more complicated for

multifactorial diseases [13].3 The potential recoverability correction factor (PRCF) enables one to bridge the

gap between rates of induced mutations at specified loci in mice and the risk of

genetic disease in humans. Its introduction into the risk equation for the first time is

one of the important outcomes of the integration of the advances in human

molecular biology into the framework of genetic risk estimation [14].

K. Sankaranarayanan, H. Nikjoo / Mutation Research 764 (2015) 1–15 3

monkeys and humans.4 Consequently, scientific committeesabandoned the use of data from studies of female mice sincethe late 1980s because of uncertainty whether mouse immatureoocytes would provide a good model for assessing mutationalradiosensitivity of human immature oocytes [25]. To err on theside of caution, the following assumption was used for the purposeof risk estimation:

mutational radiosensitivity of human stem cell spermatogonia �mutational radiosensitivity of human immature oocytes �mutational radiosensitivity of mouse stem cell spermatogonia

There does not seem to be an easy way either to validate or torefute this assumption.

2.3. Lack of evidence for radiation-induced genetic diseases in humans

A key assumption used by the scientific committees in the1950s when they began their work on genetic risk estimation wasthat radiation-induced mutations would cause genetic diseaseswith phenotypes similar to those that occur naturally as a result ofspontaneous mutations in single genes (i.e., Mendelian diseases).This assumption was based on the limited knowledge of geneticdiseases and the data then available (see for example [26–28]).However, there has been no evidence for a radiation-induced germcell mutation in humans resulting in a Mendelian disease or anyother kind of disorder formally called a ‘genetic disease’.Nonetheless, scientific reports published since then, includingthe ‘‘latest’’ ones [7,9], have continued using the assumption of‘inducible genetic disease’ in risk estimation, expanding it toinclude other classes of genetic diseases.

The view that radiation is unlikely to induce single genemutations emerged in the late 1990s from analyses (genetic,cytogenetic and molecular) of radiation-induced mutations invarious test systems, including those in mouse germ cells andmammalian somatic cells in culture carried out in the 1960sthrough the 1990s. These studies showed that most inducedmutations, although scored through the phenotypes conferred bymarker genes, are multi-gene deletions of different sizes thatinclude the marker genes under study. Clues on the potentialphenotypes of radiation-induced deletions in germ cells came from(a) accumulated knowledge on known differences betweenspontaneous mutations that underlie Mendelian diseases andradiation-induced mutations in experimental systems (in nature,mechanisms and impact on gene function) and (b) studies ofnaturally occurring microdeletion syndromes in humans in the1990s.5 Together, they paved the way for advancing the conceptthat DNA deletions induced in human germ cells, if compatible

with offspring viability, are more likely to manifest themselves asmulti-system developmental abnormalities [32]. These induced

developmental abnormalities are predicted to be phenotypicallysimilar to naturally occurring congenital abnormalities with oneimportant difference: most of the induced ones are expected toshow autosomal dominant patterns of inheritance, whereas mostof the naturally occurring congenital abnormalities still continueto be interpreted as being of multifactorial etiology.

The above concept, which challenges the basic premise ofgenetic risk estimation used thus far, was introduced into theframework of genetic risk estimation for the first time in 2000[8]. It underlies the use of mouse data on developmentalabnormalities to derive a provisional estimate risk of induceddevelopmental abnormalities with autosomal dominant patternof inheritance in humans (without using the DD method). Theestimate is presented in Table 1 (2000 cases per Gy per 106

progeny) under the heading ‘‘congenital abnormalities’’ forreasons of consistency with the scheme and headings used overthe years for presenting risk estimates.6 This estimate isindependent of and in addition to that shown under the heading‘‘autosomal dominant and X-linked diseases’’ (750–1500 cases perGy per 106 progeny), which has been arrived at using the DDmethod. Footnote c to Table 1 underscores a need for cautionabout the possibility of overlap (i.e., double counting) between thetwo estimates of risk of dominant effects estimated by differentmethods.

Why did the scientific committees continue using ‘induciblegenetic disease’ as the unit to express genetic risks in spite of thefact that none has been found? One possible reason is that overmore than three decades the risk of genetic diseases predicted withthe DD method at low doses was small compared to baseline risks,and the absence of any measurable increase in adverse effects inthe children of A-bomb survivors was consistent with theprediction of the DD method. Further, without a justifiablealternative unit to express risks, it was pointless to abandon theexisting unit. The concept of multisystem developmental abnor-malities (with predominantly autosomal dominant pattern ofinheritance) as the major phenotypes of radiation-induceddeletions emerged only in 1999. It needed to gain traction inthe scientific community (which requires a new mindset!) before aparadigm shift and redefinition of the goal of genetic riskestimation could occur. At the end of the 20th century it wasrealized that the DD method might need to be replaced bysomething different, but a new framework for genetic riskestimation was not available.

3. Publication of the draft sequence of the human genome andhopes for an impact in our field

When the draft sequence of the human genome was publishedin 2001 [33], it was hoped that ‘‘. . .the scientific work will haveprofound long-term consequences for medicine, leading to theelucidation of the underlying molecular mechanisms of diseaseand thereby facilitating the design in many cases of rationaldiagnostics and therapeutics targeted at those mechanisms.’’ Theissue of Nature in which the human genome sequence waspublished also carried nine invited data-mining articles thatinterrogate the genome from distinct biological perspectives (see[34]). These perspectives ranged from broad topics – cancer,addiction, gene expression, immunology and evolutionary geno-mics – to the more focused – membrane trafficking, cytoskeleton,

4 Mouse immature oocytes (sampled 7 weeks post-irradiation) are insensitive to

radiation-induced mutations in contrast to mature and maturing oocytes (sampled

in the first seven weeks after irradiation), which are very sensitive to mutation

induction. However, mouse immature oocytes are very sensitive to radiation-

induced killing; the LD50 being of the order of 0.15 Gy [19,20]. In contrast, immature

oocytes of the rhesus monkey and of human females (maintained in organ cultures)

are much less sensitive: the doses required for similar levels of cell killing are

estimated to be at least 100-fold higher than in the mouse [21,22]. Studies of

Wallace et al. [23,24] also show that human immature oocytes are more resistant to

killing than mouse immature oocytes.5 Microdeletion syndromes, also termed ‘contiguous gene deletion syndromes’

are conditions that result from deletions of multiple, often functionally unrelated

and yet physically contiguous, genes that are compatible with viability in

heterozygous condition. They are identified clinically through a characteristic

association of unusual appearance and defective organ development. Mental

retardation and growth retardation are often prominent features [29,30]. Many of

these deletions have been found to originate through nonallelic homologous

recombination (NAHR) between segmental duplications (SD) in meiosis [31].

6 Over the years, scientific committees have presented risk estimates under the

headings of ‘‘autosomal dominant and X-linked diseases,’’ ‘‘autosomal recessive

diseases,’’ ‘‘chromosomal diseases’’ and ‘‘multifactorial diseases’’ (which include

‘‘congenital abnormalities’’ and ‘‘chronic diseases’’). Note that the group titled

‘congenital abnormalities’ is not an aetiological category, and neither is the group

called ‘chromosomal diseases,’ which was originally used to include gross structural

and numerical anomalies of chromosomes.

To the best of our knowledge, no computer models of genomicDNA in cell nuclei of human spermatogonial stem cells or ofimmature oocytes are available. However, in the context oftheoretical biophysical studies, several investigators have usedmodels mostly of interphase nuclei of mammalian somatic cellsthat vary in complexity from cylinders representing simple linearsegments of DNA to higher order structures such as nucleosomes,chromatin fiber, DNA loops and chromosomal domains (e.g.,[37,38,48–55]). Models of mitotic nuclei have also been published[56,57].

We believe that a computer model of the mammalianinterphase nucleus with atomic resolution such as the onedeveloped by Nikjoo and Girard [38] may be a good starting pointfor spermatogonial stem cells. In this model, the nucleus consists of‘domains,’ each containing one of the two arms of a chromosome.Each arm contains ‘factories,’ and each factory has ‘scaffolds’ towhich loops of DNA are attached. The cell nucleus is represented bya sphere of 10 mm diameter, but the size of the spherical nucleuscan be changed as necessary. Each chromosome is modeled as apair of adjacent spheres, one for each arm. The human interphasenucleus thus consists of a total of 92 spheres fitted into the outersphere in such a way that they can touch but do not overlap. Themodel of canonical B-DNA that was used has been assumed to takethe form of a 30 nm solenoid fiber. The lengths of the loops of fiberare variable between 7.5 and 175 kbp. The DNA sequence has beenassigned according to the model of the human genome publishedby the National Center for Biotechnology Information in 2010 andmay need updating (see [38] for further details).

A computer model for the human oocyte nucleus in thediplotene stage of prophase 1 of meiosis remains to be developed.

4.4.2. Induction of different types of lesions in cellular DNA

Ionizing radiation induces a variety of DNA lesions in cellularDNA including damaged bases, single-strand breaks (SSBs) andsimple and complex DSBs.7 The types, frequencies and location ofthese different types of lesions have been modeled using MonteCarlo methods (statistical techniques based on the use of randomnumbers and probability statistics) using knowledge of radiationtrack structure with certain assumptions about the minimumenergy deposition required (e.g., [37]). Of note here is that it was onthe basis of computer simulations that it was predicted that about30% of DNA DSBs induced by low LET irradiation are expected to becomplex by virtue of additional breaks/damage. This proportionrises to more than 70% with high LET radiations along with anincrease in the degree of complexity [37,44,45,55,58–60].

Specific track structure computer codes such as PARTRAC(PARticle TRACks) have been developed to study DNA DSBs andfragmentation yields with different types of radiation andcomparisons with data obtained in laboratory studies. PARTRACcalculates DNA damage in human cells based on the superpositionof simulated track structures in liquid water to an ‘atom-by-atom’model of human DNA. It also takes into account the higher orderDNA organization in chromatin fiber and chromatin territories. Theinitial damage simulations in PARTRAC reproduced essentialfindings in corresponding laboratory experiments using pulsedfield gel electrophoresis (PFGE) to assay DNA fragment distribu-tions (e.g., [61,62]). Thus, it provides a reliable basis for theestimate of initial frequency and distribution of DSBs for the repairmodels developed subsequently [49,63,64]. Friedland et al. [64]have provided a comprehensive review of the PARTRAC code andits applications (see also [65]).

In our group, Nikjoo and Girard [38] tested their computermodel of the eukaryotic nucleus by generating tracks of protonsand a-particles (using Monte Carlo track structure codes PITS99[Positive Ion Track Structure] and KURBUC8) that traversed thenucleus. Damaged sites in the DNA were located and classifiedaccording to complexity. Other modeling studies by our group,using recent versions of KURBUC, have focused on DSB repair andare discussed in Section 4.4.5.

4.4.3. DNA damage response pathways and their interrelationships

Laboratory studies have provided evidence that eukaryotic cellsrespond to radiation damage by activating DNA-damage response(DDR) pathways through which signal transduction processes alertthe cell to the presence of DNA damage and trigger suchdownstream events as cell cycle arrest, repair and apoptosis(reviewed in [66–69]). The critical components that are activatedby DSBs include the MRE11-RAD50-NBS1 complex (MRN complex;also involved in the homologous recombination repair [HRR]pathway); Ku proteins and phosphatidylinositol 3-kinase-relatedkinases (PIKK); DNA-PKcs; the ATM (ataxia-telangiectasia mutat-ed) protein kinase and the ATR (ataxia-telangiectasia-related)kinase [also involved in the NHEJ pathway] [70–78].

Another early step in the response of the cell to DSBs is thetriggering of phosphorylation of the H2A histone family,9 memberX, H2AX, which can be carried out redundantly by ATM or DNA-dependent protein kinase (DNA-PKcs)[79]. H2AX phosphorylationon Ser-139 (to form g-H2AX) can extend up to several thousandnucleosomes from the actual site of the DSB, producing discretemicroscopically detectable foci as a result of the alteration ofchromatin structure. Total numbers of g-H2AX foci have beenshown to be fairly representative of the total number of DSBsproduced initially. These chromatin modifications are believed tofacilitate the recruitment and access of repair proteins involved inNHEJ and HR to the liberated chromosomal ends [80–85].

4.4.4. Synopsis of current knowledge on error-prone DSB Repair

pathways

Three error-prone pathways exist for DSB repair: non-homologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), single-strand annealing (SSA) and nonallelichomologous recombination repair (NAHR) [69,86–94]. Since mostof the modeling efforts thus far have focused on NHEJ, we discussthis pathway in slightly more detail to consider DSB complexitythat was included in the models.

(a) Non-homologous end-joining (NHEJ). NHEJ is the major DSBrejoining pathway, occurs in all cell cycle stages and is thepredominant one in the G1 phase of the cell cycle. It can join DNAends with a number of different structures with little or nohomology at the junctions. Small sequence deletions or additionsmay be introduced during the repair process. The key players inNHEJ include the heterodimer Ku70/80, the catalytic subunit of the

7 A simple DSB represents two opposing DNA ends with easily ligatable 50-

phosphate and 30-hydroxyl groups; example of complex DSBs: base dama-

ge + SSB + DSB in close proximity within one or two helical turns of DNA.

8 KURBUC stood for Kyushu University Radiobiology Unit Code. The current code

features a comprehensive suite of Monte Carlo track structure codes for simulation

of X-rays, electrons and ions in water (vapor, liquid and ice) and DNA. A unique

feature of the code includes full slowing down simulations of beams for protons

(1 keV–300 MeV), alpha-particles (1 keV/u–8 MeV/u), and carbon ions (1 keV/u–

10 MeV/u).9 In eukaryotic cells, DNA is densely packaged within chromatin, the basic

material of chromosomes, which consists of DNA complexed with basic proteins,

notably histones, and acidic proteins. At its most fundamental level, chromatin

consists of a repeating series of nucleosome core beads separated by linker DNA

strings. Each nucleosome core consists of an octamer of basic histone proteins (two

each of H2A, H2B, H3 and H4) around which a stretch of 146 bp of double-stranded

DNA is wrapped approximately 1.75 turns. Adjacent nucleosomes are connected by

histones of the H1 linker class and a short spacer DNA. The nucleosomes are

assembled into higher order structures that are inaccessible to trans factors [80–

cell cycle and circadian clock. There was the hope that in the post-genome era some of the enthusiasm and excitement that madepossible the deciphering of the human genome sequence wouldspill over to our field, applying the new knowledge to reduceuncertainties and shed light on some of the unsolved problems ingenetic risk estimation. This hope did not materialize. Further, wehave seen little evidence of funding organizations in Europepromoting research in this area for the past two decades (see[35,36] for a recent discussion of the workshop on the EuropeanCommunity’s low-dose radiation research program designated bythe acronyms MELODI/DoReMi). Presumably a similar situationholds in the USA.

Our group has conducted pilot studies on the use of genomicknowledge to address some of the problems in genetic riskestimation (e.g., [1,37–46]) but a comprehensive and integratedframework has not yet been put in place; there is still a long wayto go. Several others have published on the basic science andbiophysical modeling aspects of some of the issues consideredhere and are discussed in the text. The next section presents ourvision of a provisional framework as it now stands, and we hopethat it helps to catalyze further thinking, and more importantly,action.

4. A new provisional framework for genetic risk estimation inthe post-genome era

4.1. Scope of genetic risk estimation

Two concepts that emerged from radiation mutagenesis andDNA repair studies in the second half of the 20th century werediscussed in the NRC [7] and UNSCEAR [9,47] reports but were notfully integrated within the framework of genetic risk estimation.These are: (1) most radiation-induced germ cell mutations areDNA deletions, often encompassing more than one gene, and (2)the deletions arise as a result of the repair or misrepair of inducedDNA double-strand breaks (DSBs). These concepts help to set thestage and redefine the scope and direction of this field in the 21stcentury: the redefined scope of genetic risk estimation should notbe focused anymore on the search for ‘induced genetic diseases’(as was the case earlier) but must encompass work devoted tostudies on DNA deletions induced in human germ cells andprediction of their expected frequencies, phenotypes and theassociated clinical consequences in the descendants carryingthese deletions [39].

4.2. Approach envisaged and rationale: general aspects

We envisage a human genome-based, mechanism-drivencomputational modeling of genetic risks of ionizing radiationand believe that it may be the next frontier in genetic riskestimation. The scientific landscape for undertaking genome-based research for assessing genetic risks of radiation has neverbeen more favorable than now. One should consider the wholeissue in the context of the phenomenal advances that are beingmade in human genome research against the backdrop of thosealready made in cell biology and in theoretical studies oninteractions of ionizing radiation with DNA in chromatin. Genomicadvances have highlighted mechanisms that generate variabilityand the roles of error-prone DSB repair and of DNA-replication-based mechanisms in the origin of naturally occurring deletionsand other structural changes in the genome. On the cell biologyside, advances relating to the organization of chromatin in thechromosomes and nuclear architecture hold clues that may enableus to envision how small and large deletions might be induced atlow radiation doses.

4.3. Why computational modeling?

At least five arguments can be advanced in support of increasedreliance on computational modeling. First, the era of large-scaleexperimental radiation mutagenesis work with mice on the scalescarried out in Oak Ridge National Laboratory, Harwell or Munich inthe second half of the 20th century is over. Second, while it hadbeen possible to obtain data on radiation-induced cancers inhuman populations exposed to radiation, the amount of directinformation on radiation-induced adverse genetic effects inhumans remains very limited despite more than half a centuryof research; new large-scale studies on genetic effects inpopulations exposed to radiation seem very unlikely. Third, anyhuman genome-based research will minimize the need forextrapolation; while one may still need some experimental workwith mice, this will be geared more toward testing predictions thatemerge from human genome-based work. Fourth, over the pastdecades, we have accumulated a vast corpus of knowledge fromtheoretical studies on interactions of radiation with DNA inchromatin and from experimental studies on radiation-inducedmutations, mutational mechanisms, DSB repair, etc. All of this canbe put to good use in modeling genetic risks. Finally, for the firsttime in the history of risk estimation, we have the challengingopportunity to use the knowledge and tools that genome researchhas provided to query the human genome directly for adversegenetic effects of radiation. The question no longer is whether buthow.

4.4. The scope of models that are required, kinds of information

currently available and needed, and the way forward

Ideally, the models to be used in our ‘bottom-up’ approachshould include all the sequential events and effects that followirradiation. They should start with quantitative information forthe early steps of the interaction of radiation with genomic DNAin the cell nucleus (the target for radiation action) and theirsubsequent fates, focusing on DNA damage responses, cellsignaling cascades, DSB repair/misrepair and their interrelation-ships. The aim is a realistic prediction of resultant deletions ofdifferent sizes. This step is then followed by assessment of thegene content of the deletions and of their recoverability in theprogeny, their phenotypes and frequencies. Not all the steps maybe amenable to modeling, and plausible assumptions may need tobe formulated. As discussed below, the computational modelingstudies carried out thus far, albeit with different objectives, havealready provided substantial information ranging from earlysteps of interaction of radiation with genomic DNA in the cellnucleus to selected aspects relating to DSB repair. In a sense, theinfrastructure for the approach that we propose already exists –all we need to do is extend it to include modeling for the inductionof deletions in germ cells, their transmission to progeny, andassessment of phenotypes and the impact on health. In this paper,we discuss the trajectory from the early steps of interaction ofradiation with genomic DNA up to the induction of DNA deletionsin germ cells.

4.4.1. Models of genomic DNA in cell nuclei and radiation conditions

The germ cell stages of interest in the present context are thestem cell spermatogonia in males (most in the G0/G1 phase of thecell cycle) and immature oocytes in females (in the diplotene stageof prophase of meiosis 1) [39]. Although the radiation conditionsconventionally used in genetic risk estimation (at the populationlevel) are low dose, chronic, low LET irradiation, we suggest that itwould be instructive to also include high doses in modelingbecause of their potential applicability in radiation accidentsituations.

and characterization of lesion complexity. Repair enzyme activitiesand their association and dissociation from DNA termini weremodeled in first-order kinetics by the Monte Carlo methodcomplemented with step-by-step Brownian motion of DNAtermini. Considered overall, the results of different scenariosshowed reasonable agreement for DSB rejoining kinetics, incor-rectly joined DSBs and chromosomal aberrations. However, theyields of residual DSBs were overestimated by the model relative toexperimental results. The need to reexamine the assumption of themovement of free DNA ends according to Brownian motion wasindicated.

In their second study on stochastic modeling of DSB repair after60Co-g- and N ion radiations, Friedland et al. [49] used a designsimilar to the one in the 137Cs study with some adaptations inmodel structure, namely, inclusion of an on-going production ofDSBs by enzymatic processing of labile sites [116] and consider-ation of a limited availability of enzymes removing additionalbreaks or base damage at complex lesions. The model predictionswere compared with actual results reported in the experiments ofStenerlow et al. [100] with human fibroblasts (cell line GM5758)irradiated with 60Co-g-rays and N ions. With the stated adapta-tions, the agreement of the model calculations with the measuredDSB rejoining kinetics was improved. Further, the agreementbetween calculated and measured fragment distributions bothinitially and during repair was reasonable. However, for N-ionirradiation the model predicts the existence of a large number ofDSBs associated with very short fragment (<5 kbp) but these werenot detectable in the experiments of Stenerlow et al. [100].

Friedland et al. [49] caution that their work using the DNArepair model in PARTRAC demonstrates the feasibility of reprodu-cing the rather different repair kinetics after low- and high-LETradiations, but that the model is a first approach that combinesspatial aspects of DNA damage within cells, DNA lesion complexityand temporal development of the states of DNA ends during NHEJDSB repair. It works with cell-cycle-averaged rates of enzymeattachments and neglects any alternative or back-up pathways.The implication is that further refinements are needed before theycan be confidently used for predictive purposes.

In our group Taleei and Nikjoo [42] have presented amechanistic mathematical model for the kinetics of NHEJ repairof DSBs. Biochemical data from studies of HRR-repair-deficientcells (mouse embryonic fibroblasts mutated in Rad54; DT40chicken cells mutated in Rad54, Rad52, Rad51 and Rad51B; and CHOcells mutated in XRCC2 and XRCC3) and proficient cells (wild typeDT40 and human fibroblast cells) following high radiation doses(10–80 Gy) were used for this purpose. These data on repair weretransformed into a set of 9 nonlinear differential equations basedon the law of mass action. Additionally, the initial recruitmentkinetics of Ku80 and DNA-PKcs were modeled from real-timekinetic data of Uematsu et al. [117], obtained in experiments usingfluorescent labeling, albeit over a short time (30 s). The Deq (doseequivalent unrepaired DSBs) calculated with the NHEJ model andexperimental measurements were found to show fast and slowrepair fractions, with about 50% of the DSBs repaired in �30 min bythe fast repair process and the remainder requiring more timepresumably because of the complexity of DSBs and/or chromatinstructure.

Subsequently, Taleei et al. [43] applied their model to examinethe repair kinetics of simple and complex types of DSB breaksinduced by ultrasoft X-rays and low energy electrons (see also[41]). Both low energy electron tracks (100 eV to 4.55 keV) and CK,AlK and TiK ultrasoft X-rays were simulated using the updatedversion of the track structure KURBEC code. The target was theatomistic model of the mammalian interphase nucleus developedby Nikjoo and Girard [38]. The activity elapsed time of sequentiallyindependent steps of repair performed by NHEJ proteins was

calculated for separate DSBs. The kinetics of repair of DSBs werecomputed and compared with published data on repair kineticsdetermined using the PFGE method in Chinese hamster V79-4 cells[118,119]. There was good agreement between the modelpredictions and empirical observations for ultrasoft X-rays. Theaverage times for the repair of complex DSBs were longer thanthose for simple ones and confirm that DSB complexity is likely tobe the underlying cause for the slow component of repair observedin the hamster cells.

Taleei et al. [44,45] extended the above studies to specificallymodel DSB repair in mammalian cells irradiated in G1 and S phaseof the cell cycle, taking into account recent advances in the field,some of which were mentioned earlier. These provided therationale for the present study. Briefly, (i), during G1 and early S,NHEJ and MMEJ pathways are activated depending on the type ofDSBs. Simple DSBs are substrates for NHEJ whereas complex DSBsand DSBs in heterochromatin require further end processing; (ii)repair of all DSBs start with NHEJ presynaptic processes, whichinclude damage recognition by Ku heterodimer, recruitment ofDNA-PKcs and autophosphorylation of DNA-PKcs. The process isstraightforward for simple DSBs and leads to end ligation with fastkinetics; (iii) for simple DSBs in heterochromatin, further end-processing and opening of the chromatin are performed by Artemisand ATM proteins that phosphorylate KAP-1; and (iv) complexdamage undergoes resection, and repair continues via MMEJ in G1

and early S. Instead of acting competitively, the repair pathwaysare assumed to cooperate with each other in repairing DSBs.Comparison of the model predictions with experimental repairkinetics data in V79 Chinese hamster cells (irradiated with 45 Gy of60Co g-rays [99]) and in human dermal fibroblasts (80 Gy of X-rays[98]) measured using PFGE and CFGH, respectively, showed thatcomplex DSBs are indeed repaired with slow kinetics.

Reynolds et al. [102] had shown that the choice of NHEJ proteinsis determined by the complexity of DSBs (discussed earlier). In afollow-up of this work, Li et al. [114] formulated a mechanisticmathematical model and examined whether it could simulate thekinetics of formation of foci reported by Reynolds et al. Thequestion was answered in the affirmative (i.e., the modelsupported the experimental findings that simple DSBs undergofast repair in a Ku-dependent and DNA-PKcs-independent manner,whereas complex DSBs also require DNA-PKcs for end processing,resulting in its slow repair). An important prediction of their modelis that the rejoining of the complex DSBs is through a process ofsynapsis formation, similar to a second order reaction betweenends, rather than first order break filling/joining. The authors notethat the synapsis formation model allows for the diffusion of endsbefore synapsis formation, which is precluded in the first orderbreak-filling model. Therefore, the synapsis-formation model alsopredicts higher numbers of chromosomal aberrations with highLET radiation and an increased probability of mis-rejoiningfollowing diffusion before synapsis. We will return to this pointin Section 4.4.7.

Cucinotta et al. [111] developed a biochemical kinetic model todescribe NHEJ repair of DSBs produced by low LET radiation and itsrelationship to g-H2AX foci. The autophosphorylation of DNA-PKcs

and subsequent induction of g-H2AX foci were modeled. Analysisshowed that the model predictions were consistent withexperimental observations including DSB rejoining as measuredby PFGE at 10 min or longer post-irradiation intervals. Compar-isons of model calculations with empirical measurements of g-H2AX foci by Leatherbarrow et al. [84] in V79 and HF19 cellsshowed good agreement. The authors note that their model isbased on major NHEJ components and that it is subject toimprovement when more information becomes available.

Taleei et al. [115] published a study on the biochemical kineticmodeling of single strand annealing (SSA) for the repair of

DNA-dependent protein kinase (DNA-PKcs), Artemis and a complexof DNA ligase IV/XRCC4 and XLF/Cernunnos. After DSBs areinduced, DNA free ends are first bound by the Ku heterodimer,which forms a ring surrounding the DNA. DNA-PKcs is thenrecruited and bound to Ku to form a holoenzyme DNA-PK. OtherNHEJ factors are then recruited. Once a pair of DNA free ends boundwith NHEJ factors forms a synapsis, the kinase activity of DNA-PKcs

is activated [95] so that phosphorylation of other NHEJ factorsregulates the biochemical steps leading to end-processing andligation by ligase IV/XRCC4 and XLF/Cernunnos. Meanwhile, theDNA-PKcs also undergoes autophosphorylation that leads to itsdissociation from DNA [68,96]. The recruitment of NHEJ factors inthis order constitutes the ‘classic sequential model’ of NHEJ.

Earlier studies on DSB repair (using physical methods generallybased on DNA size changes) have demonstrated that induced DSBsare rejoined with biphasic kinetics with approximately 80% ofthem being rejoined with fast kinetics while a smaller fraction isrejoined with much slower kinetics [97–101]. The slow componentof DSB repair increases with an increase in LET [99,102,103]. Thereis evidence that slow repair is due to DSB complexity; additionally,there are data that support the inference that DSBs in heterochro-matin are repaired slowly, and this pathway requires ATM[68,96,104,105].

Based on the indications that a fraction of DSBs may be repairedby NHEJ in a Ku70/80-dependent but DNA-PKcs-independentmanner, Reynolds et al. [102] and Mari et al. [106] addressed thequestion of whether complex DSBs require a different subset ofproteins than simple DSBs. DSBs were induced in fluorescentlytagged (for Ku80, DNA-PKcs and XRCC4) Chinese hamster cells byultrasoft X-rays (USX) and multi-photon near infrared microbeam(NIR) (the latter gives a high proportion of complex DSBs). Thedoses were: 27 Gy and 137 Gy for USX and 730 nm photons for NIR.The dynamics of the NHEJ proteins involved in the repair of DSBs ofdifferent levels of complexity was followed. The results show thatsimple DSBs induced by USX or NIR microbeam irradiation arerepaired rapidly and involved KU70/Ku80 and XRCC4/Ligase IV/XLF (but not DNA-PKcs). In contrast, DSBs with greater chemicalcomplexity are repaired slowly involving DNA-PKcs in addition tothe others. Inhibition of ATM-activity retarded the repair ofcomplex DSBs.

(b) Microhomology-mediated end-joining (MMEJ). MMEJ is alsoreferred to as ‘alternative NHEJ’ (alt-NHEJ) [91] and functions in theabsence of canonical NHEJ factors. This Ku-independent pathwayinvolves PARP1 and ligase III. Unlike NHEJ, MMEJ starts with endresection by MRN (Mre11-Rad50-Nbs1) that is mediated by CtIP,especially in G1. The most distinguishing property of MMEJ is therequirement of 5–25 bp microhomology during the alignment ofbroken ends before rejoining, a process that results in deletionsflanking the original break. MMEJ utilizes two proteins thatfunction in single-strand break repair (PARP-1/XRCC1 and ligaseIII) [92,94] and is slower.

(c) Single-strand annealing (SSA). This DSB repair process isconsidered a variant form of homologous recombination repair(HRR). It is initiated when a DSB occurs (or is induced) betweentwo repeat sequences oriented in the same direction. Single-stranded regions are created adjacent to the break by 50–30

resection, which extends to the repeated sequences such that thecomplementary strands can anneal to each other. The processinvolves the loss of one of the two direct repeats and theintervening DNA. SSA is facilitated by RPA and RAD52 in a RAD51-independent manner [88,107].

(d) Current view on the relative importance of the different DSB

repair pathways. The view that seems to be gaining currency in thefield with respect to the relative importance of NHEJ, MMEJ, SSAand HRR in repairing DSBs can be summarized as follows: (i) NHEJis the major DSB rejoining process that occurs in all cell cycle

stages; (ii) HRR is the predominant pathway to repair one-endedDSBs arising in S phase when the replication fork encounterssingle-stranded breaks or base damage (fork stalling); (iii) two-ended DSBs directly induced by X or g-irradiation in late S or G2

phase are repaired predominantly by NHEJ with fast repairkinetics; (iv) when NHEJ is hindered (e.g., by complexity of DSBsor location of the DSBs in heterochromatin), an orchestrated andregulated switch to HRR occurs, and the kinetics of repair is slow[68,88]; and (v) such a switch from NHEJ to MMEJ/SSA may alsooccur with complex DSBs/non-ligatable ends.

(e) Nonallelic homologous recombination (NAHR). NAHR is anerror-prone version of the naturally occurring process of homolo-gous recombination (HR) that occurs during germ cell develop-ment (prophase I in meiosis) in sexual organisms. HR involvesprecise pairing of paternal and maternal chromosomes duringmeiosis and reciprocal exchange of genetic material between themwith no loss of genetic material (i.e., it is error-free). At themolecular level, HR is initiated by the programmed occurrence ofDNA DSBs and their repair by recombination with homologoussequences on a non-sister chromatid. Hence, HR is a DSB repairprocess that is referred to here as homologous recombinationrepair (HRR).

Unlike HR, in which pairing between homologous chromo-somes (i.e., homologous DNA sequences) is precise (i.e., allelic), inNAHR the pairing is nonallelic and occurs between misalignedrepetitive sequences such as segmental duplications (also knownas low copy repeats, LCRs) that are present in the genome. Whenthis happens, DNA sequences that lie between the repeats thatundergo NAHR are either deleted or duplicated, thus changingtheir copy number (CNV). NAHR-mediated deletions and duplica-tions arise when the LCRs are in direct orientation[31,108,109]. The enzymatic machinery involved in NAHR isassumed to be the same as that for HRR10,11 in mammalian somaticcells in which HRR has been extensively studied.

4.4.5. Published computational modeling studies of DSB repair

Several mechanistic mathematical models have been proposedto describe DSB repair with respect to the biochemical action ofDSB repair proteins. In these models, the repair activities,represented as a sequence of chemical reactions at the site ofdamage, are translated into a system of nonlinear differentialequations by applying the law of mass action. The solutions of theequations provide individual protein activity kinetics and overallrepair kinetics, and these can be compared with experimentalmeasurements (usually rate of repair of DSBs, post-irradiation,using PFGE in the dose range of 40–100 Gy). While most of themodels dealt with the NHEJ pathway (e.g., [41,42,44,49,63,64,111–114], some have also considered single-strand annealing (SSA)[115]; microhomology-mediated end-joining (MMEJ) [48]; appli-cation to high LET radiations [48]; and base-excision-repair (BER)[46].

In the work of Friedland et al. [63] involving 137Cs g-irradiation,DNA damage ‘induced’ in a target model of human fibroblast cellnucleus with the Monte Carlo track structure code PARTRACprovided the starting conditions for the spatial distribution of DSBs

10 In mammalian somatic cells, the initiating step of HRR following DSB induction

is end-resection to create a 30-ended single-stranded region in which CtIP/MRE11-

RAD50-NBS1 [MRN] is involved. This is followed by rapid binding of the region with

replication protein A and its subsequent displacement by RAD51, which mediates

the core reactions of HRR, namely, homology searching, strand exchange, Holliday

junction formation, repair synthesis (using the sister chromatid as a template),

Holliday junction resolution and ligation of the DNA ends effecting error-free DSB

repair [89,92,110].11 Although mammalian somatic cells are diploid, HRR seldom uses the

homologous chromosome as a template for DSB repair. HRR only functions in

late S/G2 when a sister chromatid is available.

and analyzing a ‘haploid’ human genome from a completehydatiform mole (CHM113) [126,127] and progressing toward atruly complete human genome sequence (christened as the‘platinum genome’) [128,129].

Second, projects that are now underway, including ENCODE(ENCyclopedia Of DNA Elements), Entrez, Ensembl, GENCODE (asubproject of the ENCODE project), and others, offer the promise ofenhanced genome annotation. For example, the GENCODEConsortium has recently released GENCODE 7 dataset version21 (called June 2014 freeze, GRCh38 – Ensembl), which providesinformation on the total number of genes, protein-coding genes,long noncoding RNA genes, small noncoding RNA genes, tran-scripts, etc. [131,132]. The time-frame for completion of thismammoth task is not known at present.

It will necessarily be some time before a complete and fullyannotated reference genome becomes available. Nonetheless, it ispossible to use the current version of the genome sequence along withother bioinfomatics resources14 that already exist to make tentativeinferences on the anticipated phenotypes conferred by deletions‘induced’ in the computational modeling studies. One can also checkand fine-tune these inferences using what had been predicted in1999 as potential phenotypes associated with DNA deletions that areinduced in germ cells and are compatible with viability. These includemulti-system developmental abnormalities affecting growth andmental development in the children of those exposed [32].

Insights from recent studies can also be instructive and provideuseful pointers. Cooper et al. [133] compared copy numbervariants (CNVs) in 15,767 children with intellectual disabilityand various congenital defects (i.e., cases) to CNVs in 8329 unaf-fected adults (i.e., controls) and created a CNV morbidity map. Theyestimated that 14.2% of disease in these children is caused by CNVs(mostly deletions and duplications) larger than 400 kb. Theyobserved a greater enrichment for CNVs in individuals withcraniofacial anomalies and cardiovascular defects than in thosewith epilepsy or autism. In a subsequent paper Coe et al. [134]created an expanded morbidity map from 29,085 children withdevelopmental delay compared to 19,584 healthy controls,identifying 70 significant CNVs. They also resequenced 26 candi-date genes in 4716 additional cases with developmental delay orautism and 2193 controls. As the authors state, the CNV morbiditymap they created is ‘‘. . . one of the largest CNV morbidity maps ofindividuals with intellectual disability, developmental delay and/or autism spectrum disorder, both as a clinical resource forpathogenic CNVs and also to identify genes potentially sensitive todosage imbalance.’’ Beaudet [135], who commented on the paper,characterized this study as marking ‘‘a CNV milestone.’’

5. Mechanism-driven modeling of risks and the LNTrelationship

The conventional way that such scientific organizations asUNSCEAR, ICRP and the BEIR VII Committee express estimates ofgenetic and carcinogenic risks is to refer to the ‘‘expected number

of affected cases per unit dose.’’ This way of expressing risksimplies that the risk increases linearly with an increase in dosewithout any threshold, and this has come to be known as the LNT(linear no-threshold) relationship. Over the years, this assumptionhas played a major role in predicting risks at low doses whenempirical data are limited or unavailable. The LNT concept is alsoused either implicitly or explicitly to assess low dose mutagenicand carcinogenic effects of chemical agents. Here we comment onthe origin of the LNT concept and how it has been important in thecontext of radiation protection, drawing attention to other viewson dose-risk relationships at low doses. We also consider thequestion of whether the proposed mechanism-driven computa-tional modeling of risks can be robust enough to be informative ofrisks at low doses, independently of the LNT assumption. Forconvenience, we use the abbreviation LNT to denote a ‘concept’,‘assumption’ or ‘a model’ interchangeably.

5.1. The LNT concept and its relevance for radiation protection

The LET concept first emerged from the genetic studies ofMuller and colleagues on radiation-induced sex-linked recessivelethal mutations in Drosophila spermatozoa in the late 1920s–early1930s (reviewed in [3,136]). In the early 1950s, the concept wasextended to quantify all adverse genetic effects as well as cancers

(implicit in statements such as ‘total risk per unit dose’). It remainsthe preferred model for predicting risk at low doses. Of interesthere is that its use for genetic risk prediction at low radiation doseshas been broadly accepted, presumably because genetic risks werealways estimated indirectly from mouse data on germ cellmutations and, in the absence of other viable options, the LNTmodel provided a convenient ‘default assumption.’

With respect to cancers, the situation has been different.UNSCEAR [47] concluded (Annex 1, paragraph 358) that ‘‘. . .theexperience of the Japanese A-bomb survivors is consistent with alinear dose–response for the risk of all solid cancers combined;therefore, as a first approximation, linear extrapolation of theestimates at 1 Sv acute dose can be used for estimating solid cancerrisks at lower doses . . .’’. Brenner et al. [137] reviewed theepidemiological evidence and the difficulties involved in quanti-fying the cancer risks of low dose radiation and addressed twospecific questions, namely (a) the lowest dose of x or g- irradiationfor which there was good evidence for increased cancer risks inhumans and (b) the most appropriate way to extrapolate to suchcancer risks at still lower doses. The answer to the first questionwas ‘‘about 10–50 mSv for an acute exposure and about 50–100 mSv for a protracted exposure.’’ The answer to the secondquestion was more complex: ‘‘given that it is supported byexperimentally grounded, quantifiable, biophysical arguments, alinear extrapolation of cancer risks from intermediate to very lowdoses currently appears to be the most appropriate methodology. . . the linearity assumption is not necessarily the most conserva-tive approach, and it is likely that it will result in an underestimateof some radiation-induced cancer risks and an overestimate ofothers. . ..’’ The BEIR VII Committee [7] concluded (page 15) that‘‘. . .current scientific evidence is consistent with the hypothesisthat there is a linear, no threshold dose-response relationshipbetween exposure to ionizing radiation and the development ofcancers in humans.’’ The Committee’s figure (Fig. ES1, page 16) forexcess relative risk for solid cancer incidence (with 10 data pointsin the range from 0 to 1.5 Sv, of which three are in the 0–0.5 Svrange) illustrates the above conclusion.

ICRP first rationalized the relevance of the LNT concept forradiation protection in Publication 9 [138] and has used it eversince. In its 2007 recommendations in Publication 103 [2], ICRPreiterated the view that ‘‘. . .the LNT model combined with a dose-and dose-rate effectiveness factor (DDREF) for extrapolation from

13 A complete hydatiform mole (CHM) is an abnormal product of conception in

which there is very early fetal demise and overgrowth of the placental tissue. Most

CHMs are androgenetic and contain only paternally derived autosomes and sex-

chromosomes resulting from either dispermy or duplication of a single sperm

genome. The absence of allelic variation in monospermic CHM makes it an ideal

candidate for producing a single haplotype representation of the human genome.

There are a number of existing sources associated with the ‘CHM1’ sample some of

which have been previously used to improve regions of the reference human

genome assembly [126].14 Database of Genomic Variants (DGV), a comprehensive catalog of structural

variation in the human genome identified in healthy individuals, International

Collaboration for Clinical Genomics (ICCG), Decipher (Database of chromosomal

imbalance and phenotypes in humans using Ensembl resources), dbVar (a database

of genomic structural variation) and OnlineMendelian Inheritance in Man (OMIM).

radiation-induced DSBs. Although this work was done earlier thanthat on NHEJ, the basic principles and modeling procedures weresimilar. The initial steps of the SSA model are those proposed forHRR by Deng et al. [120] based on experimental data. In view of thefact that SSA is considered a main pathway for DSB repair in theabsence of functioning NHEJ and HRR, enzyme kinetic dataobtained in experiments of Wang et al. [121] involving irradiated(20 Gy of X-rays) DT40 chicken B cells deficient in NHEJ and HRRwere used in the modeling. The model successfully predicted DSBresults of the DT40 cells irradiated with 80 Gy.

Finally, in our group Rahmanian et al. [46] developed amechanistic model of base excision repair (BER) to deal with therepair of non-DSB damage induced by low and high LET radiations.The repair of single strand breaks (SSB) and base damage (BD),which are governed by BER, was found to be a function of thecomplexity of DNA damage induced by ionizing radiations.

4.4.6. General conclusions from computational modeling studies

Much data on biochemical kinetic modeling of one major DSBrepair process, NHEJ, now exists supporting the feasibility ofmodeling DSB repair. However, there are uncertainties arisingfrom such sources as the number of parameters to be modeled,simplifying model assumptions, estimation of rate constants, andstatistical questions. Most real-time experiments that provideddata on enzyme kinetics and those that were used to cross-checkmodel predictions were done at very high doses. Prediction of thekinetics at biologically relevant doses is important. In our view,these problems/difficulties are not insurmountable.

4.4.7. The next step in modeling: how do deletions of different lengths

arise as a consequence of DSB repair/misrepair in irradiated germ

cells?

This question has not yet been modeled. In a recent paper [40], wereviewed human genomic studies on mechanisms of formationnaturally occurring structural variation – especially deletions – withthe aims of ascertaining their relative contribution to the ‘deletionlandscape’ of the genome and of assessing the extent to which thesemechanisms can help to explain empirical observations on deletionsin radiation mutagenesis studies. The mechanisms included NHEJand MMEJ; NAHR (discussed in Section 4.4.4); MMBIR, a micro-homology-mediated, replication-based mechanism that operates inthe S-phase of the cell cycle; and strand-slippage during replication(involved in the origin of small insertions and deletions – INDELs).The analysis revealed that these five mechanisms could explainnearly all naturally occurring deletions in the human genome, NAHRand MMBIR being potentially more versatile in this regard. Themechanistic studies suggest that deletions arising as a result of NHEJ/MMEJ processes, as currently formulated, are expected to be small.However, data on induced mutations in mouse spermatogonial stemcells (irradiation in G0/G1 phase of the cell cycle and presumablyrepaired largely by NHEJ) show that most are associated withdeletions of different sizes, some in the megabase range.

Some potentially useful clues for this discrepancy betweenwhat the basic repair studies show and what empirical data ondeletions demonstrate have emerged from research in cell biologyon the organization of chromatin and nuclear architecture. Weargued [40] that the apparent discrepancy is not real and can beresolved by considering the issue of deletions in a broader contextin conjunction with the organization of chromatin in chromosomesand nuclear architecture. We hypothesized that the repair of DSBsin the chromatin loops may offer a basis for explaining theinduction of deletions of different sizes. The assumption is that asingle track of ionization passes through a chromatin loop wherethe two arms of the loop lie next to each other. It induces a DSB ineach of the arms, and NHEJ processing leads to the excision of theloop and the formation of an interstitial deletion.

Another potentially useful idea in the above context comesfrom the paper of Li et al. [114] on the modeling of the DNA-complexity-dependent NHEJ repair pathway discussed earlier.Here, the authors state that an important prediction of their modelis that the rejoining of complex DSBs is through a process ofsynapsis formation and that such a model allows for diffusion of

ends before the synapsis formation. This, in turn, might promotechromosomal aberrations. We speculate that diffusion of ends

might also promote chromatin loop excision and the formation ofan interstitial deletion!

We turn now to the hypothesized role of NAHR in generatingdeletions in irradiated germ cells. Sankaranarayanan and Wassom[39] considered this issue in the context of radiation-induceddeletions in human oocytes and discussed the potential steps inmodeling. Theoocytes are arrested inthe dictyotene (diplotene) stageof prophase I of meiosis, a stage at which the paired chromosomalbivalents are held together by chiasmata and begin desynapsis. It washypothesized that DSB repair through NAHR between sisterchromatids (i.e., between segmental duplications [SDs] located inthem) might contribute to the occurrence of deletions, assuming thatDSBs induced in or near a segmental duplication will stimulate NAHRbetween chromatids. They discussed how one can use maps ofsegmental duplications in the human genome as primary sequence-based deletion-redisposition maps and considered possible comput-er simulation experiments. A major requirement for this is theavailability of reliable SD maps of the human genome. SD maps andsummary statistics are available at the University of Washingtonwebsite: (http://humanparalogy.gs.washington.edu/build37/build37.htm). Segmental duplication analysis is currently underwayfor the latest human reference genome (GRCh38/hg38)(Drs. JohnHuddleston and E.E. Eichler, personal communication).

4.4.8. The ultimate challenge

The ultimate challenge in the modeling approach discussed inthis paper is to be able to bridge the gap between DNA deletionsarising as a consequence of the operation of DSB repair processes inirradiated germ cells and adverse health effects in the progenyreceiving them. We have not discussed this theme in detail in thispaper because current understanding is too rudimentary to offerconcrete recommendations. We hope to address this issue in afuture paper in the ‘‘Ionizing radiation and genetic risks’’ series.Our preliminary views on the subject are outlined below.

In bridging the gap between induced DNA deletions and theiranticipated clinical phenotypes, the starting assumption inmodeling studies is that the operation of DSB repair processeson radiation-induced damage has resulted in deletions of definedlengths in genomic DNA. Therefore, given the precise location andthe extent of the deletions, comparisons of the genomes carryingthe ‘induced’ deletions with a complete reference genome that isfully annotated12 should provide us with information on genes andfunctions lost as a result of the deletion and thus the potentialclinical phenotypes. But we are not there as yet. The operativewords are the italicized ones.

First, a complete reference sequence that is fully annotatedremains one of the cherished goals toward which genomescientists are striving. Although the current human referencegenome sequence, GRCh37, is of very high quality, gaps andmisassembled sequences remain due to biological and technicalcomplexities (e.g., refs [122–125]). Ongoing research is gearedtoward identifying ‘missing sequences’ and genetic variation andclosing the gaps in the reference genome. This includes sequencing

12 Genome annotation refers to identifying all key features of the genome

sequence – in particular, the genes and their products/function, using a

combination of computational analysis, manual annotation and experimental

validation [130].

deletions are formed. It may or may not be valid up to the lastendpoint of the effect, namely, a child carrying an induced deletionwith a diagnosable phenotype.

6. Final reflections

The purpose of this paper is to review the current status of thefield of genetic risk estimation and draw attention to the fact thatthe new genomic knowledge and technologies have now providedus with unprecedented opportunities to address the uncertaintiesand unsolved problems that still remain. We believe thatcomputational modeling is a promising way forward, in partbecause the infrastructure for doing this is already in place and allthat is needed now is to build on it, and in part because it seemsunlikely that either large-scale animal studies or human epidemi-ological studies on genetic effects of radiation will be undertaken.Our inability, thus far, to find a suitable animal model for assessingthe mutational radiosensitivity of the human female has remaineda major unsolved issue; this stands a good chance of being resolvedby computational modeling. The scientists who have been mostactive in this field are those with formal backgrounds in classicalgenetics, radiobiology and population genetics. What the field nowneeds is a new generation of scientists, who can energize it and arewilling to collaborate: motivated model builders, DNA repairspecialists, genome scientists, and human geneticists, to name thecore group. Encouragement and support of their endeavors areessential. Obviously, one is not dealing with ‘low-hanging fruit,’but it will be a major scientific achievement if it becomes possibleto state in another decade or so that the genetic risks to both sexesof our species at low doses of radiation are indeed small and not acause for concern.

Conflict of interests

The authors report no conflict of interest. The authors areresponsible for the content and writing of the paper.

Acknowledgments

We wish to thank Dr. John Huddleston and Dr. Evan Eichler ofthe University of Washington, Seattle for generously providing uswith information on human segmental duplications. We thankShirin Rahmanian, Dr. Thiansin Liamsuwan, Dr. Reza Taleei, andProfessor Shuzo Uehara for helping with data and preparation ofthe manuscript. We are deeply grateful to Prof. George Hoffmannfor his very valuable suggestions and superb editorial work on themanuscript. We also wish to thank the ‘readers’ for their perceptivecomments. The work of Radiation Biophysics Group (RBG) issupported by the Swedish Radiation Safety Authority (SSM) andKarolinska Institutet.

References

[1] K. Sankaranarayanan, H. Nikjoo, Ionising radiation and genetic risks. XVI. Agenome-based framework for risk estimation in the light of recent advances ingenome research, Int. J. Radiat. Biol. 87 (2011) 161–178, http://dx.doi.org/10.3109/09553002.2010.518214.

[2] The 2007 Recommendations of the International Commission on RadiologicalProtection. ICRP publication 103, Ann. ICRP 37 (2007) 1–332, http://dx.doi.org/10.1016/j.icrp.2007.10.003.

[3] K. Sankaranarayanan, J.S. Wassom, Reflections on the impact of advances in theassessment of genetic risks of exposure to ionizing radiation on internationalradiation protection recommendations between the mid-1950s and the present,Mutat. Res. 658 (2008) 1–27, http://dx.doi.org/10.1016/j.mrrev.2007.10.004.

[4] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic risks. XI. Thedoubling dose estimates from the mid-1950s to the present and the conceptualchange to the use of human data on spontaneous mutation rates and mouse dataon induced mutation rates for doubling dose calculations, Mutat. Res. 453 (2000)107–127.

[5] J.V. Neel, W.J. Schull (Eds.), The Children of Atomic Bomb Survivors: A GeneticStudy, The National Academies Press, Washington, DC, 1991, http://www.nap.edu/openbook.php?record_id=1800.

[6] J.V. Neel, Genetic studies at the Atomic Bomb Casualty Commission – RadiationEffects Research Foundation: 1946–1997, Proc. Natl. Acad. Sci. U. S. A. 95 (1998)5432–5436.

[7] NRC, National Research Council, Health Risks of Exposure to Low Levels ofIonizing Radiation, BEIR VII, Phase 2, The National Academies Press, Washington,DC, 2006, http://www.nap.edu/openbook.php?.record_id=11340.

[8] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic risks. XIII.Summary and synthesis of papers VI to XII and estimates of genetic risks in theyear 2000, Mutat. Res. 453 (2000) 183–197.

[9] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Report to the General Assembly, with Scientific Annex, UnitedNations, New York, 2001.

[10] H.J. Muller, Radiation damage to the genetic material, in: G.A. Baitsell (Ed.),Science in Progress, Yale University Press, New Haven, 1951, pp. 93–97.

[11] H.J. Muller, The manner of dependence of the permissible dose of radiation onthe amount of genetic damage, Acta Radiol. 41 (1954) 5–20.

[12] H.J. Muller, Advances in radiation mutagenesis through studies on Drosophila,Prog. Nucl. Energy 6: Biol. Sci. 2 (1959) 146–160.

[13] R. Chakraborty, N. Yasuda, C. Denniston, K. Sankaranarayanan, Ionizing radiationand genetic risks. VII. The concept of mutation component and its use in riskestimation for Mendelian diseases, Mutat. Res. 400 (1998) 541–552.

[14] K. Sankaranarayanan, R. Chakraborty, Ionizing radiation and genetic risks. XII.The concept of ‘‘potential recoverability correction factor’’ (PRCF) and its use forpredicting the risk of radiation-inducible genetic disease in human live births,Mutat. Res. 453 (2000) 129–181.

[15] K. Sankaranarayanan, Genetic Effects of Ionizing Radiation in MulticellularEukaryotes and the Assessment of Genetic Radiation Hazards in Man, Elsevier,Amsterdam, 1982.

[16] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Report to the United Nations General Assembly, United Nations,New York, 1972.

[17] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Sources and Effects of Ionizing Radiation, Report to the UnitedNations General Assembly, United Nations, New York, 1977.

[18] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Sources and Biological Effects, Report to the United Nations GeneralAssembly, United Nations, New York, 1982.

[19] T.G. Baker, Radiosensitivity of mammalian oocytes with particular reference tothe human female, Am. J. Obstet. Gynecol. 110 (1971) 746–761.

Table 3Estimates of the average number of electron tracks generated per cell for three

different types of low LET radiation.

Sourcea At depth

(mm)bDose

Average number

of electron

tracks/cellc

30KV X 1.00 0.1 0.023

0.2 0.041

0.5 0.121

1.0 0.229

10.0 2.298

20.0 4.633

100.0 22.786

1000.0 231.193

250KV X 2.0 0.1 0.025

0.2 0.036

0.5 0.093

1.0 0.187

10.0 1.789

20.0 3.730

100.0 18.430

1000.0 184.284

60Co g-photons 5.00 0.1 0.002

1.0 0.005

2.0 0.009

10.0 0.049

20.0 0.109

100.0 0.571

1000.0 5.589

a Typical ionizing radiations used in radiobiology experiments, diagnostics,

imaging and therapy.b The attenuation of the low energy component of X-rays is much larger as the

depth increases. The reason for different depths is to keep the primary spectral

shape as much as possible.c Based on the total number of electron tracks generated in a grid of 1000 cells.

higher doses and dose-rates, remains a prudent basis forradiological protection at low doses and dose-rates. . .’’. Two pointsmerit mention here: (a) the calculation of the ‘‘effective dose,’’ thespecial radiation unit used by ICRP to quantify ‘total risk’ (i.e., thesum of absorbed doses weighted for radiation type and for tissues;the basis for radiation protection recommendations on dose limits)relies implicitly on the LNT assumption, and (b) since valuejudgments about the relative importance of different kinds of risksand the balancing of risks and benefits have always been part of theradiation protection decision-making process, there has neverbeen a one-to-one correspondence between the estimated risksand recommendations on dose limits, either to the workers or tothe population. For instance, the dose limits recommended by ICRPin 1991 and 2007 are the same despite revisions of risk estimates(see Tables 12 and 17 in Sankaranarayanan and Wassom [3]).

In contrast to the views of the scientific committees mentionedabove, the French National Academies of Science and Medicine[139,140] stated that ‘‘. . .the LNT assumption may greatlyoverestimate the carcinogenic effects of low doses (<100 mSv)and even more that of very low doses (<1.0 mSv) such as thosedelivered during X-ray examinations.’’ In a subsequent paper,Tubiana et al. [141] concluded that ‘‘. . .LNT was a useful model halfa century ago. But current radiation protection concepts should bebased on facts and on concepts consistent with current scientificresults and not on opinions. Preconceived concepts impedeprogress; in the case of the LNT model, they have resulted insubstantial medical, economic and societal harm. . .’’

A detailed discussion of the different arguments advanced infavor of or against the LNT model falls outside the scope of thisarticle. It suffices to note that we continue to believe that theapproach used by ICRP and the BEIR VII Committee is a sound onefor radiation protection purposes at present. Readers interested indiscussion of the LNT model may wish to study Trott andRosemann [142], Brenner et al. [137], and three other paperspublished in 2006 [143–145]. In the most recent paper on low doseeffects, Sasaki et al. [146] discussed a novel nonparametricstatistical procedure based on the noise cancelation process ofartificial neural networks (ANN) and its application to cancerdatabases established by the Radiation Effects Research Founda-tion in Japan. They believe that their analysis reveals (a) uniquefeatures of low dose responses (i.e., doses below 0.2 Sv), includingthe presence of a threshold that varies with organ, gender and ageat exposure, and (b) that the threshold did not conform to theconventional definition of zero effect, in that it manifested anegative excess relative risk, or suppression of background cancerrates. They explained this observation on the basis of experimentalevidence on DNA DSB repair pathway choice and its epigeneticmemory by histone marking.

5.2. Computational modeling and LNT

In addressing the question whether mechanism-driven compu-tational modeling of genetic risks can be robust enough to beinformative of risks at low doses independently of the LNT model,we wish to emphasize that the LNT model is a phenomenologicalrisk model, not a mechanistic one. However, it can be (and, in fact,has been) rationalized for low doses at the most fundamental levelof the mechanisms of radiation action, namely, the relativenumbers of electron tracks traversing the irradiated cell nuclei.The UNSCEAR report ([47], Annex G, Section I) and the paper ofBrenner et al. [137] consider the biophysical evidence based onmicrodosimetry to argue for linearity at low doses.

Here we use the illustrative example given by Brenner andcolleagues [137]. Assume that an organ dose of 10 mGy ofdiagnostic X-rays causes an increase in cancer risk. On thebiophysical premise that the number of electron tracks within cells

follows a Poisson distribution at this low dose, most irradiated cellnuclei will be traversed by one, or at most a few, physically distanttracks. Being so physically distant, it is very unlikely that these fewelectron tracks could produce DNA damage in some jointcooperative way; rather, these electron tracks will act indepen-dently to produce stochastic damage and consequent cellularchanges. Decreasing the dose, say, by a factor of 10, will simplyresult in proportionately fewer electron tracks and fewer ‘hit’ cells.It follows that those fewer cells that are hit at the 10-fold lowerdose will be subject to (i) the same type of electron damage and (ii)the same radiobiological processes as would occur at 10 mGy.Thus, decreasing the number of damaged cells by a factor of10 would be expected to decrease the biological response by thesame factor of 10, i.e., the response would decrease linearly with

decreasing dose. In other words, one should not expect qualitativelydifferent biological processes to be active at 1 mGy that were notactive at 10 mGy or vice versa. Fig. 1 and Table 3 summarizecalculations that show the relationship between absorbed dose intissue and mean number of electron tracks generated in a cellnucleus of 10 mm diameter. Because of the stochastic nature ofionizing radiation tracks, not all cells receive the same number oftracks – some receive more than others, and some receive nonedepending on the exposure dose. The calculations were carried outusing Monte Carlo track structure methods. Therefore, no prior

assumption was made of dose linearity. Brenner et al. note that thisbiophysical argument for linearity considers radiation effects dueto autonomous responses of individual cells but will also be validunder conditions of multicellular interactions as long as the rate-limiting radiation damage step is a single cell process.

For radiation-induced hereditary effects the rate-limitingdamage at the molecular level is assumed to be a radiation-induced DNA DSB, possibly a complex one, that generates a DNAdeletion of defined length as a result of the operation of error-prone repair processes. The DSB repair process that actually comesinto play may vary depending on the cell cycle stage of theirradiated cell. If it is assumed that the DSB repair processes are thesame at low doses of the magnitude discussed in this section, thenone can argue that the LNT concept may be valid up to the time

Fig. 1. Average number of electron tracks generated in a cell nucleus of 10 mm

diameter as a function of radiation dose. The indicated radiation types are used in

cellular radiobiology experiments, and they are used clinically for diagnostic

imaging and therapy. The calculations were carried out in a cubical tissue model

(100 mm � 100 mm � 100 mm), composed of a cubical lattice of

10 mm � 10 mm � 10 mm cells.

[77] D. Shechter, V. Costanzo, J. Gautier, Regulation of DNA replication by ATR:signaling in response to DNA intermediates, DNA Repair (Amst.) 3 (2004)901–908, http://dx.doi.org/10.1016/j.dnarep.2004.03.020.

[78] T.H. Stracker, J.-W.F. Theunissen, M. Morales, J.H.J. Petrini, The Mre11 complexand the metabolism of chromosome breaks: the importance of communicatingand holding things together, DNA Repair (Amst.) 3 (2004) 845–854, http://dx.doi.org/10.1016/j.dnarep.2004.03.014.

[79] T. Stiff, M. O’Driscoll, N. Rief, K. Iwabuchi, M. Lobrich, P.A. Jeggo, ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizingradiation, Cancer Res. 64 (2004) 2390–2396.

[80] E.P. Rogakou, D.R. Pilch, A.H. Orr, V.S. Ivanova, W.M. Bonner, DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139, J. Biol.Chem. 273 (1998) 5858–5868.

[81] E.P. Rogakou, C. Boon, C. Redon, W.M. Bonner, Megabase chromatin domainsinvolved in DNA double-strand breaks in vivo, J. Cell Biol. 146 (1999) 905–916.

[82] T.T. Paull, E.P. Rogakou, V. Yamazaki, C.U. Kirchgessner, M. Gellert, W.M. Bonner,A critical role for histone H2AX in recruitment of repair factors to nuclear fociafter DNA damage, Curr. Biol. 10 (2000) 886–895.

[83] O. Fernandez-Capetillo, A. Lee, M. Nussenzweig, A. Nussenzweig, H2AX: thehistone guardian of the genome, DNA Repair (Amst.) 3 (2004) 959–967, http://dx.doi.org/10.1016/j.dnarep.2004.03.024.

[84] E.L. Leatherbarrow, J.V. Harper, F.A. Cucinotta, P. O’Neill, Induction and quanti-fication of gamma-H2AX foci following low and high LET-irradiation, Int. J.Radiat. Biol. 82 (2006) 111–118, http://dx.doi.org/10.1080/09553000600599783.

[85] K. Rothkamm, M. Lobrich, Evidence for a lack of DNA double-strand break repairin human cells exposed to very low X-ray doses, Proc. Natl. Acad. Sci. U. S. A. 100(2003) 5057–5062, http://dx.doi.org/10.1073/pnas.0830918100.

[86] N. Bennardo, A. Cheng, N. Huang, J.M. Stark, Alternative-NHEJ is a mechanisti-cally distinct pathway of mammalian chromosome break repair, PLoS Genet. 4(2008) e1000110, http://dx.doi.org/10.1371/journal.pgen.1000110.

[87] S. Girirajan, C.D. Campbell, E.E. Eichler, Human copy number variation andcomplex genetic disease, Annu. Rev. Genet. 45 (2011) 203–226, http://dx.doi.org/10.1146/annurev-genet-102209-163544.

[88] T. Helleday, J. Lo, D.C. van Gent, B.P. Engelward, DNA double-strand break repair:from mechanistic understanding to cancer treatment, DNA Repair (Amst.) 6(2007) 923–935, http://dx.doi.org/10.1016/j.dnarep.2007.02.006.

[89] A. Kakarougkas, P.A. Jeggo, DNA, DSB repair pathway choice: an orchestratedhandover mechanism, Br. J. Radiol. 87 (2014) 20130685, http://dx.doi.org/10.1259/bjr.20130685.

[90] J.R. Lupski, P. Stankiewicz, Genomic disorders: molecular mechanisms for rear-rangements and conveyed phenotypes, PLoS Genet. 1 (2005) e49, http://dx.doi.org/10.1371/journal.pgen.0010049.

[91] M. McVey, S.E. Lee, MMEJ repair of double-strand breaks (director’s cut): deletedsequences and alternative endings, Trends Genet. 24 (2008) 529–538, http://dx.doi.org/10.1016/j.tig.2008.08.007.

[92] L.S. Symington, J. Gautier, Double-strand break end resection and repair pathwaychoice, Annu. Rev. Genet. 45 (2011) 247–271, http://dx.doi.org/10.1146/annurev-genet-110410-132435.

[93] L.H. Thompson, Recognition, signaling, and repair of DNA double-strand breaksproduced by ionizing radiation in mammalian cells: the molecular choreogra-phy, Mutat. Res. 751 (2012) 158–246, http://dx.doi.org/10.1016/j.mrrev.2012.06.002.

[94] M. Wang, W. Wu, W. Wu, B. Rosidi, L. Zhang, H. Wang, et al., PARP-1 and Kucompete for repair of DNA double strand breaks by distinct NHEJ pathways,Nucleic Acids Res. 34 (2006) 6170–6182, http://dx.doi.org/10.1093/nar/gkl840.

[95] L.G. De Fazio, R.M. Stansel, J.D. Griffith, G. Chu, Synapsis of DNA ends by DNA-dependent protein kinase, EMBO J. 21 (2002) 3192–3200.

[96] S. Moore, F.K.T. Stanley, A.A. Goodarzi, The repair of environmentally relevantDNA double strand breaks caused by high linear energy transfer irradiation – nosimple task, DNA Repair (Amst.) 17 (2014) 64–73, http://dx.doi.org/10.1016/j.dnarep.2014.01.014.

[97] L. Metzger, G. Iliakis, Kinetics of DNA double-strand break repair throughout thecell cycle as assayed by pulsed field gel electrophoresis in CHO cells, Int. J. Radiat.Biol. 59 (1991) 1325–1339.

[98] M. Lobrich, B. Rydberg, P.K. Cooper, Repair of X-ray-induced DNA double-strandbreaks in specific Not I restriction fragments in human fibroblasts: joining ofcorrect and incorrect ends, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 12050–12054.

[99] M. Belli, R. Cherubini, M. Dalla Vecchia, V. Dini, G. Moschini, C. Signoretti, et al.,DNA DSB induction and rejoining in V79 cells irradiated with light ions: aconstant field gel electrophoresis study, Int. J. Radiat. Biol. 76 (2000) 1095–1104.

[100] B. Stenerlow, E. Hoglund, J. Carlsson, E. Blomquist, Rejoining of DNA fragmentsproduced by radiations of different linear energy transfer, Int. J. Radiat. Biol. 76(2000) 549–557.

[101] S.J. DiBiase, Z.C. Zeng, R. Chen, T. Hyslop, W.J. Curran, G. Iliakis, DNA-dependentprotein kinase stimulates an independently active, nonhomologous, end-joiningapparatus, Cancer Res. 60 (2000) 1245–1253.

[102] P. Reynolds, J.A. Anderson, J.V. Harper, M.A. Hill, S.W. Botchway, A.W. Parker,et al., The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizingradiation is dependent on the complexity of damage, Nucleic Acids Res. 40(2012) 10821–10831, http://dx.doi.org/10.1093/nar/gks879.

[103] A. Shibata, S. Conrad, J. Birraux, V. Geuting, O. Barton, A. Ismail, et al., Factorsdetermining DNA double-strand break repair pathway choice in G2 phase,EMBO J. 30 (2011) 1079–1092, http://dx.doi.org/10.1038/emboj.2011.27.

[104] A.A. Goodarzi, A.T. Noon, D. Deckbar, Y. Ziv, Y. Shiloh, M. Lobrich, et al., ATMsignaling facilitates repair of DNA double-strand breaks associated with het-

erochromatin, Mol. Cell 31 (2008) 167–177, http://dx.doi.org/10.1016/j.mol-cel.2008.05.017.

[105] E. Riballo, M. Kuhne, N. Rief, A. Doherty, G.C.M. Smith, M.-J. Recio, et al., Apathway of double-strand break rejoining dependent upon ATM, Artemis, andproteins locating to gamma-H2AX foci, Mol. Cell 16 (2004) 715–724, http://dx.doi.org/10.1016/j.molcel.2004.10.029.

[106] P.-O. Mari, B.I. Florea, S.P. Persengiev, N.S. Verkaik, H.T. Bruggenwirth, M.Modesti, et al., Dynamic assembly of end-joining complexes requires interactionbetween Ku70/80 and XRCC4, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 18597–18602, http://dx.doi.org/10.1073/pnas.0609061103.

[107] E. Van Dyck, A.Z. Stasiak, A. Stasiak, S.C. West, Visualization of recombinationintermediates produced by RAD52-mediated single-strand annealing, EMBORep. 2 (2001) 905–909, http://dx.doi.org/10.1093/embo-reports/kve201.

[108] W. Gu, F. Zhang, J.R. Lupski, Mechanisms for human genomic rearrangements,Pathogenetics 1 (2008) 4, http://dx.doi.org/10.1186/1755-8417-1-4.

[109] P. Stankiewicz, J.R. Lupski, Genome architecture, rearrangements and genomicdisorders, Trends Genet. 18 (2002) 74–82.

[110] L. Pellegrini, D.S. Yu, T. Lo, S. Anand, M. Lee, T.L. Blundell, et al., Insights into DNArecombination from the structure of a RAD51–BRCA2 complex, Nature 420(2002) 287–293, http://dx.doi.org/10.1038/nature01230.

[111] F.A. Cucinotta, J.M. Pluth, J.A. Anderson, J.V. Harper, P. O’Neill, Biochemicalkinetics model of DSB repair and induction of gamma-H2AX foci by non-homologous end joining, Radiat. Res. 169 (2008) 214–222, http://dx.doi.org/10.1667/RR1035.1.

[112] Y. Li, F.A. Cucinotta, Modeling non-homologous end joining, J. Theor. Biol. 283(2011) 122–135, http://dx.doi.org/10.1016/j.jtbi.2011.05.015.

[113] Y. Li, H. Qian, Y. Wang, F.A. Cucinotta, A stochastic model of DNA fragmentsrejoining, PLoS ONE 7 (2012) e44293, http://dx.doi.org/10.1371/journal.-pone.0044293.

[114] Y. Li, P. Reynolds, P. O’Neill, F.A. Cucinotta, Modeling damage complexity-dependent non-homologous end-joining repair pathway, PLOS ONE 9 (2014)e85816, http://dx.doi.org/10.1371/journal.pone.0085816.

[115] R. Taleei, M. Weinfeld, H. Nikjoo, A kinetic model of single-strand annealing forthe repair of DNA double-strand breaks, Radiat. Prot. Dosim. 143 (2011) 191–195, http://dx.doi.org/10.1093/rpd/ncq535.

[116] K.H. Karlsson, I. Radulescu, B. Rydberg, B. Stenerlow, Repair of radiation-inducedheat-labile sites is independent of DNA-PKcs, XRCC1 and PARP, Radiat. Res. 169(2008) 506–512, http://dx.doi.org/10.1667/RR1076.1.

[117] N. Uematsu, E. Weterings, K. Yano, K. Morotomi-Yano, B. Jakob, G. Taucher-Scholz, et al., Autophosphorylation of DNA-PKCS regulates its dynamics at DNAdouble-strand breaks, J. Cell Biol. 177 (2007) 219–229, http://dx.doi.org/10.1083/jcb.200608077.

[118] S.W. Botchway, D.L. Stevens, M.A. Hill, T.J. Jenner, P. O’Neill, Induction andrejoining of DNA double-strand breaks in Chinese hamster V79-4 cells irradiatedwith characteristic aluminum K and copper L ultrasoft X rays, Radiat. Res. 148(1997) 317–324.

[119] C.M. de Lara, M.A. Hill, T.J. Jenner, D. Papworth, P. O’Neill, Dependence of theyield of DNA double-strand breaks in Chinese hamster V79-4 cells on the photonenergy of ultrasoft X rays, Radiat. Res. 155 (2001) 440–448.

[120] X. Deng, A. Prakash, K. Dhar, G.S. Baia, C. Kolar, G.G. Oakley, et al., Humanreplication protein A-Rad52-single-stranded DNA complex: stoichiometry andevidence for strand transfer regulation by phosphorylation, Biochemistry 48(2009) 6633–6643, http://dx.doi.org/10.1021/bi900564k.

[121] H. Wang, Z.C. Zeng, T.A. Bui, E. Sonoda, M. Takata, S. Takeda, et al., Efficientrejoining of radiation-induced DNA double-strand breaks in vertebrate cellsdeficient in genes of the RAD52 epistasis group, Oncogene 20 (2001) 2212–2224,http://dx.doi.org/10.1038/sj.onc.1204350.

[122] R. Robledo, S. Orru, A. Sidoti, R. Muresu, D. Esposito, M.C. Grimaldi, C. Carcassi, A.Rinaldi, et al., A 9.1 kb gap in the genome reference map is shown to be a stabledeletion/insertion polymorphism of ancestral origin, Genomics 80 (2002) 585–592.

[123] E.E. Eichler, R.A. Clark, X. She, An assessment of the sequence gaps: unfinishedbusiness in a finished human genome, Nat. Rev. Genet. 5 (2004) 345–354.

[124] Editorial, E pluribus unum, Nat. Methods 7 (2010) 331.[125] D.M. Church, V.A. Schneider, T. Graves, K. Auger, F. Cunningham, N. Bouk, et al.,

Modernizing reference genome assemblies, PLoS Biol. 9 (2011) e1001091.[126] K.M. Steinberg, V.A. Schneider, T.A. Graves-Lindsay, R.S. Fulton, R. Agarwala, J.

Huddleston, S.A. Shiryev, et al., Single haplotype assembly of the human genomefrom a hydatiform mole, Genome Res. (2014), http://dx.doi.org/10.1101/gr.180893.14.

[127] M.J.P. Chaisson, J. Huddleston, M.Y. Dennis, P.H. Sudmant, M. Malig, F. Hormoz-diari, F. Antonacci, et al., Resolving the complexity of the human genome usingsingle molecule sequencing, Nature (2014), http://dx.doi.org/10.1038/na-ture13907.

[128] E. Callaway, ‘Platinum’ genome takes on disease, Nature 515 (2014) 323.[129] N.I. Weisenfeld, S. Yin, T. Sharpe, B. Lau, R. Hegarty, L. Holmes, B. Sogoloff, D.

Tabbaa, L. Willimas, et al., Comprehensive variation discovery in single humangenomes, Nat. Genet. 46 (2014) 1350–1355.

[130] L. Stein, Genome annotation: from sequence to biology, Nat. Rev. Genet. 2 (2001)493–503.

[131] J. Harrow, A. Frankish, J.M. Gozalez, E. Tapanari, M. Diekhans, F. Kokocinski, B.L.Aken, D. Barrell, A. Zadissa, S. Searle, et al., GENCODE: the reference humangenome annotation for the ENCODE project, Genome Res. 22 (2012) 1760–1774.

[132] H. Qu, X. Fang, A brief review on the human encyclopedia of DNA elements,Genomics Proteomics Bioinform. 11 (2013) 135–141.

[20] E.F. Oakberg, E. Clark, Species comparisons of radiation response of the gonads,in: W.D. Carlson, F.X. Gassner (Eds.), Effects of Radiation on the ReproductiveSystem, Pergamon Press, Oxford, 1964, pp. 11–24.

[21] T.G. Baker, The sensitivity of oocytes in post-natal rhesus monkeys to X-irradia-tion, J. Reprod. Fertil. 12 (1966) 183–192.

[22] T.G. Baker, Comparative aspects of the effects of radiation during oogenesis,Mutat. Res. 11 (1971) 9–22.

[23] W.H. Wallace, S.M. Shalet, J.H. Hendry, P.H. Morris-Jones, H.R. Gattamaneni,Ovarian failure following abdominal irradiation in childhood: the radiosensitiv-ity of the human oocyte, Br. J. Radiol. 62 (1989) 995–998.

[24] W.H.B. Wallace, A.B. Thomson, T.W. Kelsey, The radiosensitivity of the humanoocyte, Hum. Reprod. 18 (2003) 117–121.

[25] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Effects and Risks of Ionizing Radiation, Report to the GeneralAssembly, United Nations, New York, 1988.

[26] National Research Council (NRC), Biological Effects of Atomic Radiation (BEIR 1),The National Academies Press, Washington, DC, 1956.

[27] A.C. Stevenson, The load of hereditary defects in human populations, Radiat. Res.(Suppl. 1) (1959) 306–325.

[28] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Report to the General Assembly with Annexes, United Nations,New York, 1958.

[29] A. Schinzel, Microdeletion syndromes, balanced translocations, and gene map-ping, J. Med. Genet. 25 (1988) 454–462.

[30] T. Strachen, A. Read, Human Molecular Genetics, Bios Scientific Publishers,Oxford, 1996.

[31] J.R. Lupski, Genomic disorders: structural features of the genome can lead toDNA rearrangements and human disease traits, Trends Genet. 14 (1998) 417–422.

[32] K. Sankaranarayanan, Ionizing radiation and genetic risks. X. The potential‘‘disease phenotypes’’ of radiation-induced genetic damage in humans: per-spectives from human molecular biology and radiation genetics, Mutat. Res. 429(1999) 45–83.

[33] E.S. Lander, L.M. Linton, B. Birren, C. Nusbaum, M.C. Zody, J. Baldwin, et al., Initialsequencing and analysis of the human genome, Nature 409 (2001) 860–921,http://dx.doi.org/10.1038/35057062.

[34] E. Birney, A. Bateman, M.E. Clamp, T.J. Hubbard, Mining the draft human genome,Nature 409 (2001) 827–828, http://dx.doi.org/10.1038/35057004.

[35] H. Nikjoo, K. Sankaranarayanan, State of the art in research into the risk of lowdose radiation exposure, J. Radiol. Prot. 34 (2014) 253–258, http://dx.doi.org/10.1088/0952-4746/34/1/L01.

[36] S. Salomaa, K.M. Prise, M.J. Atkinson, A. Wojcik, A. Auvinen, B. Grosche, et al.,Reply to ‘‘State of the art in research into the risk of low dose radiation exposure’’,J. Radiol. Prot. 34 (2014) 259–260.

[37] H. Nikjoo, P. O’Neill, W.E. Wilson, D.T. Goodhead, Computational approach fordetermining the spectrum of DNA damage induced by ionizing radiation, Radiat.Res. 156 (2001) 577–583.

[38] H. Nikjoo, P. Girard, A model of the cell nucleus for DNA damage calculations, Int. J.Radiat. Biol. 88 (2012) 87–97, http://dx.doi.org/10.3109/09553002.2011.640860.

[39] K. Sankaranarayanan, J.S. Wassom, Ionizing radiation and genetic risks XIV.Potential research directions in the post-genome era based on knowledge ofrepair of radiation-induced DNA double-strand breaks in mammalian somaticcells and the origin of deletions associated with human genomic disorders, Mutat.Res. 578 (2005) 333–370, http://dx.doi.org/10.1016/j.mrfmmm.2005.06.020.

[40] K. Sankaranarayanan, R. Taleei, S. Rahmanian, H. Nikjoo, Ionizing radiation andgenetic risks. XVII. Formation mechanisms underlying naturally occurring DNAdeletions in the human genome and their potential relevance for bridging thegap between induced DNA double-strand breaks and deletions in irradiatedgerm cells, Mutat. Res. (2013), http://dx.doi.org/10.1016/j.mrrev.2013.07.003.

[41] R. Taleei, H. Nikjoo, Repair of the double-strand breaks induced by low energyelectrons: a modelling approach, Int. J. Radiat. Biol. 88 (2012) 948–953, http://dx.doi.org/10.3109/09553002.2012.695098.

[42] R. Taleei, H. Nikjoo, The non-homologous end-joining (NHEJ) pathway for therepair of DNA double-strand breaks: I. A mathematical model, Radiat. Res. 179(2013) 530–539, http://dx.doi.org/10.1667/RR3123.1.

[43] R. Taleei, P. Girard, K. Sankaranarayanan, H. Nikjoo, The non-homologous end-joining (NHEJ) mathematical model for the repair of double-strand breaks. II.Application to damage induced by ultrasoft X-rays and low energy electrons,Radiat. Res. 179 (2013) 540–548.

[44] R. Taleei, H. Nikjoo, Biochemical DSB-repair model for mammalian cells in G1and early S phases of the cell cycle, Mutat. Res. 756 (2013) 206–212, http://dx.doi.org/10.1016/j.mrgentox.2013.06.004.

[45] R. Taleei, P. Girard, H. Nikjoo, DSB repair model for mammalian cells in early Sand G1 phases of the cell cycle: application to damage induced by ionizingradiation of different quality, Mutat. Res. (2015) (submitted for publication).

[46] S. Rahmanian, R. Taleei, H. Nikjoo, Radiation induced base excision repair (BER):a mechanistic mathematical approach, DNA Repair 22 (2014) 89–103.

[47] United Nations Scientific Committee on the Effects of Atomic Radiation(UNSCEAR), Report to the General Assembly, with Scientific Annex, UnitedNations, New York, 2000.

[48] H. Nikjoo, T. Liamsuwan, Biophysical basis of ionizing radiation, in: A. Brahme(Ed.), Comprehensive Biomedical Physics, 1st ed., vol. 9, 2014, in: A. Brahme(Ed.), Comprehensive Biomedical Physics, 2nd ed., vol. 9, 2014 (Chapter 2).

[49] W. Friedland, P. Kundrat, P. Jacob, Stochastic modelling of DSB repair afterphoton and ion irradiation, Int. J. Radiat. Biol. 88 (2012) 129–136, http://dx.doi.org/10.3109/09553002.2011.611404.

[50] W. Friedland, P. Jacob, H.G. Paretzke, T. Stork, Monte Carlo simulation of theproduction of short DNA fragments by low-linear energy transfer radiation usinghigher-order DNA models, Radiat. Res. 150 (1998) 170–182.

[51] D. Alloni, A. Campa, M. Belli, G. Esposito, A. Facoetti, W. Friedland, et al., A MonteCarlo study of the radiation quality dependence of DNA fragmentation spectra,Radiat. Res. 173 (2010) 263–271, http://dx.doi.org/10.1667/RR1957.1.

[52] D. Alloni, A. Campa, M. Belli, G. Esposito, L. Mariotti, M. Liotta, et al., Monte Carloevaluation of DNA fragmentation spectra induced by different radiation quali-ties, Radiat. Prot. Dosim. 143 (2011) 226–231, http://dx.doi.org/10.1093/rpd/ncq384.

[53] W.R. Holley, I.S. Mian, S.J. Park, B. Rydberg, A. Chatterjee, A model for interphasechromosomes and evaluation of radiation-induced aberrations, Radiat. Res. 158(2002) 568–580.

[54] W.R. Holley, A. Chatterjee, Clusters of DNA induced by ionizing radiation:formation of short DNA fragments. I. Theoretical modeling, Radiat. Res. 145(1996) 188–199.

[55] D.E. Charlton, H. Nikjoo, J.L. Humm, Calculation of initial yields of single- anddouble-strand breaks in cell nuclei from electrons, protons and alpha particles,Int. J. Radiat. Biol. 56 (1989) 1–19.

[56] Y. Nishino, M. Eltsov, Y. Joti, K. Ito, H. Takata, Y. Takahashi, et al., Human mitoticchromosomes consist predominantly of irregularly folded nucleosome fibreswithout a 30-nm chromatin structure, EMBO J. 31 (2012) 1644–1653, http://dx.doi.org/10.1038/emboj.2012.35.

[57] N. Naumova, M. Imakaev, G. Fudenberg, Y. Zhan, B.R. Lajoie, L.A. Mirny, et al.,Organization of the mitotic chromosome, Science 342 (2013) 948–953, http://dx.doi.org/10.1126/science.1236083.

[58] H. Nikjoo, P. O’Neill, D.T. Goodhead, M. Terrissol, Computational modelling oflow-energy electron-induced DNA damage by early physical and chemicalevents, Int. J. Radiat. Biol. 71 (1997) 467–483.

[59] H. Nikjoo, P. O’Neill, M. Terrissol, D.T. Goodhead, Quantitative modelling of DNAdamage using Monte Carlo track structure method, Radiat. Environ. Biophys. 38(1999) 31–38.

[60] H. Nikjoo, S. Uehera, Track structure – studies of biological systems, in: A.Mozumder, Y. Hatano (Eds.), Charged Particle and Photon Interactions withMatter: Chemical, Physicochemical, and Biological Consequences with Applica-tions, Marcel Dekker, New York, 2004.

[61] W. Friedland, H.G. Paretzke, F. Ballarini, A. Ottolenghi, G. Kreth, C. Cremer, Firststeps towards systems radiation biology studies concerned with DNA andchromosome structure within living cells, Radiat. Environ. Biophys. 47 (2008)49–61, http://dx.doi.org/10.1007/s00411-007-0152-x.

[62] A. Campa, D. Alloni, F. Antonelli, F. Ballarini, M. Belli, V. Dini, et al., DNAfragmentation induced in human fibroblasts by 56Fe ions: experimental dataand Monte Carlo simulations, Radiat. Res. 171 (2009) 438–445, http://dx.doi.org/10.1667/RR1442.1.

[63] W. Friedland, P. Jacob, P. Kundrat, Stochastic simulation of DNA double-strandbreak repair by non-homologous end joining based on track structure calcula-tions, Radiat. Res. 173 (2010) 677–688, http://dx.doi.org/10.1667/RR1965.1.

[64] W. Friedland, P. Jacob, P. Kundrat, Mechanistic simulation of radiation damage toDNA and its repair: on the track towards systems radiation biology modelling,Radiat. Prot. Dosim. 143 (2011) 542–548, http://dx.doi.org/10.1093/rpd/ncq383.

[65] D. Alloni, A. Campa, W. Friedland, L. Mariotti, A. Ottolenghi, Track structure,radiation quality and initial radiobiological events: considerations based on thePARTRAC code experience, Int. J. Radiat. Biol. 88 (2012) 77–86, http://dx.doi.org/10.3109/09553002.2011.627976.

[66] S.P. Jackson, Sensing and repairing DNA double-strand breaks, Carcinogenesis 23(2002) 687–696, http://dx.doi.org/10.1093/carcin/23.5.687.

[67] C.H. Bassing, F.W. Alt, The cellular response to general and programmed DNAdouble strand breaks, DNA Repair (Amst.) 3 (2004) 781–796, http://dx.doi.org/10.1016/j.dnarep.2004.06.001.

[68] P.A. Jeggo, V. Geuting, M. Lobrich, The role of homologous recombination inradiation-induced double-strand break repair, Radiother. Oncol. 101 (2011) 7–12, http://dx.doi.org/10.1016/j.radonc.2011.06.019.

[69] M. O’Driscoll, P.A. Jeggo, The role of double-strand break repair – insights fromhuman genetics, Nat. Rev. Genet. 7 (2006) 45–54, http://dx.doi.org/10.1038/nrg1746.

[70] C.J. Bakkenist, M.B. Kastan, DNA damage activates ATM through intermolecularautophosphorylation and dimer dissociation, Nature 421 (2003) 499–506,http://dx.doi.org/10.1038/nature01368.

[71] S. Burma, D.J. Chen, Role of DNA-PK in the cellular response to DNA double-strand breaks, DNA Repair (Amst.) 3 (2004) 909–918, http://dx.doi.org/10.1016/j.dnarep.2004.03.021.

[72] J. Falck, J. Coates, S.P. Jackson, Conserved modes of recruitment of ATM, ATR andDNA-PKcs to sites of DNA damage, Nature 434 (2005) 605–611, http://dx.doi.org/10.1038/nature03442.

[73] J. Kobayashi, A. Antoccia, H. Tauchi, S. Matsuura, K. Komatsu, NBS1 and itsfunctional role in the DNA damage response, DNA Repair (Amst.) 3 (2004) 855–861, http://dx.doi.org/10.1016/j.dnarep.2004.03.023.

[74] S. Koundrioukoff, S. Polo, G. Almouzni, Interplay between chromatin and cellcycle checkpoints in the context of ATR/ATM-dependent checkpoints, DNARepair (Amst.) 3 (2004) 969–978, http://dx.doi.org/10.1016/j.dnarep.2004.03.010.

[75] E.U. Kurz, S.P. Lees-Miller, DNA damage-induced activation of ATM and ATM-dependent signaling pathways, DNA Repair (Amst.) 3 (2004) 889–900, http://dx.doi.org/10.1016/j.dnarep.2004.03.029.

[76] J.H.J. Petrini, T.H. Stracker, The cellular response to DNA double-strand breaks:defining the sensors and mediators, Trends Cell Biol. 13 (2003) 458–462.

[133] G.M. Cooper, B.P. Coe, S. Girirajan, J.A. Rosenfeld, T.H. Vu, C. Baker, C. Williams, H.Stalker, et al., A copy number variation morbidity map of developmental delay,Nat. Genet. 43 (2011) 838–846.

[134] B.P. Coe, K. Witherspoon, J.A. Rosenfeld, B.W.M. van Bon, A.T. Vulto-van Silfhout,P. Bosco, K.L. Friend, C. Baker, et al., Refining analyses of copy number variationidentifies specific genes associated with developmental delay, Nat. Genet. 46(2014) 1063–1071.

[135] A.L. Beaudet, Reaching a CNV milestone, Nat. Genet. 46 (2014) 1046–1048.[136] K.L. Mossman, A brief history of radiation bioeffects, in: W.R. Hendee, F. Edwards

(Eds.), Health Effects of Exposure to Low Level Ionizing Radiation, IOP Publishing,Bristol, PA, 1996, pp. 495–525.

[137] D.J. Brenner, R. Doll, D.T. Goodhead, E.J. Hall, C.E. Land, J.B. Little, et al., Cancerrisks attributable to low doses of ionizing radiation: assessing what we reallyknow, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13761–13766, http://dx.doi.org/10.1073/pnas.2235592100.

[138] ICRP, Recommendations of the International Commission on Radiological Pro-tection, Publication 9 (Adopted September 17, 1965), Pergamon Press, Oxford,1966.

[139] M. Tubiana, Dose-effect relationship and estimation of the carcinogenic effectsof low doses of ionizing radiation: the joint report of the Academie des Sciences(Paris) and of the Academie Nationale de Medecine, Int. J. Radiat. Oncol. Biol.Phys. 63 (2005) 317–319, http://dx.doi.org/10.1016/j.ijrobp.2005.06.01.

[140] M. Tubiana, A. Aurengo, D. Averbeck, R. Masse, Recent reports on the effect oflow doses of ionizing radiation and its dose–effect relationship, Radiat.Environ. Biophys. 44 (2006) 245–251, http://dx.doi.org/10.1007/s00411-006-0032-9.

[141] M. Tubiana, L.E. Feinendegen, C. Yang, J.M. Kaminski, The linear no-thresholdrelationship is inconsistent with radiation biologic and experimental data,Radiology 251 (2009) 13–22, http://dx.doi.org/10.1148/radiol.2511080671.

[142] K.R. Trott, M. Rosemann, Molecular mechanisms of radiation carcinogenesis andthe linear, non-threshold dose response model of radiation risk estimation,Radiat. Environ. Biophys. 39 (2000) 79–87.

[143] J. Breckow, Linear-no-threshold is a radiation-protection standard rather than amechanistic effect model, Radiat. Environ. Biophys. 44 (2006) 257–260, http://dx.doi.org/10.1007/s00411-006-0030-y.

[144] D.J. Brenner, R.K. Sachs, Estimating radiation-induced cancer risks at verylow doses: rationale for using a linear no-threshold approach, Radiat. En-viron. Biophys. 44 (2006) 253–256, http://dx.doi.org/10.1007/s00411-006-0029-4.

[145] A.A. Friedl, W. Ruhm, LNT: a never-ending story, Radiat. Environ. Biophys. 44(2006) 241–244, http://dx.doi.org/10.1007/s00411-006-0028-5.

[146] M.S. Sasaki, A. Tachibana, S. Takeda, Cancer risk at low doses of ionizingradiation: artificial neural networks inference from atomic bomb survivors, J.Radiat. Res. 55 (2014) 391–406, http://dx.doi.org/10.1093/jrr/rrt133.

Reflections on the development and application of FISH whole chromosome painting ☆James D. Tucker

Department of Biological Sciences, 5047 Gullen Mall, Wayne State University, Detroit, MI 48202-3917, USAReflections in Mutation Research

Reflections on the development and application of FISH wholechromosome painting§

James D. Tucker

Department of Biological Sciences, 5047 Gullen Mall, Wayne State University, Detroit, MI 48202-3917, USA

1. Before my time: forerunners of molecular cytogenetics

The first localization of nucleic acids by in situ hybridization tometaphase chromosomes was described by Pardue and Gall in 1970[1]. Hybridization of [3H]-labeled mouse satellite DNA and itscomplementary RNA were found to localize to the pericentromericregions of every chromosome except the Y. I remember readingabout this work while I was in graduate school at the Oregon Health& Science University, and I realized that in situ hybridization had thepotential to be a truly special and powerful method. I was intriguedby the ability to query chromosomes and entice them into yieldingtheir deepest secrets. Everyone wanted to know what DNAsequences existed in the euchromatic and heterochromatic regionsof specific human chromosomes, and there was keen interest indetermining the map locations of specific genes. However, theresolution of the radiolabeling method was inherently low and thetechnique was cumbersome, with some hybridizations requiringweeks to obtain enough [3H]-disintegrations to evaluate. Tocomplicate matters, chromosome banding methods were in theirinfancy and it was clear that further technological advances wouldbe needed before in situ hybridization would see widespread use. Iwas occupied with other interests but the field of cytogenetics had

begun an inexorable tug on me. I finished graduate school andmoved to West Virginia University (WVU) in Morgantown.

2. The very early years

My first job after completing my Ph.D. was in clinical genetics,which did not work out well. Soon I was looking for another job.Joginder Nath, Chair of the Genetics & Developmental Biologyprogram at WVU, was a plant cytogeneticist who took specialinterest in my career. He threw a party at his house where I metsome of the key scientists in Morgantown. Years would pass beforeI learned that one of the reasons for this social event was to help mefind another job. I greatly enjoyed myself at that party. I met manypeople and was soon working for Tong-man Ong at the NationalInstitute for Occupational Safety and Health (NIOSH), which waslocated immediately adjacent to WVU. Tong-man was an excellentscientist and he turned out to be a fabulous mentor.

At NIOSH I was charged with getting a human cytogeneticslaboratory up and running. I had my own small research space,which was a room that had previously been used for Neurosporamutagenesis. The refrigerator still had vials of specific Neurosporastrains, and I could only imagine how many spores were lyingeverywhere, just waiting to grow explosively in my tissue cultureflasks. How was I to perform mammalian cell culture in anenvironment that (I presumed) was heavily contaminated withNeurospora? The last thing I wanted was fungal-contaminatedcultures. I spent hours spraying and wiping clean every surface

Article history:

Accepted 11 November 2014

Available online 22 November 2014

Keywords:

Chromosome painting

Fluorescence in situ hybridization

Molecular cytogenetics

Human cytogenetics

Rodent cytogenetics

Historical review

A B S T R A C T

This review describes my personal reflections on the development of whole chromosome painting using

fluorescence in situ hybridization and how my laboratory applied the technology in humans and in

animal models. The trials and triumphs of the early years are emphasized, along with some of the

scientific surprises that were encountered along the way. Scientific issues that my laboratory addressed

using chromosome painting technologies are summarized and related to questions in radiation

dosimetry, chemical clastogenesis, translocation persistence, and translocation frequencies in

unexposed people. A description is provided of scientific controversies that were encountered and

how they were resolved. I hope this paper will encourage young scientists to follow their passions and

pursue their scientific dreams even if the task seems daunting and the circumstances appear exceedingly

difficult. In my case the journey has been challenging, exciting, and richly rewarding on many levels.

§ This article is part of the Reflections in Mutation Research series. To suggest

topics and authors for Reflections, readers should contact the series editor, G.R.

Hoffmann (ghoffmann@holycross.edu).

E-mail address: jtucker@biology.biosci.wayne.edu.

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ rev iew sm rCo mm un i ty ad dr es s : w ww.els evier . co m/lo c ate /mu t r es

http://dx.doi.org/10.1016/j.mrrev.2014.11.006

or 1985 I brought up this possibility with a senior person at LLNLwho shall remain nameless. I was told that the signals generated byfluorochrome-conjugated in situ hybridized DNA ‘‘would never bebright enough to detect, and I can show you the math.’’ Being stillrather early in my career, I was unaware of the results of a Dutchgroup [4] that argued to the contrary, and I made the mistake ofbelieving this naysayer who claimed that my idea wouldn’t work.

In 1985 the Dutch group showed that fluorescence in situ

hybridization (FISH) could be used to map unique genes [5]. And in1986, Joe Gray’s group, working down the hall from my laboratory,used FISH on metaphase cells to identify human chromosomes insomatic cell hybrids [6]. Two years later they used chromosome-specific DNA libraries to paint chromosomes 4 and 21 in humanperipheral blood metaphase cells [7]. Importantly, they identifiedtranslocations in chromosome 4 by obvious color junctions,making it abundantly clear that fluorescence in situ hybridizationworked and that structural chromosome aberrations could bereadily identified. Paints for additional chromosomes were beingdeveloped by the Gray group. I was firmly convinced that paintingwould soon become routine and that it could be used to assaystructural and numerical chromosome aberrations in a widevariety of applications. I was specifically interested in developing aworkable assay for translocations, initially in human cells and laterin common laboratory animals. Painting one chromosome pair wasa major accomplishment, but a truly effective bioassay wouldrequire simultaneous labeling of multiple chromosomes. Joe Grayand his colleagues were kind enough to share some of theirvaluable probes with me. As the technology continued to improve,my laboratory was given enough probe to establish the methodfirmly in our hands and to obtain preliminary data that wereessential for grant applications. However, several years would passbefore my laboratory had its first publication with the method.

4. A fortuitous EMS meeting leading to a parallel path withmicronuclei

While the whole chromosome paints were being developed, Ihad other research projects. In 1985, Fenech and Morley hadpublished their seminal paper on micronuclei in cytokinesis-blocked cells [8], now known widely as the cytokinesis-blockmicronucleus (CBMN) assay. This method was simple in itselegance and was used to address many key scientific questions. In1988 I had the idea of using various molecular methods toinvestigate the contents of individual micronuclei. Micronucleiwere widely believed to consist of chromosome fragments andeven whole chromosomes, but no one had established a method todistinguish between these possibilities in individual cells. Havingbecome aware of anti-kinetochore antibodies, I reasoned that itshould be possible to apply them to cells on microscope slides in amanner that was directly analogous to the anti-BrdU antibodywork a few years previously.

The early results with an anti-kinetochore antibody were quitepromising and I was excited. A few months after I began thepreliminary experiments to make this assay work, a youngpostdoctoral fellow by the name of Dave Eastmond, who hadbeen awarded an Alexander Hollaender fellowship to come toLLNL, joined my laboratory for a few months. The original plan wasto have Dave work with several Livermore investigators during thecourse of his 2-year fellowship. Dave was interested in aneuploidyand saw the potential of the anti-kinetochore antibody assay withmicronuclei. He helped me finish the developmental aspects of thismethod, and soon the two of us were busy scoring coded slides todetermine whether aneugenic and clastogenic agents producedthe predicted types of micronuclei. They did, and several paperswere quickly published, e.g. [9] (Fig. 2). Although the developmentof the anti-kinetochore antibody assay was not directly related to

FISH-painting, it would soon prove to have a significant impact onthis nascent field. Of course I didn’t know this, and I had no ideawhat lay ahead.

The anti-kinetochore antibody papers caught the attention ofthe organizers of the 1991 meeting of the Environmental MutagenSociety (EMS, now called the Environmental Mutagenesis andGenomics Society – EMGS), which was held in Orlando, Florida. Iwas asked to anchor a session on micronuclei at a Workshop called‘‘Micronuclei as an Index of Cytogenetic Damage: Past, Present andFuture.’’ My talk was titled ‘‘The use of molecular probes tocharacterize the contents of micronuclei.’’ At this time the only‘‘probing’’ of micronuclei had been done with antibodies, yet FISHclearly worked on metaphase cells, and applying the same probesto binucleated cells was an obvious next step. The gist of my talkwas that molecular methods and cytology can and should bemerged, and that by doing so, compelling scientific questions couldbe asked and much would be learned. I ended the talk with a list ofscientific questions and possible future directions. I had lots ofideas and nowhere near enough money to carry them out (somethings never change!).

I needed collaborators and was hoping to find at least one atthat meeting. Much to my delight, my friend Joginder Nath fromWVU, who had been so instrumental in redirecting my career 9years previously, approached me and asked what it would take topursue some of the ideas I had shared. I said, ‘‘People. I have thelaboratory space and all the equipment, and I have money for

Fig. 2. Human peripheral blood lymphocyte cultured in the presence of

Cytochalasin B to block cytokinesis and obtain binucleated cells. Top panel:

DAPI staining to label the nuclei and simultaneous phase contrast to show the cell

membranes. Bottom panel: same cell, with the kinetochores labeled as described

[9]. (Figure from reference [9].)

J.D. Tucker / Mutation Research 763 (2015) 2–144

with a disinfectant. The hood, refrigerator, incubator, and all thehorizontal surfaces received my most utmost attention whichmust have paid off because I never had any fungal contaminationin my cultures. However, the laboratory smelled of disinfectant forweeks.

I began by culturing human peripheral blood lymphocytes. Theprevailing cytogenetic assays at the time were sister chromatidexchanges (SCEs) and structural chromosome aberrations inunbanded cells. There were several interesting projects to workon, and I loved every minute of it. One of the more challengingprojects involved evaluating diesel exhaust for cytogeneticactivity. I learned how to expose cultured cells to diesel fumesthat were bubbled through the flasks by way of plastic tubes thatran directly from the exhaust pipe of a large truck. The flasks had tobe maintained at 37 8C and protected from light because thebromodeoxyuridine (BrdU) that was required for seeing the SCEswas subject to photoactivation. To complicate matters, theexperiments were performed in winter, outside in the snow. Asluck would have it, the experiments always seem to be scheduledfor bright sunny days. Happily, water baths function effectivelyoutdoors and the control cultures showed us that aluminum foil isexcellent at keeping out bright sunlight. And yes, diesel exhaustinduces SCEs, at least in blood from some subjects [2].

Unfortunately the NIOSH job was only available for two years,and I needed to look for another position. In December 1983 therewas a symposium on SCEs at the Brookhaven National Laboratory.Tong-man and I drove there, and I planned to network with asmany people as possible to find another job. Highest on my list ofpeople to talk with was Tony Carrano who was at the LawrenceLivermore National Laboratory (LLNL). He was constantly sur-rounded by people and, try as I might, I couldn’t get near him toinitiate a conversation about working for him. As much as I enjoyedthe meeting, I left without any real job leads. Several months later Isaw an advertisement for a postdoctoral fellowship in cytogeneticsat LLNL. I immediately wrote the cover letter, attached my CV, andput the materials in an envelope. In those days there were no faxmachines or email, and no web sites for uploading applications.Snail mail was the only way to send the materials. I weighed theletter and found it needed two stamps. Alas, we had only one stampat home, which I licked and stuck on the envelope. I planned to stopby the post office and buy more stamps, but in the meantime Iplaced the letter on our desk. My wife saw the letter and thought Iforgot to mail it, so she put it in the mailbox for me. Miraculouslythe letter arrived because I was contacted a few weeks later andoffered an interview. A job offer ensued which I immediatelyaccepted. By July I was an LLNL employee. I never did learn whetherLLNL had to pay the postage due or if the US Postal Service hadn’tweighed the letter.

3. The first molecular cytogenetic success, but ‘‘your idea won’twork’’

At LLNL I began by working with the familiar endpoints of SCEsand structural chromosome aberrations. The science was interest-ing, and the answers to the scientific questions were relevant toour understanding of human responses to chemical and radiationexposures. However, the tools of molecular biology were rapidlybeing acquired by many laboratories, and more and moreinvestigators were incorporating these methods into their work.How, I wondered, might cytogenetics and molecular biology be‘‘married’’? I had no experience in anything molecular, but thefuture seemed clear. Tony came up with the idea of using anti-BrdUantibodies, which had been developed by another group in ourbuilding, to detect SCEs. BrdU was needed to visualize SCEs withthe fluorescence-plus-Giemsa technique, but BrdU also inducedSCEs. The scientific question was whether SCEs were an inherent

part of cell division or were an artifact of the culturing method. Weput serial dilutions of BrdU in the culture medium, then denaturedthe chromosomes and added the anti-BrdU antibody that wasdetected with a second antibody conjugated with fluorescein. Theresults showed that the frequencies of SCEs were independent ofBrdU at very low BrdU concentrations, indicating that SCEs were anatural cellular event and not induced by the culturing method.The results were esthetically pleasing, and we included colorphotographs of the labeled cells in the paper [3]. The publisherliked the photos and put them on the cover of the issue thatcontained our paper. This was my first publication to beaccompanied by an image on the journal cover, and perhaps moreimportantly, this was my first foray into ‘‘molecular’’ cytogenetics(Fig. 1).

The results of this work also indicated that fluorochromelabeling works at the molecular level and that our microscopescould see the signals very clearly. So why not do in situ

hybridization with fluorochrome-labeled DNA, rather than radio-actively-labeled DNA? I had not forgotten the work of Pardue andGall [1], and the next step seemed obvious, at least to me. In 1984

Fig. 1. Human (upper panel) and mouse (lower panel) peripheral blood

lymphocytes grown for one cell division in the presence of BrdU and a second

cell division without BrdU. Slides were stained with an anti-BrdU antibody followed

by a second antibody that was conjugated with fluorescein [3]. Propidium Iodide

(red) was used to visualize the unlabeled chromatids. The unlabeled mouse

chromosome is the late-replicating Y. (Figure from reference [3].)

J.D. Tucker / Mutation Research 763 (2015) 2–14 3

reagents and supplies.’’ He said, ‘‘We should think seriously about acollaboration in which some of our Ph.D. students can come workin your lab.’’ Thus began a long and very fruitful collaboration. Thefirst Ph.D. student, John Hando, used the anti-kinetochore antibodyand FISH probes for the sex chromosomes to show that the age-related increase in micronuclei in lymphocytes from women wasmostly due to the inactive X, whereas in males the age-relatedincrease could largely be explained by malsegregation of the Ychromosome. Molecular cytogenetics had now been used toanswer some fundamental biological questions, and we hadanother journal cover image [10].

As part of John’s Ph.D. work, we had collected dozens of bloodsamples from adults aged about 20 to nearly 80, and we also hadumbilical cord blood samples from about 20 newborns. In additionto preparing slides for the CBMN assay, I took a step of faith andcultured some of each blood sample to obtain cells in metaphase. Iwanted to perform whole chromosome painting on these sameindividuals to quantify their translocation frequencies. However,there was no money for painting, and everyone in my laboratorywas busy on other projects.

5. The development of whole chromosome painting

By now it was abundantly clear that the method of FISH wholechromosome painting worked well. However, some fundamentalquestions first had to be addressed if the method was to gain wide-spread acceptance. Not everyone thought the idea was worth-while. Some of the questions colleagues asked me were: ‘‘There are46 chromosomes in a normal human cell. What possible goodcould come from evaluating just a few of them?’’ ‘‘Howconsistently will the method work when it has to be applied tovaluable blood samples from exposed people?’’ ‘‘The probes are soexpensive, how can anybody afford to use them?’’ And the questionthat really raised my blood pressure was: ‘‘We already have thedicentric assay which has worked very well for so many years. Doyou really intend to replace that method?’’ Fortunately, by now Ihad learned not to take seriously the unsolicited advice andcriticisms that were freely offered by so many people. I held myscientific ground and pursued funding. Fortunately most of thereviewers of my proposals were positive, and grant money for FISHpainting started coming in.

5.1. The early days of whole chromosome painting with FISH

High on my list of priorities was the need to validate wholechromosome painting as an assay for radiation-induced chromo-some damage. The key questions were: ‘‘How did FISH transloca-tion frequencies induced by ionizing radiation compare with (a)dicentric frequencies observed by FISH painting on the samemicroscope slides, (b) dicentric frequencies scored with Giemsa-stained, i.e., unbanded, slides from the same cultures, and (c)translocation frequencies identified by G-banding?’’ I knew thatacceptance of FISH painting by the radiation biology communitywas absolutely essential; if the assay could not be validated to thesatisfaction of radiobiologists, then the future for the method as abiological dosimeter would be bleak. We began by irradiatinghuman whole blood and making dozens of slides from each dose.One of the first whole chromosome paints developed by the Graygroup was chromosome 4 [7] and so we began with thatchromosome (Fig. 3) [11]. In parallel we scored Giemsa-stainedcells for dicentrics, rings, and fragments, and G-banded cells for alltypes of rearrangements including translocations. Fortunately, thedose–response curves obtained by these different methods turnedout to be very similar once the smaller target size of the paintedchromosomes was considered [12,13]. Encouraged by theseresults, we decided to increase the fraction of translocations

detected by adding two more whole chromosome paints to thecocktail of probes. Chromosome 1 was added because it was thelargest chromosome, and we added the probe for chromosome 3because at that time it worked better than the probe forchromosome 2. We were now painting chromosomes 1, 3, and 4simultaneously, and detecting 32.9% of all the simple exchanges[11]. We also tried a nonfluorescent method of detecting thehybridized probe, which was a technological success except thatcentromere identification was quite difficult so we abandoned thatapproach. Had it worked, the problem of fading of the fluorescencesignal would have been avoided.

One of the key concerns about FISH painting was the cost of theprobes. However, we quickly learned that the amount ofchromosome painting data that could be generated in a givenperiod of time sitting at a microscope greatly exceeded the amountof data that could be obtained by conventional cytogeneticmethods. The faster throughput significantly offset the cost ofthe probes, meaning that FISH painting turned out to be lessexpensive, not more expensive, than classical cytogenetics becausethe personnel costs were substantially lower. Our trained slidereaders could routinely evaluate 1000 metaphase cells per day, andsometimes twice this number. With the ability to detect nearly 33%of all translocations, this corresponded to 330–660 whole genomeequivalents per day, which was 10- to 20-fold faster than G-banding analyses, and nearly double or even triple the rate atwhich many laboratories could evaluate unbanded cells fordicentrics.

5.2. Refinement of the painting methods

At about this same time, paints for chromosomes 1, 2, and 4became commercially available as one set, which we usedsuccessfully for several years, e.g., [14] (Fig. 4). I was happy thatwe no longer had to make our own probes as this saved usconsiderable effort. One of my laboratory’s funded projectsinvolved cytogenetic evaluations of the ‘‘cleanup’’ workers whowere exposed to ionizing radiation resulting from the Chernobylaccident (see Section 6 below). We were now several years into thisproject and we were also evaluating other exposed populations.

Fig. 3. Normal human cell in metaphase labeled with a whole chromosome paint for

chromosome 4.

The commercial availability of the probes meant that anybodycould now do what we were doing, and I wanted to stay ahead ofthe competition. To accomplish this we needed to reduce the costof the assay, which meant we had to improve the speed of themicroscopy, and this meant that we needed to add additionalpaints to the cocktail in a second color. I wanted to add the nextthree largest chromosomes so that we could detect as manychromosome exchanges as possible. I also wanted to paintchromosomes 4 and 5 in different colors because they areindistinguishable by their arm ratios. We resumed the productionof chromosome paints, and by 1998 we had enhanced the assay toconsist of chromosomes 1, 2, and 4 painted red, and 3, 5, and 6painted simultaneously in green [15] (Fig. 5). This combination ofwhole chromosome paints enabled us to detect 56% of all simpleexchanges. Adding the three additional chromosome probes waschallenging because we had to optimize the ratios of the individualpaints in the cocktail, but the additional efficiency was worth theeffort. Eventually this combination of probes became commercial-ly available, and is still sold today by several companies. At onetime we even tried adding chromosomes 7, 8, and 9 painted inyellow to the cocktail mix, but this turned out to make the slidereading harder rather than easier, so we abandoned that approach.

Within a few years it became possible to paint every humanchromosome in its own unique color [16,17]. This technologyprovided a major improvement in the ability to evaluatechromosomes in tumor cells, but the time-consuming nature ofthe analyses and the high cost of the probes precluded their routineuse in human exposure studies. These probes, however, werecommercially available which allowed investigators to ordercustomized sets of paints. One day a few years ago I asked agraduate student to order more painting probe for chromosomes1–6, anticipating receipt of our usual combination of chromosomes1, 2, and 4 painted red, and 3, 5, and 6 painted green. The probesarrived and were soon hybridized to a slide. A day or two later thestudent viewed the results and reported to me that ‘‘only some ofthe chromosomes were painted’’. Puzzled, I looked at thehybridization results myself and we soon realized that chromo-somes 1–6 were indeed painted, but that each chromosome pairwas labeled in its own unique color, and not every color was visiblewith our usual filter set. The student was devastated because we

had spent several thousand dollars on the probes, which were nowuseless for our intended purpose. However, I saw an opportunity.Mutation Research Reviews had for some years published on itscover an image from my laboratory of a cell with chromosomes 1,2, and 4 painted red, and 3, 5, and 6 painted green. I had an openinvitation to provide an updated image for the cover and I saw thissituation as an opportunity to do exactly that. I submitted a few ofthe images for consideration and was very pleased when one ofthem was accepted. The 6 chromosome pairs in their own uniquecolors provided a nice esthetic image which has now been on thecover for 4 years (Fig. 6).

After years of work establishing a multi-chromosome paintingmethod, we finally had an optimized and validated assay in whichsix pairs of chromosomes were painted, three pairs in two distinctcolors. It was reliable, faster, and less expensive than Giemsa-baseddicentric analyses, and it yielded equivalent results. The exquisitedetail of the structurally rearranged chromosomes was amazing,and as it turned out, problematic.

Fig. 4. Human cell in metaphase in which chromosomes 1, 2, and 4 are labeled with

Spectrum Orange [14]. The unpainted chromosomes are labeled with DAPI (blue).

Arrows indicate a reciprocal translocation between chromosome 4 and an

unpainted (blue) chromosome.

Fig. 5. Human cells in metaphase in which chromosomes 1, 2, and 4 are painted red

and chromosomes 3, 5, and 6 are simultaneously painted green. The unpainted

chromosomes are labeled with DAPI (blue). Top: arrows indicate a reciprocal

translocation between chromosome 6 (green) and an unpainted (blue)

chromosome. Bottom: multiple complex aberrations, only some of which are

indicated by arrows.

me because of another NIH Program Project we had, in which weinvestigated the cytogenetic effects of food mutagens known asheterocyclic amines. Our earlier work on these compounds withclassical cytogenetic methods indicated that they were clastogenicin mice [26] and in vitro [27], and I was keenly interested inknowing whether these compounds induced translocations.Fourth, I wanted to know whether translocation frequenciesdeclined with time following a single acute exposure to ionizingradiation.

Some people thought translocations would show completepersistence, i.e., not decline with time, e.g., [28]. However,Littlefield and Joiner [29] showed that at least some translocationspersist for years after accidental radiation exposure in humans,while the work by Buckton et al. [30] in irradiated ankylosingspondylitis patients suggested that some translocations showcomplete persistence and that others are lost with time. Takentogether, this evidence suggested, at least to me, that not alltranslocations persisted, but no one had subjected this importantquestion to rigorous experimentation. If translocations were lostover time, it would be important to quantify the kinetics of thatloss for biodosimetry purposes. Fifth, I thought it would beimportant to characterize the extent to which the presence ofdicentrics and other unstable aberrations might kill cells that alsocontained translocations, and thus contribute to the loss oftranslocations with time. A robust computational model ofaberration survival was needed to address this question. Sixth,determining the approximate breakpoint locations in paintedchromosomes could tell us two things – whether such breaksoccurred at random or nonrandom locations within a chromo-some, and whether individual chromosome types differed in theirsusceptibility to damage. Seventh, due to the stability oftranslocations through cell division, clones of otherwise normalcells with translocations should exist. How frequent were thoseclones? And how large were they? Finally, I wanted to knowwhether people of different ages differed in their susceptibility toionizing radiation. The answer to this last question would haveobvious implications for risk assessment following exposure. Eachof these eight issues is addressed below.

7.1. The frequencies of translocations are higher than those for

dicentrics

From a theoretical point of view, some investigators thoughtthe frequencies of translocations and dicentrics in a population ofacutely irradiated cells should be equal [31], because both resultfrom the random rejoining of two breaks in two chromosomes. Ourvalidation data on human peripheral blood lymphocytes clearlyindicated the presence of more translocations than dicentrics, andthis inequality really bothered me. I thought there might be aproblem with the assay. We decided to take photographs of everyabnormal cell so that the aberrations could be independentlyverified without concern that the fluorochrome signals would fade.I joked with the slide readers, telling them to follow the saying ofthe old American West, to ‘‘shoot (photos) first and ask questionslater.’’ This was hard because the slide readers wanted tocharacterize each aberration type before taking the pictures, butsometimes the paints would fade before the analyses could becompleted. With simple aberrations this did not pose a problem,but with heavily damaged cells a fully detailed and very carefulanalysis would be essential. Digital cameras did not yet exist, so weused Ektachrome slide film, and the 2-inch by 2-inch slide imagesof every chromosomally abnormal cell were projected on a screenand evaluated very carefully. The Ektachrome slides were thenstored in plastic pages in 3-ring binders that we placed verticallyon the top shelves in the laboratory. Over the years weaccumulated a line of binders that was perhaps 40 feet long. Even

with these very careful and time-consuming analyses, thefrequencies of translocations remained higher than dicentrics.We verified that the vast majority of the cells we analyzed were intheir first mitotic division, so the lower number of dicentrics couldnot be explained by their loss through mitosis. I worried greatlyabout this problem for 6 months and was not sure whether I shouldattempt to publish the data. Would anyone believe these results?Everyone ‘‘knew’’ the frequencies of translocations and dicentricswere equal, but as far as I knew, my laboratory was the only onewith data that argued otherwise. I shared the problem with severalother cytogeneticists to get their opinions, including GayleLittlefield, whose views I greatly respect. She convinced me tosubmit the results for publication. I took her advice and the workwas accepted for publication relatively quickly [11]. This findingwas subsequently confirmed by others, e.g., [32].

In retrospect, the inequality of translocation and dicentricfrequencies was not as big a problem as I thought it would be, forseveral reasons. Few of my colleagues seemed to care about thisissue as much as I did. Some research groups reported differencesin the frequencies of translocations and dicentrics (e.g., [33,34], butothers did not (e.g., [35,36]). Eventually I realized that the problemcould be explained as follows. The theoretical prediction ofequality of translocations and dicentrics is likely to be true for cellswith exactly two double-strand breaks, because the DNA double-strand-break repair enzymes have no mechanism to identify thelocations of the centromeres in the damaged chromosomes.However, in cells with more than two breaks there may be moreacentric fragments than in cells with only two breaks, as somechromosome arms will be broken in two or more places. In suchsituations the fragments will outnumber the chromosomes withterminal deletions, which means there will be more opportunitiesfor forming translocations than dicentrics. Thus in heavilydamaged cells there tended to be more individual events thatwere being judged as translocations than dicentrics. This led to theidentification of a nomenclature problem.

7.2. Complex aberrations and cytogenetic nomenclature

The twin problems of inequality in the frequencies oftranslocations and dicentrics, and the occurrence of complexaberrations, appeared to be related because these phenomenawere most pronounced at acute doses of 2 Gy and above, althoughoccasional cells at lower doses exhibited complex rearrangements.How could cytogenetic theory be dose-dependent? Could repair ofDNA double-strand breaks be different at low and high doses? Didrepair somehow get ‘‘sloppy’’ at high doses? Certainly it wasreasonable to think that heavily damaged cells might be so stressedthat many of their biochemical functions were impaired, but howcould functional impairment be related to the positions ofcentromeres located many megabases away? Eventually I quitworrying about these theoretical questions and instead focusedmy efforts on the practical aspects of describing the complexstructural rearrangements we were seeing. Conversations withmany of my cytogenetic colleagues indicated they were also seeingcomplex damage. Discussing these events was complicatedbecause all of us used different terminology to describe whatwe were seeing, which only made matters worse. We decided toconvene an ad hoc meeting to resolve this issue. In October 1993,an international group of 9 people met in Boston to come up with anomenclature system. Over the period of two days we discussedthe problem and debated various approaches for resolving it. Weagreed that the nomenclature should be purely descriptive, kept assimple as possible, capable of describing even the most compli-cated rearrangements, flexible enough to work in different speciesand adaptable to future improvements in painting technology. Iknew that having a simple, descriptive name for the nomenclature

6. Application of painting to human populations

As the probe technology was being developed to the pointwhere it worked reliably, and while the validation effort wasshowing that the assay gave results consistent with conventionalcytogenetic methods, a group of investigators at LLNL includingmyself had written an NIH Program Project grant application toevaluate the Chernobyl cleanup workers. These workers wereindividuals who had been involved in mitigating the levels ofradioactivity in the area around the reactor. The Soviet Union hadseparated into different countries, and we hoped that the end ofthe Cold War would allow Americans and Russians to collaborateon evaluating some of the consequences of this tragic incident. In1991 a group of LLNL people including myself went to Russia andUkraine. As guests in their countries we were treated exceptionallywell. The warmth and hospitality were overwhelming, and theseeds for a decade-long and mutually beneficial collaboration wereestablished.

As part of the Chernobyl grant application I felt it was importantto show that chromosome painting worked well enough togenerate data from blood samples provided by a large group ofsubjects. The donors I had in mind were those we had evaluated forthe anti-kinetochore CBMN assay, whose metaphase cells were onslides waiting to be hybridized. By now it was possible to purchasesome of the paints commercially, and chromosomes 1, 2, and 4were available as a set. Testing showed these probes workedreliably and consistently, and they were less expensive to purchasethan to produce ourselves.

The first basic science question I wanted to address concernedthe frequency of translocations with age in healthy people. Sincetranslocations were widely believed to be stable through mitosis, Ireasoned they should accumulate with age as a result of chronicenvironmental exposures and spontaneous aberrations occurringduring everyday life. The nearly 100 samples we had obtainedduring the micronuclei anti-kinetochore antibody project werearchived specifically to test this hypothesis. Gradually I foundsmall amounts of money, and my laboratory worked on this effortas time allowed. Marilyn Ramsey was quite interested in what wenow called the ‘‘baseline translocation’’ project and took the leadon it. The first few subjects that we evaluated were carefully

selected to represent a wide range of ages. Being naturallyimpatient (as well as scientifically curious), I wanted an answersooner rather than later. I also wanted preliminary data for theChernobyl Program Project grant application. Of course we had noidea whether there would be an age effect, or how big it might be. Iwould have been happy with any statistically significant age-related increase. As I recall, we broke the code after scoring cellsfrom the first 9 donors, and there was an obvious effect of age. Thatsimple graph went into the Chernobyl grant application. Encour-aged, we increased the effort to score slides from more donors. Wepublished our initial findings [14], and then a year later Marilynwrote a more definitive paper with a larger sample size [18]. Inthose days people requested reprints by mailing postcards to thecorresponding author, and I think we received more requests forthese papers than almost any of the others that had been producedby my laboratory up to that time. Other laboratories confirmed thisrather profound age effect, and some years later an internationaleffort led to a publication of the world’s data on baselinetranslocation frequencies in healthy people [19].

The data showing an age effect for translocations turned out tobe one of several key components of the Chernobyl ProgramProject grant application, and to everyone’s delight the grant wasfunded. We finally had our first opportunity to use chromosomepainting for radiation biodosimetry. The blood samples startedcoming in from the former Soviet Union. We saw a radiationresponse in the Chernobyl cleanup workers [20,21], and thankfullythe doses derived from cytogenetics were generally lower than hadbeen estimated based on field-dosimetry [22]. We also receivedfunding for a separate project evaluating the Sellafield Nuclear FuelWorkers and this also showed a significant effect of radiationexposure [23]. This latter finding was significant because many ofthese exposures had occurred over several decades, whereas someof the Chernobyl liquidators’ exposure durations were compara-tively short, i.e., hours to weeks long. In a later study we sawincreased frequencies of translocations in airline flight crews [24],again showing that translocations accumulate with chronicexposure. By now it appeared that translocations were anintegrating biodosimeter, i.e., capable of providing exposureestimates for chronic as well as sub-acute exposures. From atheoretical perspective this was not surprising, although confir-mation with an animal model was needed.

7. Additional scientific issues to address

The initial results of the human population studies weregratifying but there were other compelling scientific questions thatneeded to be addressed. These issues arose from our validationwork as well as my desire to address apparent discrepanciesbetween our data and cytogenetic theory, and my general scientificcuriosity. I felt that moving beyond the applied science ofpopulation studies into more basic work would be moreinteresting and provide a nice balance to my laboratory’s scientificefforts. Here I highlight eight such scientific issues and questions,although perhaps twice this many could have been selected.

The first scientific issue arose when the initial validation workshowed that the frequencies of translocations were higher than thefrequencies of dicentrics, which contradicted theory [25]. Thesecond problem also arose from the validation work, whichrevealed the existence of very complex aberrations, especially (butnot entirely) at the high doses. It wasn’t clear what theseaberrations should be called or how they should be categorized,and some investigators doubted that such aberrations evenexisted. The third issue was that I desperately wanted wholechromosome paints for mice and rats so we could conductvalidation work in these animal models. However, no such probeswere available. Paints for the mouse were of particular interest to

Fig. 6. Human peripheral blood lymphocyte. Chromosomes 1–6 are painted red,

green, purple, yellow, blue, and aqua, respectively. There is a reciprocal

translocation involving chromosome 6 and an unpainted chromosome. This

image is also on the cover of this journal.

I told my laboratory members that I needed irrefutableevidence that each probe painted only the Robertsonian chromo-some from which that probe had been developed, i.e., the entireRobertsonian chromosome had to be painted, and that noadditional regions of the genome should be labeled. My concernwas that the chromosomes had rearranged during the in vitro

culturing, as established cell lines were well-known to do. Thisproof required hybridization back to chromosomes from periph-eral blood lymphocytes from the same strains of animals. Ourmoment of triumph came when the first such hybridization clearlyand unambiguously labeled only the Robertsonian chromosome.

Some months later (I can’t remember exactly when, maybe itwas my birthday), the people in my laboratory gave me a coffeemug on which there was a picture of a mouse peripheral blood cellin metaphase (Fig. 7). Both Robertsonian chromosomes werelabeled with one of the probes we had developed. I was deeplytouched by this gift, and drank coffee out of that mug every day fora long time. Then the inevitable happened. I dropped the mug andit broke. I was devastated, but managed to pick up the pieces andcarefully glue them back together. Afraid to drink out of it anymore, the mug has now served as my pencil and pen holder formany years, sitting on my desk and gently reminding me that Idon’t have to believe everything that others say, that naysayers aresometimes best ignored, and that perseverance can overcomemany adversarial situations.

We subsequently went on to make additional mouse wholechromosome paints and demonstrated that combinations ofoverlapping Robertsonian chromosomes could be used to achievemulticolor paints [44]. The success of this type of approach isshown in Fig. 8, where two chromosome pairs are painted red, twopairs are painted green, and two pairs are painted both green andred, which appear as yellow.

7.3.2. Successes with mouse and rat whole chromosome paints

Once the mouse probes were working well [45], we beganaddressing a series of scientific questions. Alison Director wantedto investigate the in vivo cytogenetic effects of food mutagens

known as heterocyclic amines. Many of these compounds hadalready been shown to be mutagenic or clastogenic in vitro, e.g.,[26,27,46,47], and she wanted to know whether chronic feeding ofthese chemicals to mice would lead to elevated levels oftranslocations. The first food mutagen she investigated was knownas PhIP [48]. Much to our surprise, PhIP did not induce significantincreases in translocations, even in animals that for 6 months werecontinuously fed chow that contained the compound. However,the same animals showed nonpersistent but statistically signifi-cant increases in SCEs and micronuclei in normochromaticerythrocytes, which provided a clear indication that the chemicalwas cytogenetically active. We also evaluated a related compoundcalled MelQx using a similar 6-month long protocol. This time,elevated SCEs were observed in the same mice but there were noincreases in micronuclei, and again, no increases in translocationfrequencies [49]. Puzzled, we wondered whether more powerfulclastogenic chemicals would induce translocations in vivo.Accordingly we evaluated two very potent clastogens, cyclophos-phamide and urethane (ethyl carbamate), both of which wereadministered for up to 12 weeks in the drinking water. Once again,neither compound resulted in statistically significant increases inthe frequencies of translocations in blood or bone marrow,although micronuclei in normochromatic erythrocytes wereinduced [50].

Why would known powerful clastogens not induce transloca-tions in vivo, even in the presence of other clear cytogeneticresponses in the same animals? The only reasonable explanation Icould think of was that chemicals do not induce enough DNAdouble-strand breaks that are close enough together in 4dimensions (3-dimensional space plus time) to result in chromo-some exchanges. This made sense in light of other in vivo work,which mostly showed chromatid rather than chromosome breaksfollowing a single i.p. injection [26]. Still, this was disappointing tome, as I had expected a very different cytogenetic outcome forthese chronic exposures.

In parallel with the work on food mutagens, Michelle Spruillwas using mice to evaluate the persistence of translocationsinduced by acute exposures to Cesium-137 gamma rays. Sheirradiated a large number of animals with doses of 0 (control) to4 Gy, and euthanized the mice at a series of post-exposure times upto two years. As we had expected, large numbers of translocationswere induced in both blood and bone marrow cells, and thesetranslocations showed much greater persistence than dicentrics or

Fig. 7. The coffee mug with a photograph of a peripheral blood lymphocyte from a

mouse homozygous for a Robertsonian (2;8) translocation. The original image,

which we published in Chromosoma [45], shows both Robertsonian chromosomes

painted in full, and none of the other chromosomes show any hybridization signals.

This cell was one of many demonstrating that flow-sorting of mouse Robertsonian

chromosomes was a viable method for developing whole chromosome paints.

Fig. 8. Normal mouse metaphase cell painted with probes for Robertsonian

chromosomes 1.3 and 4.6 (red), and 2.8 and 4.6 (green). The yellow color

(chromosome pairs 4 and 6) is a blend of the red and green probes.

would be needed to help it obtain acceptance. After muchdiscussion and debate, we conceived the system called PAINT(Protocol for Aberration Identification and Nomenclature Termi-nology) [37,38], which met all these criteria.

PAINT was not welcomed by everyone. Some people held fast tothe belief that all translocations had to be reciprocal (‘‘2-way’’only), and that exchanges that appeared to be multi-way were dueto poor staining or less-than-adequate scoring. Others disagreedstrongly with observations of apparently nonreciprocal (‘‘1-way’’)translocations. The authors of the PAINT paper had anticipated thisproblem and included figures that explained how apparentlynonreciprocal translocations can occur. Fortunately, most inves-tigators did not struggle with the existence of any of these events,and complex rearrangements and nonreciprocal translocationsbecame well-accepted, not only because of the PAINT paper butalso by the work of others [39]. In the meantime John Savage hadbeen working on his own nomenclature system, which was moremechanistically based than PAINT [40]. The two systems werequite complementary, and shortly after PAINT was published thetwo systems were compared and contrasted [41].

Perhaps the biggest criticism of PAINT stemmed from the factthat its method of describing individual rearranged chromosomeswas interpreted by some as being a recommendation for radiationdosimetry, e.g., [32]. The authors of the PAINT paper hadanticipated this criticism, and for this reason included in thepaper the statement, ‘‘Thus, an unavoidable consequence of ourproposed system is that even single exchange events, such as asimple dicentric with an associated acentric fragment, must bedescribed separately. The same holds for other complete forms ofchromosome interchange. Of course, we do not wish to imply from a

mechanistic standpoint that a simple exchange represents two

separate events.’’ (emphasis added). Several years would passbefore PAINT was accepted as being only a nomenclature systemand not a dosimetry recommendation. Most investigators readilywelcomed PAINT and used it in their work. The PAINT paper hasbeen widely cited, and the nomenclature has even been used inpapers without citing the original reference, which to me is theultimate measure of its widespread recognition and acceptance.

7.3. Painting probes for laboratory animals

The human whole chromosome paints were working well and wewere addressing important questions with them. However, therewere other important questions that could not be addressed inhumans. For example, do translocation frequencies accumulate withchronic or highly fractionated radiation exposure, i.e., are transloca-tion frequencies truly an integrating biodosimeter? The observationsin airline flight crews [24] and the age-related increase in humanssuggested this would be true, but experimental data on animalswere needed to confirm this finding. I also wanted to determinewhether in vivo chemical exposures induced translocations.Addressing this issue would be much easier in rodents than inchemotherapy-exposed people who have underlying major healthproblems. The prevailing answer at the time was ‘‘of coursetranslocations are induced by chemicals,’’ but again, no one had anydata. The LLNL Food Program Project provided an ideal platform toanswer this question with compounds that were known to becarcinogenic, mutagenic, and clastogenic in vivo using conventionalcytogenetic assays, e.g., [26,27]. Determining whether chemicalscould induce translocations had obvious implications for riskassessment, because unlike the kinds of chromosome damage thatare identified in classical cytogenetic assays, translocations persistthrough cell division and thus may confer long-term risks.

For all these reasons I wanted whole chromosome paints forlaboratory animals and especially for the mouse. Unfortunatelythese probes were not yet commercially available. However, Joe

Gray’s group had flow-karyotyped mouse chromosomes and foundone distinctive peak of chromosomes. They sorted that peak andmade a probe for it, and found that it labeled one chromosome pair.However, they did not know which chromosome it was, so mylaboratory was asked to identify it, and it turned out to be #11 [42].Obtaining other mouse chromosome paints by this method wasnot possible because the mouse flow-karyotype did not have anyother distinct peaks, for the simple reason that mouse chromo-somes are much more similar in size than human chromosomes. Adifferent approach for isolating mouse chromosomes was needed.

I knew that there were strains of mice with a wide range ofRobertsonian translocations [43]. For maximum scoring efficiencywe needed mouse paints for more than one chromosome, andideally I wanted paints for several of the largest chromosomes. Ireasoned that the Robertsonian chromosomes should flow-sortinto distinctive positions due to their larger size, and therebyrender them amenable to the production of paints. Such paintswould label two chromosome pairs simultaneously, which wouldsimplify the process of making paints for multiple chromosomes.Furthermore, it occurred to me that Robertsonian chromosomesfrom different strains of mice might have one chromosome incommon, which meant we could use combinatorial labeling topaint multiple chromosomes each in their own unique color. Wepurchased 5 different strains of Robertsonian mice (no mouseRobertsonian cell lines were available at that time), some of whichhad one chromosome in common. We made cell lines from the lungfibroblasts, and we were ready to sort those chromosomes. Thenwe waited, because in the meantime another naysayer hadappeared.

7.3.1. It won’t work...

Sometimes it only takes one person to stop a research project.This time it was an influential person in the flow-sorting operation.He was not the least bit interested in making mouse wholechromosome paints. I tried to convince him that this project wassignificant and that the probes would have numerous usefulapplications. He thought the Robertsonian chromosomes wouldsort into the same location as the ‘‘debris peak,’’ an area outside therange where normal-sized chromosomes sorted. I countered withthe idea that Robertsonian chromosomes of different sizes wouldbe used, and that at least one of them would not co-sort with thedebris peak, which would then result in paints for two autosomepairs. He responded by saying, ‘‘It won’t work, and even if it did,you don’t have the skills to make it happen.’’ It’s hard to reasonwith a person who has a closed mind.

That person eventually left LLNL, and Rich Langlois, who knewof my dilemma, was put in charge of flow cytometry. When Richtook over, he immediately walked to my end of the building andsaid the words I had been waiting to hear: ‘‘You are cleared to sort.’’And we were ready. We thawed the cells, grew them for a fewpassages, then isolated the metaphase chromosomes and sortedthem. Rich was assisted by Jerry Eveleth, and working closely withboth individuals was absolutely wonderful. We soon had a series offlow-sorted Robertsonian chromosomes, and in a matter of monthswe had multiple excellent whole chromosome paints – more thanwe needed. And no, not one of the Robertsonian chromosomesflow-sorted under the debris peak.

I was immensely proud of this achievement for several reasons.Roy Swiger and John Breneman, two people in my laboratory, hadworked very hard to make the paints from the flow-sortedmaterial. By now there were two of Joginder Nath’s Ph.D. studentsin my laboratory, Michelle Spruill and Alison Director. Both werepatiently waiting to apply this new technology to their projects.Everyone in my laboratory knew that we had been told this project‘‘wouldn’t work,’’ and this negative assessment turned out to bevery motivational.

answer the question herself, and I was eager to let her do exactlythat. By that time our archive of Ektachrome slides of abnormalcells was large enough to address this question. Jamie meticulouslywent through numerous 3-ring binders of photographic slides,projecting each image and making exceedingly careful measure-ments of the chromosome breakpoint locations in more than 900cells. While her initial hypothesis of a hotspot near the centromereof chromosome 2 was not upheld, the breakpoint location datawere relevant and subsequently published [64].

A related problem was whether the frequencies of breaks in thepainted chromosomes were proportional to their size. Investiga-tors including myself thought this was true, but no one hadspecifically tested this assumption. This was a critically importantquestion because dosimetry requires that aberration frequenciesin the painted chromosomes be extrapolated to the whole genome[12,13]. Another of Joginder Nath’s graduate students by the nameof Kirby Johnson came to do his Ph.D. work in my laboratory. Kirbyshowed that this assumption was in fact valid, both for some of thehuman chromosomes that we were routinely painting [65], and formany different autosomes that had been painted by others [66].Some small areas of the genome showed exceptions to this norm,e.g., the short arms of the acrocentric chromosomes as well as someheterochromatic and centromeric regions. However, in general, theproportionality of damage to chromosome size was upheld, whichwas very good news because it allowed investigators who useddifferent chromosome paints to make direct comparisons of theirresults.

7.7. Clones of cells with translocations

One of the key arguments supporting the long-term stability ofat least some of the induced translocations is the presence of clonesof cells with these aberrations. Cancer cells are widely recognizedas clonal and are well known to have numerous translocations, e.g.,[67,68]. We saw our first clone in the peripheral lymphocytes ofone of the baseline subjects [14]. This clonal aberration wasactually an insertion, not a translocation. Our archives ofphotographs of abnormal cells continued to pay off by allowingus to perform detailed analyses of rearranged chromosomes longafter the fluorochromes on the microscope slides had faded. KirbyJohnson was very interested in clones of abnormal cells and usedthe archived photographic slides to assess their frequencies in ourChernobyl population [69] as well as our baseline population [70].Kirby showed that clones were common enough that dosimetryestimates in a modest fraction of subjects could be skewed unlessthe frequencies of translocated cells were adjusted to remove theeffects of clones. We obtained similar results in our analyses ofMichelle Spruill’s irradiated mice [52]. The size of some of theclones was quite large and would have led to substantial errors indosimetry if not taken into account.

7.8. Age and susceptibility to ionizing radiation

One of the key assumptions for radiation dosimetry is thatpeople acquire the same amount of cytogenetic damage inresponse to the same type and amount of exposure. The exceptionis people who have certain rare genetic conditions that limit theirDNA repair capacity or restrict their ability to respond to damagingevents [71]. This assumption is not so much a formal finding butrather a simplifying notion that is not founded on strongsupporting data. For example, children are widely believed to bemore susceptible to radiation than adults, yet until recently thisassumption had not been formally tested, at least with acytogenetic endpoint. Marina Bakhmutsky tested this assumptionwhen she was a Ph.D. student in my laboratory. She used FISHpainting to show that peripheral lymphocytes from the umbilical

cord of newborns are significantly more prone to radiation-induced chromosome damage than peripheral lymphocytes fromadults. She also showed that adults do not appear to change insensitivity to radiation as they age [72], but confirmed again thatthe frequencies of aberrations increase with age owing to theaccumulation of exposures over time. These findings may beimportant when making radiation risk estimates in people ofdifferent ages.

8. Conclusions

The development and application of whole chromosomepainting was a challenging and exceptionally exciting time. Oncemany of the key issues involving painting were resolved, and oncewe established the ability to enumerate translocations quickly andeasily, it became fairly easy to obtain external funding to addresskey scientific problems. Sometimes I didn’t even have to writegrant applications. Instead, the phone would ring and I would beinvited to write a mini-proposal or to apply for an inter-agencyagreement that sometimes involved only the submission of a fewparagraphs. Those were amazing days, but times have changed. Inretrospect it is hard to believe how many different fundedmolecular cytogenetic projects my laboratory sometimes had atone time. However, as with many investigators, there were timeswhen I did nothing but write proposals full time for many monthsin a row. And yes, there were days when I wondered whether thenext grant would get funded before I had to lay off personnel. I wastruly fortunate to have been in the right places at the right times topursue my passion for cytogenetics. I have also conducted othertypes of research, and I enjoyed that work tremendously too, butcytogenetics has been my main scientific love.

I could tell many other stories concerning the development andapplication of FISH painting – stories of more radiation-exposedpopulations, of chemical-exposed people including cigarettesmokers, of the influence of genotype on translocation frequencies,and of modeling studies in mice and monkeys. There are also FISHstories that do not involve whole chromosome painting; forexample, stories of human gene mapping, of identifying whichmice carry a transgene, and of detecting a single base mutation in achromosome. And yes, there were times when my ideas of newapproaches for FISH-related technologies did not work in spite ofthe tremendous efforts by many people. Unfortunately, space doesnot permit a recounting of all these stories, wonderful as I thinkthey are.

Conflict of interest statement

The author states that there is no conflict of interest.

Acknowledgments

I thank Dr. George Hoffmann for his excellent suggestions andeditorial help on this paper.

None of the experiences described in this paper would havebeen possible without the strong and consistent support of mywife Barb for 37 years. Being married to a scientist isn’t easy. Thankyou for being so close by my side all these years. I also want tothank our three daughters; although I tried to shield them from mycrazy schedule as much as possible, in many ways they did have toendure some of the side effects of my career during their formativeyears.

It is not an exaggeration to say that my career as a cytogeneticistwould never have begun without the overwhelming kindness ofDr. Joginder Nath at West Virginia University. His involvementwith me in the pre-molecular cytogenetic years and in the first

other types of aberrations [51,52]. As we had previously seen inirradiated human cells, complex aberrations were observed at thehigher doses. We also began to observe animals that had multiplecells with apparently identical types of translocations. Analyzingthese potential clones in detail was possible because we hadcontinued our practice of taking and carefully evaluating photo-graphs of every abnormal cell. The results showed that doseestimates could be skewed significantly by clonal expansion, butthat reasonable dose estimates could be achieved if the cloneswere removed from the analyses [52]. Michelle’s work also showedthat translocation frequencies increase with age, an observationthat was later substantiated in a larger group of mice [53].

The success with mouse whole chromosome paints led us topursue similar work with rats. While flow-sorting chromosomes tomake paints was not always straightforward, we enjoyedconsiderable success with the method. Fortunately the ratkaryotype has chromosomes of sufficiently different sizes thatindividual chromosome types are readily identifiable and can beflow-sorted. We made three rat whole chromosome paints, each ina different color, and quantified the persistence of translocations inblood that had been irradiated in vitro [13]. And these resultsconfirmed that we had another problem.

7.4. The persistence of translocations

Michelle Spruill’s initial work with irradiated mice clearlyindicated that at least some translocations persist for manymonths following exposure, and she subsequently showed thattranslocations persist over the lifespan of the mouse [52].However, her results also showed that translocation frequencies,at least at the highest doses, declined with time. Similar losses ofcells with translocations were observed in irradiated rat blood [13].

The next scientific issue that I wanted to address was quitespecific: How persistent are translocation frequencies? Showingthat some induced translocations lasted a lifetime in mice was onething, but quantifying that persistence, especially in humans, wasanother. Factors such as the persistence of subpopulations of long-lived lymphocytes, or the presence of translocations in clones ofstem cells, could play important roles in translocation persistence.Quantifying translocation persistence is significant for performingradiation biodosimetry in putatively exposed people. However,identifying human subjects who have no underlying disease suchas cancer, and who also have well-characterized radiationexposures, is difficult. Furthermore, it would be most straightfor-ward if those exposures were acute and whole-body, and of courserecent exposures would be essential so the initial frequencies ofaberrations could be characterized. Fortunately, such exposedpeople are rare, although Natarajan et al. evaluated individualsexposed as a result of a radiation accident in Goiania, Brazil [54].Their results indicated that translocation frequencies could beused for dosimetry years after exposure but that those frequenciesdeclined substantially with time.

Since I didn’t have access to a recently-irradiated humanpopulation with well-characterized acute doses, we did the nextbest thing – we exposed human peripheral blood from normalhealthy subjects in vitro. We used a wide range of doses andharvested cells at multiple time intervals out to a week, which wasthe maximum time the cells would stay healthy. The results clearlyindicated that translocation frequencies declined significantlywith time, but of course not as fast as dicentrics, rings, andfragments [55,56]. Coupled with the results that we saw in mice in

vivo and in rat blood irradiated in vitro, it was now apparent thattranslocation frequencies decline following acute exposure, atleast for doses of 2 Gy and above, a finding that is consistent withresults from other studies, e.g., [54,57]. Some investigators haveshown that restricting the analyses to cells that have only

reciprocal translocations results in translocation frequencies thatdo not decline with time [58]. However, this approach involvesignoring some damaged cells, which may be problematicespecially at high doses where most aberrant cells have multipledamage events. There is no one optimal solution to this problem,and the different ways of evaluating translocations has beendiscussed in detail [55].

Perhaps most importantly, many of the studies on translocationpersistence showed that translocation frequencies decline to anon-zero plateau, i.e., not every cell containing a translocation willdie. For acute gamma doses of 2 Gy and higher, about 40% of cellswith translocations are lost [55,56,59], whereas following doses of1 Gy and below, translocations show nearly complete persistence[54,60]. Therefore, dosimetry that is performed with translocationsis often superior to dosimetry performed with dicentrics, but someconsideration must still be given to the amount of time elapsedsince exposure [61]. Knowing when the non-zero plateau isreached relative to the exposure time is important.

7.5. Influence of dicentrics on translocation persistence

Numerous experiments in cells from humans, mice, and ratsshowed that translocations did not exhibit complete persistence. Akey question in my mind was whether this loss of translocationsover time was due to instability of some of the translocationsthemselves, or to selective pressure against other aberrations suchas dicentrics in the same cells, or perhaps both. Since it is notpossible to induce translocations without also inducing otherstructural aberrations, a mathematical modeling approach wasneeded. My laboratory had the data, all we needed was someonewho knew how to do the modeling. A postdoctoral fellow by thename of Shea Gardner had the requisite skills, and I convinced herthat the analyses were worth doing. Shea used Monte Carlosimulations and fitted models to the frequencies of translocationsand dicentrics induced by gamma rays in human peripherallymphocytes from two donors. She showed that inherent lethalityof translocations and selection against dicentrics in the same cellscan both contribute to the loss of cells with translocations [62].This was a significant finding for two reasons – first, because itshowed that multiple independent types of selective pressure caninfluence the frequency of induced translocations, and second,because it demonstrated that the commonly used term ‘‘stabletranslocation’’ is not entirely accurate, in that not all translocationsare actually stable.

7.6. Breakpoint distributions and frequencies in painted chromosomes

In my entire career I brought only one high school student towork in my laboratory. Jamie Senft was hired and trained to scorestructural chromosome aberrations in painted human cells. Iroutinely told my slide readers to ‘‘keep their eyes open’’ and to askquestions if they saw anything interesting or unusual. I didn’t wantrobots at the microscope; I wanted young scientists who werethinking about what they were doing, even though the slides werealways coded to prevent observer bias. Jamie took my advice andone day she made an observation that interested me greatly. Shethought she was observing an unusually high frequency of breaksin the centromeric region of human chromosome 2. I was intriguedbecause this chromosome actually occurs as two separatechromosomes in the gorilla and chimpanzee [63]. Could thisregion of the human genome be a hot spot for chromosomebreakage? Might there be some evolutionary significance to thisobservation? This latter question was a long shot, but the questionof breakpoint locations had not yet been investigated in paintedchromosomes. Of course we first needed to determine whetherJamie’s observation held up to formal scrutiny. Jamie wanted to

[45] J.W. Breneman, M.J. Ramsey, D.A. Lee, G.G. Eveleth, J.L. Minkler, J.D. Tucker, Thedevelopment of chromosome-specific composite DNA probes for the mouse andtheir application to chromosome painting, Chromosoma 102 (1993) 591–598.

[46] L. Thompson, A. Carrano, E. Salazar, J. Felton, F. Hatch, Comparative genotoxiceffects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria andcultured mammalian cells, Mutat. Res. 117 (1983) 243–257.

[47] J.S. Felton, M.G. Knize, C. Wood, B.J. Wuebbles, S.K. Healy, D.H. Stuermer, L.F.Bjeldanes, B.J. Kimble, F.T. Hatch, Isolation and characterization of new mutagensfrom fried ground beef, Carcinogenesis 5 (1984) 95–102.

[48] A.E. Director, J. Nath, M.J. Ramsey, R.R. Swiger, J.D. Tucker, Cytogenetic analysis ofmice chronically fed the food mutagen 2-amino-1-methyl-6-phenylimida-zo[4,5b]pyridine, Mutat. Res. 359 (1996) 53–61.

[49] J.W. Breneman, J.F. Briner, M.J. Ramsey, A. Director, J.D. Tucker, Cytogeneticresults from a chronic feeding study of MeIQx in mice, Food Chem. Toxicol. 34(1996) 717–724.

[50] A.E. Director, J.D. Tucker, M.J. Ramsey, J. Nath, Chronic ingestion of clastogens bymice and the frequency of chromosome aberrations, Environ. Mol. Mutagenes. 32(1998) 139–147.

[51] M.D. Spruill, M.J. Ramsey, R.R. Swiger, J. Nath, J.D. Tucker, The persistence ofaberrations in mice induced by gamma radiation as measured by chromosomepainting, Mutat. Res. 356 (1996) 135–145.

[52] M.D. Spruill, D.O. Nelson, M.J. Ramsey, J. Nath, J.D. Tucker, Lifetime persistenceand clonality of chromosome aberrations in the peripheral blood of mice acutelyexposed to ionizing radiation, Radiat. Res. 153 (2000) 110–121.

[53] J.D. Tucker, M.D. Spruill, M.J. Ramsey, A.D. Director, J. Nath, Frequency of sponta-neous chromosome aberrations in mice: effects of age, Mutat. Res. 425 (1999)135–141.

[54] A.T. Natarajan, S.J. Santos, F. Darroudi, V. Hadjidikova, S. Vermeulen, S. Chatterjee,M. Berg, M. Grigorava, E.T. Hojo-Sakamoto, F. Granath, A.T. Ramalho, M.P. Curado,137Cesium-induced chromosome aberrations analyzed by fluorescence in situhybridization: eight years follow up of the Goiania radiation accident victims,Mutat. Res. 400 (1998) 299–312.

[55] J.D. Tucker, J. Cofield, K. Matsumoto, M.J. Ramsey, D.C. Freeman, Persistence ofchromosome aberrations following acute radiation: II, does it matter how trans-locations are scored? Environ. Mol. Mutagenes. 45 (2005) 249–257.

[56] J.D. Tucker, J. Cofield, K. Matsumoto, M.J. Ramsey, D.C. Freeman, Persistence ofchromosome aberrations following acute radiation: I, PAINT translocations,dicentrics, rings, fragments, and insertions, Environ. Mol. Mutagenes. 45(2005) 229–248.

[57] M.P. Hande, J.J. Boei, F. Granath, A.T. Natarajan, Induction and persistence ofcytogenetic damage in mouse splenocytes following whole-body X-irradiationanalysed by fluorescence in situ hybridization. I. Dicentrics and translocations,Int. J. Radiat. Biol. 69 (1996) 437–446.

[58] J. Moquet, A. Edwards, D. Lloyd, P. Hone, The use of FISH chromosome painting forassessment of old doses of ionising radiation, Radiat. Prot. Dosim. 88 (2000) 27–33.

[59] M.P. Hande, A.T. Natarajan, Induction and persistence of cytogenetic damage inmouse splenocytes following whole-body X-irradiation analysed by fluores-cence in situ hybridization. IV. Dose response, Int. J. Radiat. Biol. 74 (1998) 441–448.

[60] J. Lucas, W. Deng, Views on issues in radiation biodosimetry based on chromo-some translocations measured by FISH, Radiat. Prot. Dosim. 88 (2000) 77–86.

[61] M.L. Camparoto, A.T. Ramalho, A.T. Natarajan, M.P. Curado, E.T. Sakamoto-Hojo,Translocation analysis by the FISH-painting method for retrospective dose re-construction in individuals exposed to ionizing radiation 10 years after exposure,Mutat. Res. 530 (2003) 1–7.

[62] S.N. Gardner, J.D. Tucker, The cellular lethality of radiation-induced chromosometranslocations in human lymphocytes, Radiat. Res. 157 (2002) 539–552.

[63] D. Miller, L. Firschein, V. Dev, R. Tantravahi, O. Miller, The gorilla karyotype:chromosome lengths and polymorphisms, Cytogenet. Cell Genet. 13 (1974) 536–550.

[64] J.D. Tucker, J.R. Senft, Analysis of naturally occurring and radiation-inducedbreakpoint locations in human chromosomes 1, 2 and 4, Radiat. Res. 140(1994) 31–36.

[65] K.L. Johnson, J.D. Tucker, J. Nath, Frequency, distribution and clonality of chro-mosome damage in human lymphocytes by multi-color FISH, Mutagenesis 13(1998) 217–227.

[66] K.L. Johnson, D.J. Brenner, J. Nath, J.D. Tucker, C.R. Geard, Radiation-inducedbreakpoint misrejoining in human chromosomes: random or non-random? Int.J. Radiat. Biol. 75 (1999) 131–141.

[67] F.J. Kaye, Mutation-associated fusion cancer genes in solid tumors, Mol. CancerTher. 8 (2009) 1399–1408.

[68] P.A. Edwards, Fusion genes and chromosome translocations in the commonepithelial cancers, J. Pathol. 220 (2009) 244–254.

[69] K.L. Johnson, J. Nath, J.M. Pluth, J.D. Tucker, The distribution of chromosomedamage, non-reciprocal translocations and clonal aberrations in lymphocytesfrom Chernobyl clean-up workers, Mutat. Res. 439 (1999) 77–85.

[70] K.L. Johnson, D.J. Brenner, C.R. Geard, J. Nath, J.D. Tucker, Chromosome aberrationsof clonal origin in irradiated and unexposed individuals: assessment and implica-tions, Radiat. Res. 152 (1999) 1–5.

[71] L.H. Thompson, Recognition, signaling, and repair of DNA double-strand breaksproduced by ionizing radiation in mammalian cells: the molecular choreography,Mutat. Res. 751 (2012) 158–246.

[72] M.V. Bakhmutsky, M.C. Joiner, T.B. Jones, J.D. Tucker, Differences in cytogeneticsensitivity to ionizing radiation in newborns and adults, Radiat. Res. 181 (2014)605–616.

decade of chromosome painting is summarized in this paper.Words alone can never express the depth and breadth of mygratitude to him.

This paper focuses mostly on the early years of chromosomepainting, when it wasn’t clear whether the resources would beavailable, whether the technology would work, whether the keyscientific questions would be resolved, and whether some of thecontroversies would ever go away. In the end, chromosomepainting enjoyed its success because of the people who made ithappen. In my laboratory over the years there have been severalscientists who visited or took their sabbatical in my laboratory, aswell as postdoctoral fellows, many graduate students, roughly 100undergraduate students, and numerous staff scientists whoworked diligently on projects that are not mentioned in thispaper. To tell all the stories, both scientific and personal, wouldrequire a book. However, I would like to give four people specialrecognition by name: Marilyn Ramsey and Roy Swiger at LLNL,without whom the work in the early years might have gone in atotally different direction, and Dr. Robert A. Thomas and Dayton M.Petibone at Wayne State University, without whom the work in thelater years would never have been possible. Thank you all for yourmany years of dedication and commitment.

Finally, I thank the Lord for His guidance and direction in mycareer, especially during the many difficult times, only some ofwhich are described here.

References

[1] M.L. Pardue, J.G. Gall, Chromosomal localization of mouse satellite DNA, Science168 (1970) 1356–1358.

[2] J.D. Tucker, J. Xu, J. Stewart, P.C. Baciu, T.M. Ong, Detection of sister chromatidexchanges induced by volatile genotoxicants, Teratogenes. Carcinogenes. Muta-genes. 6 (1986) 15–21.

[3] J.D. Tucker, M.L. Christensen, C.L. Strout, A.V. Carrano, Determination of thebaseline sister chromatid exchange frequency in human and mouse peripherallymphocytes using monoclonal antibodies and very low doses of bromodeox-yuridine, Cytogenet. Cell Genet. 43 (1986) 38–42.

[4] A. Van Prooijen-Knegt, J. Van Hoek, J. Bauman, P. Van Duijn, I. Wool, M. Van derPloeg, In situ hybridization of DNA sequences in human metaphase chromosomesvisualized by an indirect fluorescent immunocytochemical procedure, Exp. CellRes. 141 (1982) 397–407.

[5] J.E. Landegent, N.J. in de Wal, G.-J.B. van Ommen, F. Baas, J.J.M. de Vijlder, P. vanDuijn, M. van der Ploeg, Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization, Nature 317 (1985) 175–177.

[6] D. Pinkel, S. Straume, J.W. Gray, Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization, Proc. Natl. Acad. Sci. 83 (1986) 2934–2938.

[7] D. Pinkel, J. Landegent, C. Collins, J. Fuscoe, R. Segraves, J. Lucas, J. Gray, Fluores-cence in situ hybridization with human chromosome-specific libraries: detectionof trisomy 21 and translocations of chromosome 4, Proc. Natl. Acad. Sci. 85 (1988)9138–9142.

[8] M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res.147 (1985) 29–36.

[9] D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents usingcytokinesis-blocked human lymphocytes and an antikinetochore antibody, En-viron. Mol. Mutagenes. 13 (1989) 34–43.

[10] J.C. Hando, J. Nath, J.D. Tucker, Sex chromosomes, micronuclei and aging inwomen, Chromosoma 103 (1994) 186–192.

[11] J.D. Tucker, M.J. Ramsey, D.A. Lee, J.L. Minkler, Validation of chromosome paintingas a biodosimeter in human peripheral lymphocytes following acute exposure toionizing radiation in vitro, Int. J. Radiat. Biol. 64 (1993) 27–37.

[12] J. Lucas, T. Tenjin, T. Straume, D. Pinkel, D. Moore II, M. Litt, J. Gray, Rapid humanchromosome aberration analysis using fluorescence in situ hybridization, Int. J.Radiat. Biol. 56 (1989) 35–44, Erratum: Int. J. Radiat. Biol. 56:201 (1989).

[13] J.D. Tucker, J.W. Breneman, J.F. Briner, G.G. Eveleth, R.G. Langlois, D.H. Moore 2nd,Persistence of radiation-induced translocations in rat peripheral blood deter-mined by chromosome painting, Environ. Mol. Mutagens. 30 (1997) 264–272.

[14] J.D. Tucker, D.A. Lee, M.J. Ramsey, J. Briner, L. Olsen, D.H. Moore 2nd, On thefrequency of chromosome exchanges in a control population measured bychromosome painting, Mutat. Res. 313 (1994) 193–202.

[15] K. Matsumoto, M.J. Ramsey, D.O. Nelson, J.D. Tucker, Persistence of radiation-induced translocations in human peripheral blood determined by chromosomepainting, Radiat. Res. 149 (1998) 602–613.

[16] E. Schrock, S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M.A. Ferguson-Smith,Y. Ning, D.H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini, T. Ried, Multicolorspectral karyotyping of human chromosomes, Science 273 (1996) 494–498.

[17] M. Speicher, S. Ballard, D. Ward, Karyotyping human chromosomes by combina-torial multi-fluor FISH, Nat. Genet. 12 (1996) 368–375.

[18] M.J. Ramsey, D.H. Moore 2nd, J.F. Briner, D.A. Lee, L. Olsen, J.R. Senft, J.D. Tucker,The effects of age and lifestyle factors on the accumulation of cytogenetic damageas measured by chromosome painting, Mutat. Res. 338 (1995) 95–106.

[19] A.J. Sigurdson, M. Ha, M. Hauptmann, P. Bhatti, R.J. Sram, O. Beskid, E.J. Tawn, C.A.Whitehouse, C. Lindholm, M. Nakano, Y. Kodama, N. Nakamura, I. Vorobtsova, U.Oestreicher, G. Stephan, L.C. Yong, M. Bauchinger, E. Schmid, H.W. Chung, F.Darroudi, L. Roy, P. Voisin, J.F. Barquinero, G. Livingston, D. Blakey, I. Hayata, W.Zhang, C. Wang, L.M. Bennett, L.G. Littlefield, A.A. Edwards, R.A. Kleinerman, J.D.Tucker, International study of factors affecting human chromosome transloca-tions, Mutat. Res. 652 (2008) 112–121.

[20] D.H. Moore II, J.D. Tucker, I.M. Jones, R.G. Langlois, P. Pleshanov, I. Vorobtsova, R.Jensen, A study of the effects of exposure on cleanup workers at the Chernobylnuclear reactor accident using multiple end points, Radiat. Res. 148 (1997) 463–475.

[21] D.H. Moore II, J.D. Tucker, Biological dosimetry of Chernobyl cleanup workers:inclusion of data on age and smoking provides improved radiation dose estimates,Radiat. Res. 152 (1999) 655–664.

[22] I.M. Jones, H. Galick, P. Kato, R.G. Langlois, M.L. Mendelsohn, G.A. Murphy, P.Pleshanov, M.J. Ramsey, C.B. Thomas, J.D. Tucker, L. Tureva, I. Vorobtsova, D.O.Nelson, Three somatic genetic biomarkers and covariates in radiation-exposedRussian cleanup workers of the chernobyl nuclear reactor 6–13 years afterexposure, Radiat. Res. 158 (2002) 424–442.

[23] J.D. Tucker, E.J. Tawn, D. Holdsworth, S. Morris, R. Langlois, M.J. Ramsey, P. Kato,J.D. Boice Jr., R.E. Tarone, R.H. Jensen, Biological dosimetry of radiation workers atthe Sellafield nuclear facility, Radiat. Res. 148 (1997) 216–226.

[24] L.C. Yong, A.J. Sigurdson, E.M. Ward, M.A. Waters, E.A. Whelan, M.R. Petersen, P.Bhatti, M.J. Ramsey, E. Ron, J.D. Tucker, Increased frequency of chromosometranslocations in airline pilots with long-term flying experience, Occup. Environ.Med. 66 (2009) 56–62.

[25] J.A. Heddle, Randomness in the formation of radiation-induced chromosomeaberrations, Genetics 52 (1965) 1320–1334.

[26] J.D. Tucker, A.V. Carrano, N.A. Allen, M.L. Christensen, M.G. Knize, C.L. Strout, J.S.Felton, In vivo cytogenetic effects of cooked food mutagens, Mutat. Res. 224(1989) 105–113.

[27] L.H. Thompson, J.D. Tucker, S.A. Stewart, M.L. Christensen, E.P. Salazar, A.V.Carrano, J.S. Felton, Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity, Mutagenesis 2 (1987)483–487.

[28] A. Carrano, J. Minkler, D. Piluso, On the fate of stable chromosomal aberrations,Mutat. Res. 30 (1975) 153–156.

[29] L. Littlefield, E. Joiner, Cytogenetic follow-up studies in six radiation accidentvictims, Int. At. Energy Agency 1 (1978) 297–308.

[30] K.E. Buckton, G.E. Hamilton, L. Paton, A.O. Langlands, Chromosome aberrations inirradiated ankylosing spondylitis patients, in: H.J. Evans, D.C. Lloyd (Eds.), Muta-gen-induced Chromosome Damage in Man, University Press, Edinburgh, 1978, pp.142–150.

[31] J.R. Savage, D.G. Papworth, Frequency and distribution studies of asymmetricalversus symmetrical chromosome aberrations, Mutat. Res. 95 (1982) 7–18.

[32] P. Finnon, D.C. Lloyd, A.A. Edwards, Fluorescence in situ hybridization detection ofchromosomal aberrations in human lymphocytes: applicability to biologicaldosimetry, Int. J. Radiat. Biol. 68 (1995) 429–435.

[33] A.T. Natarajan, R.C. Vyas, F. Darroudi, S. Vermeulen, Frequencies of X-ray-inducedchromosome translocations in human peripheral lymphocytes as detected by insitu hybridization using chromosome-specific DNA libraries, Int. J. Radiat. Biol. 61(1992) 199–203.

[34] F. Darroudi, J. Formina, M. Meijers, A.T. Natarajan, Kinetics of the formation ofchromosome aberrations in X-irradiated human lymphocytes, using PCC andFISH, Mutat. Res. 404 (1998) 55–65.

[35] M.S. Kovacs, J.W. Evans, I.M. Johnstone, J.M. Brown, Radiation-induced damage,repair and exchange formation in different chromosomes of human fibroblastsdetermined by fluorescence in situ hybridization, Radiat. Res. 137 (1994) 34–43.

[36] R. Kanda, I. Hayata, Comparison of the yields of translocations and dicentricsmeasured using conventional Giemsa staining and chromosome painting, Int. J.Radiat. Biol. 69 (1996) 701–705.

[37] J.D. Tucker, W.F. Morgan, A.A. Awa, M. Bauchinger, D. Blakey, M.N. Cornforth, L.G.Littlefield, A.T. Natarajan, C. Shasserre, A proposed system for scoring structuralaberrations detected by chromosome painting, Cytogenet. Cell Genet. 68 (1995)211–221.

[38] J.D. Tucker, W.F. Morgan, A.A. Awa, M. Bauchinger, D. Blakey, M.N. Cornforth, L.G.Littlefield, A.T. Natarajan, C. Shasserre, PAINT: a proposed nomenclature forstructural aberrations detected by whole chromosome painting, Mutat. Res.347 (1995) 21–24.

[39] J.N. Lucas, R.K. Sachs, Using three-color chromosome painting to test chromosomeaberration models, Proc. Natl. Acad. Sci. 90 (1993) 1484–1487.

[40] J.R.K. Savage, P.J. Simpson, FISH painting patterns resulting from complexexchanges, Mutat. Res. 312 (1994) 51–60.

[41] J.R. Savage, J.D. Tucker, Nomenclature systems for FISH-painted chromosomeaberrations, Mutat. Res. 366 (1996) 153–161.

[42] K. Miyashita, M.A. Vooijs, J.D. Tucker, D.A. Lee, J.W. Gray, M.G. Pallavicini, A mousechromosome 11 library generated from sorted chromosomes using linker-adapt-er polymerase chain reaction, Cytogenet. Cell Genet. 66 (1994) 54–57.

[43] S. Adolph, J. Klein, Robertsonian variation in Mus musculus from Central EuropeSpain, and Scotland, J. Hered. 72 (1981) 219–221.

[44] J.W. Breneman, R.R. Swiger, M.J. Ramsey, J.L. Minkler, J.G. Eveleth, R.A. Langlois,J.D. Tucker, The development of painting probes for dual-color and multiplechromosome analysis in the mouse, Cytogenet. Cell Genet. 68 (1995) 197–202.

The comet assay: Reflections on its development, evolution and applications ☆Narendra P. Singh *

Department of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195, USAReflections in Mutation Research

The comet assay: Reflections on its development, evolution andapplications$

Narendra P. SinghDepartment of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195, USA

Article history:Accepted 14 May 2015Available online 29 December 2015

Keywords:Comet assayMicrogel electrophoresisSingle cell gel electrophoresisDNA damage and repairCancerAging

A B S T R A C T

The study of DNA damage and its repair is critical to our understanding of human aging and cancer. Thisreview reflects on the development of a simple technique, now known as the comet assay, to study theaccumulation of DNA damage and its repair. It describes my journey into aging research and the need for amethod that sensitively quantifies DNA damage on a cell-by-cell basis and on a day-by-day basis. Myinspirations, obstacles and successes on the path to developing this assay and improving its reliabilityand sensitivity are discussed. Recent modifications, applications, and the process of standardizing thetechnique are also described. What was once untried and unknown has become a technique used aroundthe world for understanding and monitoring DNA damage. The comet assay’s use has grownexponentially in the new millennium, as emphasis on studying biological phenomena at the single-celllevel has increased. I and others have applied the technique across cell types (including germ cells) andspecies (including bacteria). As it enters new realms and gains clinical relevance, the comet assay mayvery well illuminate human aging and its prevention.

ã 2016 Published by Elsevier B.V.

1. Introduction

By the time I began research, it was already accepted that tounderstand the process of aging and its causes, one had to see DNA.The scientists who laid the foundations for our field were scientistswho found a way to see: whether Sutton [1] and Boveri who sawthat genes had to be located on chromosomes, Franklin and Gosling[2] who saw the structure of DNA, or Tjio and Levan [3] who sawthe true number of human chromosomes. The desire to see humanaging with as much clarity as I could was always my main mission,and the development of the comet assay was a result of this desire.I always felt that, once seen, the secret of aging and its preventioncould be found.

2. Scientific foundation in India

As a child, I thought that I would find the secret to aging andmake my parents immortal, but I had no knowledge about researchand no intention to pursue it. In July 1967, when I entered KingGeorge’s Medical College (KGMC) in Lucknow, India, it was with

the goal of becoming a family doctor in a village like the one that Ihad just left or a small town clinic. But KGMC was a unique place.Set on the Gomti River, it is a famously beautiful campus in a cityknown for its culture and courtliness. At the time, it was the topmedical college in India, and its alumni, called Georgians, were topphysicians, surgeons and researchers. It was also very well funded.I was exposed to new fields, taught by experts, and I had theopportunity to be in a lab. I stayed there for nearly ten years as astudent, then post-graduate and finally as faculty.

During my post-graduate studies in the Department ofAnatomy, I had the privilege of establishing a laboratory where Icould study chromosomes under the microscope. My childhooddesire to find the secret of aging was within my reach! I used tosoak red kidney beans in water for 2 to 3 h, then blend andcentrifuge them. I would remove the top supernatant layer usingan ordinary pipet and syringe. This solution was rich inphytohemagglutinin and was used to stimulate human lympho-cytes to divide. After using colchicine to arrest the cell cycle atmetaphase, I could see a cell frozen in the midst of division. Finally,I had a chance to look at chromosomes, 46 of them. I ended upwriting my thesis on chromosomal aberrations observed aftertreatments with hormones and antibiotics. During my Master'sprogram, my supervisor, Professor Avinash Chandra Das, Chair ofthe Department of Anatomy, found funding to create a cytogenetics

$ This article is part of the Reflections in Mutation Research series. To suggesttopics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann (ghoffmann@holycross.edu).

E-mail address: narendra@uw.edu (N.P. Singh).

http://dx.doi.org/10.1016/j.mrrev.2015.05.0041383-5742/ã 2016 Published by Elsevier B.V.

journal homepage: www.elsevier .com/ locate / rev iewsmrCommunit y address : www.elsevier .com/ locat e/mutres

laboratory and I was only too eager to set it up. This was thebeginning of my journey into DNA damage and aging research.

Our conditions were not perfect: the room was a convertedprocessing area for anatomy specimens and body parts. We weremissing some key equipment but we found substitutes—I took apressure cooker from our kitchen at home and this served as theautoclave for our glassware. Without having fully sterile con-ditions, I used to lose 90% of my cultures to contamination. I had aUV light and a glass chamber that I sterilized using the light. I had awater bath, light microscope and electric centrifuge but noincubator. Electricity outages were common, almost everydayoccurrences, and they interrupted many experiments. Still, byaspirating rabbit bone marrow directly, using colchicine to arrestcell division in metaphase, and staining with Wright’s or Giemsastain, we were able to visualize chromosomes. I found effects ofantibiotics (tetracycline, chloramphenicol) but not of hormones(testosterone, estrogen and progesterone) on rabbit chromosomesafter 7 days of daily injections [4].

Eventually, I wanted to see DNA, not just chromosomes, but thisgoal exceeded the resources and knowledge at KGMC. In the fall of1977, I visited the labs of Drs. Geeta Talukedar and Archana Sharmain Calcutta to learn autoradiography and unscheduled DNAsynthesis (UDS). The incorporation of radioactive bases intodamaged DNA during UDS allowed for the estimation of repairin DNA by visual grain counting. In 1978, I traveled to BhabhaAtomic Research Center in Bombay to learn mutagenesis inbacteria—the Ames test—with Drs. A.S. Aiyar and P.S. Chauhan. Thisallowed me to quantify the number of mutations induced byenvironmental chemicals. Still, even at Bhabha, they were notstudying DNA damage directly. By the time I left Bombay, I had thenotion that I would try to make an assay to directly measure a cell’sDNA damage.

Wanting to work with DNA directly, I read any article that I couldfind on DNA damage, sister-chromatid exchange (SCE), alkalineelution and chromosomal aberrations. The medical library at KGMChad few scientific journals, so I used to read articles in the well-stocked archives of the National Botanic Garden and Central DrugResearch Institute, both in Lucknow. On countless occasions, my wifewould copy the articles by hand so that I could read and replicateexperiments in the lab. After I had left Lucknow and arrived inAmerica, I showed these handwritten copies of articles to theiroriginal authors. Ronald Hart was incredulous and amusedly tookthese papers around the labs at the National Center for ToxicologicalResearch (NCTR). Painstakinglycopiedinblue ink were the articles ofDrs. Nathan Shock, Ed Schneider, George Martin and Dr. Hart himself.I gained a lot of knowledge from this published work, and it inspiredme toward new research directions and even life style changes.While I was still in India, Lester Packer’s work on vitamin E’s effect onWI-38 cells,making them immortal [5], inspired me to buya bottle ofvitamin E oil for daily ingestion.

3. Research training in the United States

Having taken advantage of all the resources available in Indiafor studying DNA damage, I began to look for a post-doctoralfellowship. I wrote letters to every author outside of India whosework I had read and respected. Two positive responses came: onefirst from Dr. Hart and then one from Dr. Ed Schneider. I acceptedDr. Hart’s offer as he was more of a basic researcher. The airplaneticket was equivalent to six months of my salary as a demonstratorin KGMC's anatomy department, where teaching medical studentswas my main job. I had to borrow money from my father and afellow “Georgian,” the co-author of my first publication, Dr. M.K.Tolani. I had never left India before, but a month after Dr. Hart’sletter arrived, I traveled 12,500 miles—exactly half way around theearth.

I arrived at Ohio State University on the 10th of October,1979, asa post-doctoral fellow. I had less than a hundred dollars in cash, aletter from Dr. Hart, and a suitcase filled with cashew nuts andraisins. As a vegetarian, I had no idea what I would find to eat in theUnited States. I was fortunate to have the best possible guide intoAmerican life; like a kindly grandmother, Mrs. Helen Dixon hostedmany foreign postdocs in her large home near campus. She was mygood friend and host for my entire time at OSU. My supervisor andthe head of our lab, Dr. Hart was tall and vibrant with a boominglaugh that conveyed positivity and progress. My main project atOSU was studying the effects of known carcinogens in rat tissue.The animals were sacrificed to estimate DNA damage in variousorgans. My approach was initially limited to mincing the organswith scalpels in a crisscrossing motion on frosted glass to getsingle-cell suspensions of the tissues that were then used for avariety of assessments. I spent many contented hours in the lab. Iemerged to use OSU’s playing fields and swimming pools, tryingAmerican-style football, diving or tennis. Many weekends werespent in the immigration offices of Cincinnati, where I struggled toobtain a temporary or permanent status that would allow me tostay in the country.

As I was finishing my postdoctoral fellowship at OSU, I wasoffered a position in Jefferson, Arkansas, in January of 1981. Dr. Harthad been appointed director of the NCTR, and he asked me to bepart of his team. He had ambitious goals. Alongside Drs. MingChang and Angelo Turturro, I worked on assessing the effects ofasbestos in vivo and in vitro [7]. I also developed a novel techniqueto infuse BrdU using an intraperitoneal catheter in utero in rats [8].We then found stage-dependent effects of toxic agents on fetaldevelopment by studying SCEs in various tissues in embryos atvarious stages of development [9].

4. Formative ideas for the comet assay

When my appointment as a visiting scientist at NCTR ended,Dr. Steve D’Ambrosio offered me a position as Visiting AssistantProfessor back at OSU in 1982. Returning to OSU resulted in mylong-lasting research collaboration and friendship with Dr. RalphStephens. I learned more about staining DNA working with Dr.Stephens than I had ever known and that was a starting point fordeveloping a new technique. We even published a methods papershowing differences in staining between live and dead cells [10].By this time, I was familiar with several techniques for assessingDNA damage, including the alkaline sucrose gradient technique,which I had learned from Dr. Hart, and the UDS assay. As apostdoctoral fellow, I also became proficient in the nucleoidsedimentation technique, thanks to the guidance of Drs. PhilipLipetz and Ralph Stephens. In this technique, the nonionicdetergent Triton X-100 was added to a high salt (2.5 M) solutionfor rapid lysis of cells.

Learning these techniques and knowing their drawbacks laidthe foundation of ideas for a new technique. While I was still apostdoc, Dr. Douglas Brash, by chance, gave me a book chapter byRydberg and Johanson [6]. Rydberg and Johanson’s techniqueinvolved embedding lymphocytes in agarose gel, lysing cells with asolution of detergent (SDS) and EDTA on microscope slides, airdrying cells in agarose, treating with an alkaline solution, and thenimmersing cells and gels in a neutralizing solution before stainingwith acridine orange. I studied the work overnight, and the nextday Dr. Brash told me how to make agarose, mix it with the cellsand solidify it on microscope slides. In this technique, the alkalinesolution unwinds the DNA, which, after staining, appears as a haloin damaged cells. The intercalation of dye in double-stranded DNAis responsible for the green fluorescence, and the red fluorescenceis due to the association of acridine orange along the singlestranded DNA. Quantification of the ratio between green and red

24 N.P. Singh / Mutation Research 767 (2016) 23–30

25 rads (250 mGy) of X-rays. Taken from these early experiments,Fig. 1 shows control and irradiated human lymphocytes aftermicrogel electrophoresis.

When I had completed a draft of my manuscript, Dr. Tice, whooften came up from Integrated Laboratory Systems in ResearchTriangle Park, NC, to visit NIA, informed me that Ostling andJohanson had published similar work a few years earlier, in 1984. Iwent to the library soon after the meeting to read their paper.Ostling and Johanson [18] had added a novel step, electrophore-sis, to the Rydberg and Johanson technique described earlier.Their new method, however, had two major disadvantages. First,due to the significant amount of RNA, estimation of the correctamount of DNA was not possible. When high quality agarose isproperly made and layered with sufficient thickness on top of alayer of cells, the matrix retains DNA strands, RNA and small,broken fragments of DNA. I wanted to see DNA strands andbroken pieces of DNA but not RNA. Second, sensitivity was limitedby the conditions used for dissociation of the chromatin, whichallowed DNA to maintain its tertiary and quaternary structures.Ostling and Johanson had used a neutral solution for cell lysis.DNA, with tertiary and quaternary structure intact, does not movein a predictable manner.

In the work that we were about to submit for publication, wehad electrophoresed lysed cells under alkaline conditions topartially disrupt secondary structure and to remove the DNA’stertiary and quaternary structure. This allowed more predictablemovement of DNA in the agarose. Alkaline conditions also degradeRNA and reveal more DNA lesions, including single-strand breaks,double-strand breaks, alkali-labile sites, etc., so they are moresensitive than neutral conditions that reveal only double-strandbreaks. This is the basis of the comet assay’s sensitivity. Ostling andJohanson were unable to detect less than 100 rads of damage, whilewe had detected significant changes at 25 rads. Finally, Ostling andJohanson had stained DNA with acridine orange and used afluorescence ratio calculation at two points (nucleus and tail) as anindex of DNA damage rather than migration distance. For thesereasons, I knew that the technique that we were about to publishwould be unique and sensitive. Some years later, after ourpublication of the 1988 paper, Dr. Karl-Johan Johanson came tomy lab at the University of Washington with his colleague Dr. Britt-Marie Svedenstål to see the kind of research we were doing. He wasa man of few words, but he was kind and tolerant and showed atrue love of science.

6. Applications of the comet assay

Our 1988 paper on this technique [11] was, I felt, a big step inthe right direction. My goal had always been to develop a techniqueto visualize aging but my larger aim was to elucidate the causes andmechanisms of aging. At this point, I integrated my original aimwith the new technique. I thought that maybe the technique wouldbe sensitive enough to see changes caused by aging. Using bloodsamples from NIA’s Baltimore Longitudinal Study on Aging, wecompared DNA damage levels in young and old individuals andfound significant differences [13]. For the first time, I was able toobserve changes in the DNA of a single cell due to aging. This hadbeen the driving force behind my leaving my home institution inLucknow, and I felt I had finally found my path.

I was thrilled by seeing the evidence of aging but therelationship was not as overwhelming as I had hoped, and Iwanted to do a better study with more samples and different celltypes. I thought of more experiments. It occurred to me that spermshould not be aging and that there should be zero damage. I lookedat other cell types that, like sperm, had condensed chromatin and Ifound that chicken erythrocytes would offer similar condensation.So I drove from Baltimore to a farm in rural Maryland to get some

fresh chicken blood. After finding extensive DNA breaks, wetheorized that alkali-labile sites are a characteristic of condensedchromatin [14,15]. This was confirmed when we compared levels ofDNA damage in mouse and human sperm [12].

Perhaps because I now had a newborn at home, onephenomenon particularly interested me: two adults, with rela-tively old cells, can produce a new baby with perfect, intact DNA.How does this happen? After seeing how many breaks werepresent in sperm cells, I speculated that the breaks could berepaired by meiotic proteins before fertilization in order toproduce healthy new offspring. I became interested in recombina-tional repair and was particularly interested in the work of aJapanese scientist, Dr. Yasuo Hotta, who had isolated a recombi-nase protein. I wrote to Dr. Hotta to ask whether I could visit his labto learn more about recombinases. He responded favorably andwas kind enough to suggest a source of support. Through thegenerosity of the Japanese Society for the Promotion of Science, Iwas able to stay in Japan for two months. This was a wonderfulexperience both in the lab and outside of it. Dr. Hotta, his team andDr. Takahiro Kunisada were ideal hosts, and I went away withfriendships, a great deal of knowledge and some new ideas.

In 1989 I left NIH to be with my wife and young children in ruralWashington State. At nearby Eastern Washington University, Icontinued to do DNA damage research [16,17], explored therelationship between DNA damage and disease, and observed DNAdamage in an Alzheimer’s model cell line. As an adjunct professor, Ihad a lab but no salary or budget for supplies or equipment. I wroteseveral unfunded grant proposals on aging, and after a year I waslooking for a new position.

In 1991, with the help of Dr. Schneider, I moved to USC where Iperformed modifications of the technique (e.g., trypsinized andnontrypsinized cells) with various kinds of agarose (e.g., lowmelting point but high resolution). None of the adaptationsprovided enough sensitivity. My goal was to detect the minutechanges of human life: exercise, X-rays, even deep inhalation. Wemade several technical modifications to further enhance sensitivi-ty [19]. To free nuclear DNA of proteins, we introduced aproteinase-K step that could be applied after or during regularlysis. To apply a uniform electric field, which minimizes variationin DNA migration from cell to cell and slide to slide, we modifiedthe electrophoretic unit and used a recirculating antioxidant-richalkaline electrophoretic solution. I tried many different kinds ofdyes that might make the technique more sensitive. I used to goaround the nearby labs, looking to get a few drops of any unusualdye – anything I could get my hands on – “Are you using that? No?Can I borrow it?” Anything that I could not find, I ordered from theSigma catalog. I tried 21 different dyes before settling on YOYO-1,an intense fluorescent dye that detects electrophoreticallymigrated DNA extremely well. These changes enabled us to detectsignificant DNA damage at doses as low as 5 rads (50 mGy) ofgamma-rays [19].

I then wanted to see whether the assay could detect the effectsof an extremely-low frequency (60-Hz) field. My family was now inSeattle, so I telephoned researchers and department heads at theUniversity of Washington (UW) trying to find someone studyingthe effects of extremely low-frequency radiation. Dr. Arthur Guy,who was head of the Bioelectromagnetics Research Laboratory,referred me to Dr. Henry Lai. Dr. Lai told me that it was unlikely thata 60-Hz field could affect DNA because its energy level was so low,but he proposed that we look at radiofrequency radiation becauseits energy is higher. Enthusiastic about this possibility, I decided toleave USC and work with Dr. Lai without pay until we could securefunding. In 1994, we finished our first experiments. I preparedslides and flew with them back to USC to perform the analysisbecause we still did not have a fluorescence microscope with imageanalysis at UW. Using the comet assay, we were able to detect

was done with a special microscope that measured their intensity.The technique estimates DNA damage using a ratio of green to redfluorescence; it cannot quantify the number of DNA strand breaks,but it can be used as an index of DNA damage. However, thevariability was so great that I could never properly visualize orassess induced DNA damage. I repeated the technique to the pointof exhaustion, but the results seemed to be pH-dependent,concentration-dependent and time-dependent. I spent manyhours in the zoology labs at OSU because Dr. Hart’s lab, with itsfocus on alkaline sucrose gradient, had no fluorescence micro-scope. I liked the idea of embedding cells in agarose, but I stillwanted a way to directly quantify DNA damage.

In May of 1982, I attended the First World Congress onToxicology and Environmental Health in Washington, D.C. At theposter session of my work, I saw Dr. Raymond Tice. I was surprisedto see his name-tag, and I asked him, “Are you the same Tice?” Hesmiled and said, “Yes, I’m the same Ray Tice.” Incredulous that theman whose work I had read for so long would be visiting my poster,I asked again and got the same answer. Dr. Tice had been a Ph.D.student under Dr. Schneider, and we had common researchinterests. Thus began our collaboration. We exchanged phone callsand letters, and over the next ten years we would publish severalpapers [11–17], beginning with the 1988 paper that forms the basisof what is now known as the comet assay.

5. The path to the comet assay

In 1985, for several months after my appointment at OSU ended,I was jobless and I spent the time thinking of the ideal technique toassess DNA damage. I already knew I would embed cells in agaroseas Rydberg and Johanson had done. At that time, I realized that Ihad three problems: isolation of living cells, embedding of cells,and lysis of cells. During this otherwise infertile, idle period, theidea came to me to electrophorese the cells in order to move thesmall, negatively-charged DNA pieces outside of the nucleus.Frustratingly, I had no lab or resources to test it. In a lucky stroke,Dr. Schneider called me from the University of Southern California(USC) in the fall of 1985 to tell me that he was going to the NationalInstitutes of Health (NIH), and he asked me to join him there in theNational Institute on Aging (NIA).

Dr. Schneider wanted someone in his lab to be trained inalkaline elution. He had found a perfect place and so for the lasttwo months of the year, I went to Lausanne to learn alkaline elutionin the laboratory of Dr. Peter Cerutti at the Swiss Institute forExperimental Cancer Research. Dr. Cerutti was a thorough teacher.At the end of my visit, he gave a dinner for me at his house. He had aspread of cheeses, breads and special foods. He offered me aspoonful of something very shiny, gray-white in color. He put itdirectly on my plate and I promptly ate it, inquiring only after it wasin my mouth what it was. Caviar, he told me. I kept chewing andasked, “What is caviar?” Fish eggs, he replied. As a vegetarian, I washorrified and had to ask for the restroom! Dr. Cerutti was equallyhorrified. He thought he was offering me a real treat! What Ilearned in the lab, however, was an inspiration for me, and Dr.Cerutti would later make several visits to NIA to see our progress. Imust have spoken of him often at home because my youngdaughter, when given a little yarn doll as a gift, promptly named itPeter Cerutti.

From Switzerland, I went back to NIA and published a paper onalkaline elution of sperm [12]. Still, I could see drawbacks to thealkaline-elution technique: it could have up to 30% variation in thesame sample, even with the same cells under the same conditions.Although I was not satisfied with the technique, I did pick up theidea that sorting DNA according to molecular weight was viableand could be informative. Even while setting up Dr. Schneider’s labfor alkaline elution in 1986, I remained eager to start working on

the idea of alkaline microgel electrophoresis. I did many experi-ments applying current to cells in agarose, but I was not able to getrid of RNA or get the right resolution. Slowly, I was refining themethod. I made microgels after isolating lymphocytes, lysing thecells in high salt with two detergents, and doing electrophoresis inhighly alkaline solution. Lacking samples during these early days ofdevelopment, I used my own blood, sometimes pricking my fingerseveral times a day. I thought to precipitate the DNA after lysis andelectrophoresis because localized DNA could be detected andmeasured more easily. I worked on precipitating DNA usingammonium acetate and ethanol combinations, spermine andethanol combinations, and later, cetrimonium bromide (CTAB) toprecipitate small amounts of DNA. I then washed the DNA inethanol and dried the slides. In previous attempts, I had used aneutral solution with acridine orange. Now I tried an alkalinesolution of ethidium bromide. It proved to be the most stable andsensitive.

I was gaining more knowledge about the structure of DNAunder neutral and alkaline conditions, and I thought it would bemore sensitive to use alkaline electrophoresis. As a bonus, RNA isdegraded under alkaline conditions. The conditions also denaturedDNA, revealing the breaks. I could easily see damage from X-rays,and for the first time I saw comet-like images with a streaming tailrather than a faint break here or there. I could not believe it! I wasjubilant to see the tail, which I knew signified DNA (not RNA). I ranto tell everyone in the lab: Mike McCoy, Dr. Tice and Dr. Schneider.They had some concerns about whether the technique could bereproduced, and I started new experiments straightaway. Isucceeded in showing a difference between controls and cellstreated with 200 rads (2 Gy) of X-rays, but the goal remained tomake the technique sensitive enough to detect damage caused by

Fig 1. Comet assay. (A) shows two human leukocytes, representing an untreatedcontrol after single-cell gel electrophoresis. (B) shows two human leukocytes thathad been irradiated with 100 rads (1 Gy) of X-rays in one minute. The comet-like tailconsists of small fragments of DNA that arose by DNA strand breakage (dye: YOYO-1; magnification 400x).

N.P. Singh / Mutation Research 767 (2016) 23–30 25

fertilization. Our findings led to new research directions that Iwould still like to pursue, specifically the fetal origins of adultdisease.

Many researchers, including myself, had by this time shownrelationships between mutagens and DNA strand breaks using thecomet assay. However, my real work in environmental chemicalsand DNA damage began with my collaborations with Dr. RussHauser at the Harvard School of Public Health who was principalinvestigator on a large study of phthalates (a class of chemicalsfound in a variety of household plastic products). Our ultimategoal was to study the effects of phthalates, PCBs and insecticideson sperm DNA. We found that urinary levels of these chemicalswere associated with increased levels of sperm DNA damage [41].Other studies with Drs. Hauser, Susan Duty and Zuying Cheninvestigated the comet assay in relation to fresh and flash-frozensemen samples [42], semen parameters [43] and insecticides[44]. A collaboration with Dr. John Wise [45] on environmentaland occupational exposures to chemicals also contributed totoxicological applications of the technique. Several CDC andNIOSH studies have recently used the comet assay to studyoccupational exposures. In collaboration with Dr. Mark Toraason,we found increased DNA damage in the leukocytes of factoryworkers exposed to spray adhesive chemicals, such as bromo-propane [46]. In collaboration with Dr. Mark Boeniger, we studiedpolycyclic aromatic hydrocarbons (e.g., benzopyrene; dimethyl-benzanthracene) and DNA damage in auto repair workers. Thesestudies prompted me to develop a protocol for the collection ofsamples in the field, their storage, and their shipment from theagency conducting the study (in our case, CDC and NIOSH) to alaboratory for freezing, thawing and assessment of DNA damage.This protocol was used by the CDC for a project headed by Dr.Mary Ann Butler to study workers exposed to Jet Fuel at US AirForce bases [47].

7. Refinement and new directions for the assay

Real refinement of the comet assay came through customiza-tion of the equipment. After experimenting with the electropho-retic units used in other techniques, in the mid-1990s I decided tomake my own. In consultation with Ralph Stephens, I began todesign a specialized unit. Early on, I would saw flat sheets ofLucite and glue them together in order to realize my designs butthey had problems due to their inexpert construction. We found askilled manufacturer and designer, Clive Ellard (Ellard Instru-mentation). The new unit solved some of the recurrent problemsin the technique and allowed greater sensitivity. I then started tomodify slides, because frosted slides caused background withYOYO dye. We had used frosted slides for better attachment ofagarose, but the uneven background from the frosting made itdifficult to analyze the migrated DNA using an image analysissystem. Two changes were made to address this problem: the useof a tray to simultaneously process eight slides and the use ofnewly designed slides with a clear window and frosted borders[30]. These changes enhanced the sensitivity of the technique tothe point that we could visualize an individual DNA double-strand break in E. coli [30].

Finally, I have worked to attain ultimate sensitivity for assessingthe extent of DNA damage. Considering the comet as only a headand tail may be simplistic. I had to consider the comet in threeparts: head, body and tail. The body consists of relaxed loops ofDNA, and the tail consists of broken pieces of DNA. Our latestrefinement of the comet assay is designed to retain these brokenpieces of DNA. The earliest comet assay studies used a singleparameter: comet length. However, the most complete picture ofDNA damage is offered by the inclusion of a variety of parameters.Dr. Peggy Olive developed the parameter “Tail Moment” to assess

intensity of broken DNA fluorescence. We developed the parame-ter “Integrated Intensity” to account for the three-dimensionalaspects of DNA migration. I have worked to incorporate suchparameters in computerized image analysis programs. I once hadto rely on my own macros and a camera hooked up to a fluorescentmicroscope and computer. Now a variety of advanced imageanalysis systems have been developed and a reliable, automatedsystem for use in labs and clinics is on the horizon.

8. The comet assay comes of age

The comet assay has been modified, adapted and adopted forvarious purposes over the past 25 years. Even the name haschanged through the years. Ostling and Johanson [18] called theirtechnique “Microelectrophoresis.” In our 1988 paper [11], wenamed the assay “the Microgel Electrophoresis technique.” Soonafter the publication of this paper, I was invited to North Carolina tohelp set up Ray Tice’s lab at Integrated Laboratory Systems. Dr. Tice,his versatile and gentlemanly technician Paul Andrews, and I cameup with a better name. We called the technique Single Cell GelElectrophoresis or just Single Cell Gel (SCG). Shortly afterward, Dr.Peggy Olive and colleagues introduced the term “comet assay”[48], and that has rightly stuck for the last 25 years.

In this span, researchers have applied the comet assay to avariety of fields. Dr. Andrew Collins and colleagues introduced theassay’s use in human biomonitoring, studying the possibleamelioration of DNA damage by nutritional supplements [49]and repair enzymes such as endonuclease and formamidopyr-imidine DNA glycosylase [50]. Dr. Awadhesh Jha and others haveinnovated ecotoxicological applications of the assay for use inwildlife and environmental monitoring [51]. My early collaborator,Ray Tice, has taken the lead, along with Drs. Diana Anderson,Emilio Rojas, Yu Sasaki and others, in validating the assay’s use ingenotoxicology [52]. There have been concerted and ongoingefforts to develop international standards for the assay, includingthose of the American, Japanese and European Centers for theValidation of Alternative Methods and principally of the EuropeanComet Assay Validation Group. On the basis of work by thesecenters and the collaborative efforts of several internationalworking groups on the comet assay, the Organisation for EconomicDevelopment and Co-operation (OECD) adopted test guidelines forthe comet assay in 2014 (https://ntp.niehs.nih.gov/iccvam/supp-docs/feddocs/oecd/oecd-tg489-2014.pdf). The assay is now anaccepted method for human biomonitoring according to FDA(http://www.fda.gov/downloads/Drugs/Guidances/ucm074931.pdf) and WHO guidelines. The comet assay has long been an onlinepresence. An NIH list-serve group was established more than 20years ago by Dr. T.S. Kumaravel (comet-assay@list.nih.gov), whobrought knowledge of the assay to thousands of experienced andnovice researchers. Dr. Alok Dhawan established an onlinerepository of protocols, discussion and research related to theassay (http://www.cometassayindia.org/).

If the comet assay had a birthplace, it would be the labs of theNational Institutes, the hub of basic science research in America.Yet, the reach of the technique has quickly expanded beyond theseborders, and I have been able to observe its application in England,Hong Kong, Germany, India, Italy, and Korea. Seeing the techniqueused in many different kinds of labs was evidence to me of itssimplicity and an indicator of its future.

From arachnids [53] to zebra mussels [54], the comet assay hasbeen used in plants, animals and microorganisms of all types. It hasbeen applied to every kind of research that I could have imaginedand at least one that I would never have imagined – preciselyestimating the time of death in homicides [55]. This post-mortemapplication never occurred to me! My original impetus for thedevelopment of the technique was the study of aging and the

increased DNA damage in brain cells of rats exposed to radio-frequency radiation at as low as 0.6 W/kg. The standard for cellphones in North America at that time was a maximum of 1.6 W/kg.The experiments with Dr. Lai on the effects of electromagneticfields [21] and radiofrequency radiation [22] were the beginning ofmy longest scientific partnership, and Dr. Lai became both friendand mentor in my new environment at UW. On the basis of ourstudies on radiofrequency radiation, we obtained funding to dofurther studies and found that 60-Hz fields caused DNA damage[23–25] at a similar frequency to that used by cell phones.Unintentionally controversial, our findings were regarded as achallenge to the growing cell phone industry.

In 1995, we introduced the use of ethanol precipitation ofmigrated DNA in agarose to enhance the sensitivity of detection ofDNA in microgels. This method also allowed slides to be preservedfor future use. Our experimental design for these studies wassimple. I taped microfuge tubes of lymphocytes to a wooden rulerat the 5, 10 and 20 cm marks. I placed the ruler next to a gamma raysource (technetium-99) and the data showed a clear dose responserelationship [20]. Exposure at 4�C prevented DNA repair, resultingin unmitigated accumulation of DNA damage for the duration ofthe exposure. We were able to detect a significant increase in DNAsingle-strand breaks at a dose as low as 1 rad (10 mGy).

I also wanted to use the technique to study the effects of variouscommon substances. Alcohol works as an antioxidant in leukocytecultures and does not cause DNA damage; the story is verydifferent in vivo where ethanol is metabolized into toxicacetaldehyde. In our work, we intubated rats with alcohol anddissected their brains to find the time kinetics of DNA damage. Wefound significant DNA damage from ethanol [26]. We also observedthat the metabolite acetaldehyde is genotoxic [27] in humanlymphocytes in vitro. I then thought that the same substances orexperiences can be oxidant (damaging to DNA) or antioxidantdepending on the existing defenses of an individual. I investigatedthe effects of antioxidants, such as vitamin C, on humanlymphocytes, human diploid fibroblasts and MOLT-4 humanleukemia cells and found a significant DNA damaging effect frommoderate doses of sodium ascorbate [28].

At this point I felt the assay was sensitive enough to detect theminute changes that lead to aging and simple enough to be aregular part of my routine. In fact, I had incorporated the techniqueinto my daily life. I would make small changes in lifestyle and testtheir effects; I did the comet assay on myself almost every day, afterplaying tennis, swimming, eating half a dozen carrots or trying anew vitamin regimen.

In 1995, a collaboration allowed us to see beyond the number ofDNA breaks: Dr. A. T. Natarajan at Leiden University, an expert inchromosome hybridization, led a study combining the neutralcomet assay with the FISH technique. This successful combinationof techniques allowed us to see genes, centromeres and telomeres,and we were able to visualize the location of gene segments. Forthe first time I could see specific genes in the halo of the comet,where we identified the O6-methylguanine-DNA methyltransfer-ase gene [29].

After working for so many years with human chromosomes andDNA, in 1999 I directed my research toward bacteria. There weretwo reasons: (1) I wanted to know whether replicating Escherichiacoli, having a theta (u) shaped chromosome, would have onestraight chromosome if broken. Only one double-strand breakwould be needed to do this and therefore, (2) I wanted to know thesensitivity of detecting only one double-strand break for testingantibiotics or chemicals. Neutral conditions were used to revealonly double-strand breaks, which are lethal in bacterial cells. Theneutral comet assay revealed a simple and elegant demonstrationof these breaks: an E. coli nucleoid with a single tail of DNAstreaming behind it [30].

Our next iteration of the comet assay was only peripherallyrelated to DNA damage: a sensitive method for visualization ofapoptosis on a cell-by-cell basis. In the DNA diffusion assay [31],cells are lysed in alkaline detergent solution, embedded in agarose,and stained using my very favorite dye, YOYO. The technique alsotakes advantage of the numerous alkali-labile sites in DNA ofdamaged cells. Under alkaline conditions, these fragments of DNAdiffuse outward from the nucleus and give apoptotic cells theappearance of a halo. Studying apoptosis, I realized, was crucial instudying how damaged cells are eliminated and thus, critical tostudying healthy aging.

The versatility of a technique lies in its adaptability to a varietyof tissues. Using the comet assay in collaboration with Dr. NormanWolf of the Department of Pathology at UW, we were able to showincreasing DNA damage with age and with light exposure in lensepithelial cells [32],which Dr. Wolf showed was related to cataractformation [33]. We also used an innovative method of dispersing avariety of tissues into single cell suspensions, including the kidney(one of the hardest tissues). Dispersion of tissues into single cells isrequired in many biological assays but the procedure often causesdamage (e.g., the mincing method that I used as a post-doc!), andthere was a need for a device to minimize DNA damage while stilleffectively dispersing tissue. I had earlier worked with a gentlemannamed Tim Hopkins, who designed a specialized and novel system,the Tissue Press [34]. A few years later he called me up with anunusual offer. He had a new device which was intended for use inimmunizations and he wondered if this device, the Biojector, couldbe adapted for use with the comet assay. The CO2 cartridge, whichwas the source of pressure in the syringe, rapidly dispersed anytissue into single cells through a narrow (<50 micron wide) holewith minimal procedural damage. Using this dispersion method, in2001, we were able to show an increase in DNA damage with age inmouse kidney cells in collaboration with Dr. Wolf and Dr. GeorgeMartin. Dr. Martin was the first to correlate lifespan with cloningefficiency in the rat model [35] and one of the authors I looked upin the libraries in Lucknow, India. We were also able to quantify andcalibrate this increase with such sensitivity that we could show theequivalent of 12 months of aging in terms of rads of X-rays andnumber of DNA double-strand breaks [36].

Yet, I still had not answered critical questions about the agingprocess. I had tried to assay DNA damage in human sperm since Ihad first developed the assay. No matter how much I tried, it didnot move during electrophoresis. Even after 24 h and 400 rads ormore of X-rays, I saw no DNA migration. Searching the literature, Iread that sperm chromatin was highly condensed. The process ofchromatin condensation requires crosslinks between DNA andproteins, such as protamines but also some histones. UsingProteinase-K in lysing solution to decondense chromatin finallyallowed me to see an X-ray dose response in sperm exposed toradiation. In 1997, Dr. Stephens and I had introduced a neutralversion of the assay to detect X-ray induced DNA damage inhuman lymphocytes [37]. In 1998, we used this neutral version ofthe assay to detect DNA double-strand breaks in sperm cells [38].This neutral comet assay, using proteinase-K, sensitively detectedDNA damage in sperm and I continue to use it in a variety ofstudies. For example, with Drs. Bhaskar Gollapudi and Sue Marty,we were able to show a relationship between p53 and levels ofDNA damage in mouse sperm [39]. In collaboration with Dr.Charles Muller of the UW’s Male Fertility Clinic, we showed asignificant increase in DNA damage and a surprising decrease inapoptosis after the age of 35 [40]. This meant that men older than35 had sperm with high levels of DNA damage that would not beeliminated by apoptosis and might go on to fertilize an ovum. Thisfinding, labeled a “male biological clock,” attracted high levels ofscientific and media interest. For me, our work contradicted myearlier theory that gametes repair their DNA damage before

[15] N.P. Singh, D.B. Danner, R.R. Tice, J.D. Pearson, L.J. Brant, C.H. Morrell, E.L.Schneider, Basal DNA damage in human lymphocytes with age, Mutat. Res. 256(1991) 1–6.

[16] N.P. Singh, R.R. Tice, R.E. Stephens, E.L. Schneider, A microgel electrophoresistechnique for the direct quantitation of DNA damage and repair in individualfibroblasts cultured on microscope slide, Mutat. Res. 252 (1991) 289–296.

[17] R.R. Tice, P.W. Andrews, O. Hirai, N.P. Singh, The single cell gel (SCG) assay: anelectrophoretic technique for the detection of DNA damage in individual cells,Adv. Exp. Med. Biol. 283 (1991) 157–164.

[18] O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation-induced DNAdamages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123(1984) 291–298.

[19] N.P. Singh, R.E. Stephens, E.L. Schneider, Modification of alkaline microgelelectrophoresis for sensitive detection of DNA damage, Int. J. Radiat. Biol. 66(1994) 23–28.

[20] N.P. Singh, M.M. Graham, V. Singh, A. Khan, Induction of DNA single strandbreaks in human lymphocytes by low doses of gamma rays, Int. J. Radiat. Biol.68 (1995) 563–570.

[21] H. Lai, N.P. Singh, Acute low-intensity microwave exposure increases DNAsingle-strand breaks in rat brain cells, Bioelectromagnetics 16 (1995) 207–210.

[22] H. Lai, N.P. Singh, Single and double-strand DNA breaks in rat brain cells afteracute exposure to radiofrequency electromagnetic radiation, Int. J. Radiat. Biol.69 (1996) 513–521.

[23] H. Lai, N.P. Singh, Acute exposure to a 60-Hz magnetic field increases DNAsingle strand breaks in rat brain cells, Bioelectromagnetics 18 (1997) 156–165.

[24] H. Lai, N.P. Singh, Melatonin and spin-trap compound block radiofrequencyradiation-induced DNA strand-breaks in brain cells of rat, Bioelectromagnetics18 (1997) 446–454.

[25] N.P. Singh, H. Lai, 60 Hz magnetic field exposure induces DNA crosslinks in ratbrain cells, Mutat. Res. 400 (1998) 313–320.

[26] N.P. Singh, H. Lai, A. Khan, Ethanol-induced single-strand DNA breaks in ratbrain cells, Mutat. Res. 345 (1995) 191–196.

[27] N.P. Singh, A. Khan, Acetaldehyde: genotoxicity and cytotoxicity in humanlymphocytes, Mutat. Res. 337 (1995) 9–17.

[28] N.P. Singh, Sodium ascorbate induces DNA single-strand breaks in human cellsin vitro, Mutat. Res. 375 (1997) 195–203.

[29] S.J. Santos, N.P. Singh, A.T. Natarajan, Fluorescence in situ hybridization withcomets, Exp. Cell Res. 232 (1997) 407–411.

[30] N.P. Singh, R.E. Stephens, H. Singh, H. Lai, Visual quantification of DNA double-strand breaks in bacteria, Mutat. Res. 429 (1999) 159–168.

[31] N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. CellRes. 256 (2000) 328–337.

[32] N.P. Singh, P.E. Penn, W.R. Pendergrass, N.S. Wolf, White light-mediated DNAstrand breaks in lens epithelial cells, Exp. Eye Res. 75 (2002) 555–560.

[33] N. Wolf, W. Pendergrass, N. Singh, K. Swisshelm, J. Schwartz, Radiationcataracts: mechanisms involved in their long delayed occurrence but thenrapid progression, Mol. Vis. 14 (2008) 274–285.

[34] N.P. Singh, A rapid method for the preparation of single cell suspension fromsolid tissues, Cytometry 31 (1998) 229–232.

[35] G.M. Martin, C.E. Ogburn, T.N. Wight, Comparative rates of decline in theprimary cloning efficiencies of smooth muscle cells from the aging thoracicaorta of two murine species of contrasting maximum life span potentials, Am.J. Pathol. 110 (1983) 236–245.

[36] N.P. Singh, C.E. Ogburn, N.S. Wolf, G. van Belle, M.G. Martin, DNA double-strandbreaks in mouse kidney cells with age, Biogerontology 2 (2001) 261–270.

[37] N.P. Singh, R.E. Stephens, Microgel electrophoresis: sensitivity, mechanisms,and DNA electrostretching, Mutat. Res. 383 (1997) 167–175.

[38] N.P. Singh, R.E. Stephens, X-ray induced DNA double-strand breaks in humansperm, Mutagenesis 13 (1998) 75–79.

[39] M.S. Marty, N.P. Singh, M.P. Holsapple, B.B. Gollapudi, Influence of p53 zygocityon select sperm parameters of the mouse, Mutat. Res. 427 (1999) 39–45.

[40] N.P. Singh, C.H. Muller, R.E. Berger, Effects of age on DNA double strand breaksand apoptosis in human sperm, Fertil. Steril. 18 (2003) 1420–1430.

[41] R. Hauser, J.D. Meeker, N.P. Singh, M.J. Silva, L. Ryan, S. Duty, A.M. Calafat, DNAdamage in human sperm is related to urinary levels of phthalate monoesterand oxidative metabolites, Hum. Reprod. 22 (2007) 688–695.

[42] S.M. Duty, N.P. Singh, L. Ryan, Z. Chen, C. Lewis, T. Huang, R. Hauser, Reliabilityof the comet assay in cryopreserved human sperm, Hum. Reprod. 17 (2002)1274–1280.

[43] S.M. Duty, N.P. Singh, M.J. Silva, D.B. Barr, J.W. Brock, L. Ryan, R.F. Herrick, D.C.Christiani, R. Hauser, The relationship between environmental exposure tophthalates and DNA damage in human sperm using the neutral comet assay,Environ. Health Perspect. 111 (2003) 1164–1169.

[44] J.D. Meeker, N.P. Singh, L. Ryan, S.M. Duty, D.B. Barr, R.F. Herrick, D.H. Bennett,R. Hauser, Urinary levels of insecticide metabolites and DNA damage in humansperm, Hum. Reprod. 19 (2004) 2573–2580.

[45] H. Xie, S.S. Wise, A.L. Holmes, B. Xu, T.P. Wakeman, S.C. Pelsue, N.P. Singh, J.P.Wise Sr., Carcinogenic lead chromate induces DNA double-strand breaks inhuman lung cells, Mutat. Res. 586 (2005) 160–172.

[46] M. Toraason, D.W. Lynch, D.G. DeBord, N. Singh, E. Krieg, M.A. Butler, Toennis C.A. and Nemhauser J. B;1. DNA damage in leukocytes of workers occupationallyexposed to 1-bromopropane, Mutat. Res. 603 (2006) 1–14.

[47] E.F. Krieg, P.I. Mathias, C.A. Toennis, J.C. Clark, K.L. Marlow, C. B’Hymer, N.P.Singh, P. Egeghy, R.L. Gibson, M.A. Butler, Detection of DNA damage in workersexposed to JP-8 jet fuel, Mutat. Res. 747 (2012) 218–227.

[48] P.L. Olive, J.P. Banáth, R.E. Durand, Heterogeneity in radiation-induced DNAdamage and repair in tumor and normal cells measured using the comet assay,Radiat. Res. 122 (1990) 86–94.

[49] A.R. Collins, V. Harrington, J. Drew, R. Melvin, Nutritional modulation of DNArepair in a human intervention study, Carcinogenesis 24 (2003) 511–515.

[50] A.R. Collins, I.M. Fleming, C.M. Gedik, In vitro repair of oxidative andultraviolet-induced DNA damage in supercoiled nucleoid DNA by human cellextract, Biochim. Biophys. Acta 1219 (1994) 724–727.

[51] A. Jha, Ecotoxicological applications and significance of the comet assay,Mutagenesis 23 (2008) 207–221.

[52] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guidelinesfor in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen. 35(2000) 206–221.

[53] M. Stalmach, G. Wilczek, P. Wilczek, M. Skowronek, M. Medrzak, DNA damagein haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spidersexposed to food contaminated with cadmiumdamage in haemocytes andmidgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to foodcontaminated with cadmium, Ecotoxicol. Environ. Saf. 113 (2015) 353–361.

[54] A. Binelli, C. Riva, D. Cogni, A. Provini, Assessment of the genotoxic potential ofbenzo(a) pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel(Dreissena polymorpha), Mutat. Res. 649 (2007) 135–145.

[55] L.A. Johnson, J.A. Ferris, Analysis of postmortem DNA degradation by single-cell gel electrophoresis, Forensic Sci. Int. 126 (2002) 43–47.

extension of healthy human lifespan. I have worked mostly onstudies in humans or animal models, but a variety of fascinatingand significant research has been done in unusual organisms,wildlife and plants.

The past has been bright: the comet assay has detected DNAdamage in a variety of organisms, tissues and cell types as a resultof aging, disease and exposures. The recent emphasis on studyingphenomena at the single-cell level will ensure its continuingrelevance. As seen in Fig. 2, the number of publications using thetechnique has grown rapidly since 1988 and most rapidly in thelast ten years. No other technique offers the same level ofinformation in the same dramatic fashion: under the microscopewe see those individual strands of DNA that form the basis of ourexistence, and we see their fragility as they break and trail outbeyond their nucleus. It is a striking picture and one that isessential to understanding the health of our own species and avariety of others. As we develop ways to improve health and extendour lifespan, the future of the comet assay looks brighter still.

Conflict of interest

The author states that there are no conflicts of interest.

Acknowledgments

This journey would not have been possible without the supportof my fellow Georgians, Drs. Naresh C. Goel, Umesh C. Goel andMadhava K. Tolani. I am forever grateful for their friendship. Iextend my sincere thanks to Dr. Raymond Tice for the greatcollaboration and many years of dedication to the technique. I amindebted to Dr. Henry Lai for his continued advice and support. Thismanuscript would not have been possible without the

encouragement and editorial direction of Dr. George Hoffman.I thank him and the editors of Mutation Research, past and present.

References

[1] W.S. Sutton, On the morphology of the chromosome group in Brachystolamagna, Biol. Bull. 4 (1902) 24–39.

[2] R. Franklin, R.G. Gosling, Molecular configuration in sodium thymonucleate,Nature 171 (1953) 740–741.

[3] J.H. Tjio, A. Levan, The chromosome number of man, Hereditas 42 (1956) 1–6.[4] N.P. Singh, A.C. Das, M.R. Jha, M.K. Tolani, V. Singh, Chloramphenicol and rabbit

chromosomes, J. Anat. Sci. 1 (1980) 17–19.[5] L. Packer, J.R. Smith, Extension of the lifespan of cultured normal human

diploid cells by vitamin E, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 4763–4767.[6] B. Rydberg, K.J. Johanson, Estimation of DNA strand breaks in single

mammalian cells, in: E.C. Friedberg, C.F. Fox (Eds.), DNA Repair Mechanisms,Academic Press, New York, 1978, pp. 465–468.

[7] M.J.W. Chang, N.P. Singh, A. Turturro, R.W. Hart, Characterization of thecytotoxic effect of asbestos on normal human fibroblasts: proceedings of thesecond international workshop on the in vitro effects of mineral dusts,Environ. Health Perspect. 51 (1983) 237–239.

[8] N.P. Singh, A. Turturro, M.J.W. Chang, R.W. Hart, The measurement of sisterchromatid exchanges induced In Utero utilizing intraperitoneal infusion ofBrdU: a novel technique, Cytogenet. Cell Genet. 35 (1983) 81–86.

[9] N.P. Singh, A. Turturro, R.W. Hart, Stage-specific induction of sister-chromatidexchanges in utero, Mutat. Res. 128 (1984) 17–24.

[10] N.P. Singh, R.E. Stephens, A novel technique for viable cell determinations,Staining Technol. 61 (1986) 315–318.

[11] N.P. Singh, M. McCoy, R.R. Tice, E. Schneider, A simple technique forquantitation of low levels of DNA damage in single cells, Exp. Cell Res. 175(1988) 184–191.

[12] N.P. Singh, D.B. Danner, R.R. Tice, M.T. McCoy, G.D. Collins, E.L. Schneider,Abundant alkali-sensitive sites in DNA of human and mouse sperm, Exp. CellRes. 184 (1989) 461–470.

[13] N.P. Singh, D.B. Danner, R.R. Tice, L. Brant, E.L. Schneider, DNA damage andrepair with age in individual human lymphocytes, Mutat. Res. 237 (1990) 123–130.

[14] R.R. Tice, P.W. Andrews, N.P. Singh, The single cell gel assay: a sensitivetechnique for evaluating intercellular differences in DNA damage and repair,Basic Life Sci. 53 (1990) 291–301.

Fig. 2. Increasing numbers of publications using the microgel electrophoresis technique widely known as the comet assay. The numbers are publications in journals indexedby the National Library of Medicine’s PubMed database since the description of the assay by Singh et al. in 1988 [11]. The search includes papers found using the search terms“comet assay,” “microgel electrophoresis,” or “single cell gel electrophoresis.” Total numbers of publications are also shown for the exact search term “comet assay’ in PubMedand Google Scholar.

Reflections on a career and on the history of genetic toxicity testing in the National Toxicology Program ☆, ☆ ☆Errol Zeiger

Errol Zeiger Consulting, 800 Indian Springs Road., Chapel Hill, NC 27514, USA

Reflections in Mutation Research

Reflections on a career and on the history of genetic toxicity testing inthe National Toxicology Program$,$$

Errol ZeigerErrol Zeiger Consulting, 800 Indian Springs Road., Chapel Hill, NC 27514, USA

Article history:Accepted 9 February 2017Available online 22 March 2017

Keywords:National Toxicology ProgramNTPNCIGene-tox testingMutation

A B S T R A C T

One of the highly visible aspects of the U.S. National Toxicology Program (NTP) has been its genetictoxicity testing program, which has been responsible for testing, and making publicly available, in vitroand in vivo test data on thousands of chemicals since 1979. What is less well known, however, is that thisNTP program had its origin in two separate testing programs that were initiated independently at theNational Cancer Institute (NCI) and the National Institute of Environmental Health Sciences (NIEHS)before the NTP was established. The NCI program was in response to the 1971 National Cancer Act whichdramatically increased the NCI budget. In contrast, the NIEHS testing program can be traced back to apublication by Bruce Ames, not the one describing the mutagenicity assay he developed that becameknown as the Ames test, but because in 1975 he published an article showing that hair dyes weremutagenic. The protocols developed for these NCI contracts became the basis for the NTP Salmonellatesting contracts that were awarded a few years later. These protocols, with their supporting NTP data,strongly influenced the initial in vitro OECD Test Guidelines. The background and evolution of the NTPgenetic toxicity testing program is described, along with some of the more significant milestonediscoveries and accomplishments from this program.

1. My introduction to genetic toxicology

I had the good fortune to become involved with mutagenicitytesting at the time when genetic toxicity was a nascent area oftoxicology, and just beginning to gain recognition as a necessaryendpoint for human health and safety considerations as part of theevaluation of the toxicity of new chemicals. This involvementpredated the Ames test and came about through a number ofsynchronous, unplanned occurrences. In late 1968 I was finalizingmy Masters thesis in microbiology (mycobacterial cell wallchemistry) at George Washington University (GWU) inWashington, DC. I had already taken enough coursework for myPh.D. with a primary interest in clinical microbiology andinfectious diseases. I was looking for a part-time laboratory jobbecause my wife was planning to quit work to remain home after

the birth of our second son, but was having trouble finding a job ina clinical microbiology laboratory with a work schedule restrictedto evenings, nights, and weekends. I had just been turned down fora night technician job at a local hospital (one of a number of turn-downs) because, although they said I was highly qualified, theywould not hire someone with a beard, which I had grown theprevious year (at this time in Washington DC, I was told that “onlyhippies and communists had beards”). When I got back to theschool lab I commented to one of the other graduate students that Iwasn’t able to find a job. Suddenly, a voice behind me said “I knowsomebody who is hiring.” It was Rosalie Donnelly, a microbialgeneticist who had just begun a part-time appointment in thedepartment and whom I had not yet met. She was also doingresearch part-time at the US Food and Drug Administration (FDA)for Marvin Legator. I called Marvin for an appointment and metwith him the following week. The interview went well and heoffered to hire me, not for the part-time position I had requested,but as a full-time FDA employee. In addition, because he hadrecently gotten an adjunct appointment in the GWU MicrobiologyDepartment, any FDA research project that I designed andconducted could be used as the basis for my Ph.D. dissertation,with him serving as my advisor (I added Rosalie Donnelly as my

$ This article is part of the Reflections in Mutation Research series. To suggesttopics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann (ghoffmann@holycross.edu).$$ A number of events described here were described previously [1].

E-mail address: zeiger@nc.rr.com (E. Zeiger).

journal homepage: www.else vie r .com/ locate / rev iewsmrCommunity address : www.elsevier .com/ locate /mutre s

co-advisor because of her training and experience in microbialgenetics). I was so relieved at getting what I thought would be apart-time interim job, which suddenly became a full-time, salariedjob that would also allow me to finish my Ph.D., that I never askedfor details about what I would be doing.

On my first day on the job in July 1969, Marvin gave me a rack ofSalmonella cultures labeled G46, C3076, D3052, and C207, that hehad received from Bruce Ames at UC Berkeley, and introduced me tothe Host-Mediated Assay (HMA), a procedure that his lab haddeveloped and which he had just published [2]. In this procedure,indicator bacteria are injected intraperitoneally into mice and thetest chemical is administered orally or intramuscularly. This allowedfor the bacteria to respond to mutagenic metabolites formed fromthe in vivo metabolism of the test chemical. Three hours aftertreatment the animals were sacrificed and the bacteria wereremoved from the peritoneal cavity and plated for determinationof survival and mutation so that the mutation frequency could bedetermined. Now I knew why Marvin had been so enthusiastic abouthiring me. During our initial interview, he recognized that theprocedures involved in the HMA were identical to procedures I hadused 5 years earlier when I was doing peritonitis research in mice atthe Walter Reed Army Institute of Research where I was assignedafter my Army basic training. The only difference between my Armyresearch procedures and the HMA was that, although I was usingenteric bacteria (E. coli, Salmonella spp., etc.) at Walter Reed, I wasnot looking for mutants, only at total cell counts. Mike Gabridge,who was instrumental in developing the HMA, had left the FDA andMarvin was looking for somebody to run the assay just as I walkedinto his office.

At the time he interviewed me in January 1969, Marvin andothers were heavily involved in forming the EnvironmentalMutagen Society (EMS) and trying to persuade the FDA and otherregulatory agencies to test food additives, drugs, and pesticides formutagenicity [e.g.,3]. The primary impetus for this testing was aconcern for heritable (germ cell) mutation and, secondarily, forcancer. Over the next few years, I performed HMA experiments infulfillment of my dissertation research project on dietary effects onin vivo metabolism of N-nitrosamines to mutagens, in addition toin vitro mutagenicity studies with chemicals not requiringmetabolic activation.

The first three of the Salmonella strains were engineered byAmes to become TA1535 and TA100 (G46), TA1537 (C3076), andTA98 (D3052). C207 (which produced TA1536) was a frameshiftstrain that was discontinued because it did not appear to add to theinformation provided by the other three strains. I becameacquainted with Bruce Ames shortly after starting work at FDA,and he would send me the latest versions of his tester strains (e.g.,the TA1500 series; TA100; TA98; TA97/97a) as they becameavailable, and I used them for the HMA and in vitro procedures.However, Marvin was less than pleased when, after completing mystudies, I concluded that the HMA was a good research tool, butwas too insensitive for use in routine screening [4], since Marvinand others had envisioned the HMA as a primary in vivo test [3,atpg. 603]. My conclusion regarding the HMA’s lack of sensitivity as ascreening assay was confirmed a few years later by the results froman early NCI carcinogen screening project [5].

Entering the field of mutagenicity research at that particulartime in those very early years was especially fortuitous becauseduring my first week on the job I was introduced to HeinrichMalling and Fred de Serres from Oak Ridge National Laboratory,and Gary Flamm from the National Cancer Institute (NCI). They hadcome to FDA at Marvin’s invitation to present a mini-symposiumon mutagenesis and DNA repair, which served as my introductionto these topics. My involvement with these three individuals whowere highly influential in the field turned out to be very importantfor my career; I worked for each of them at one time or another.

Gary succeeded Marvin as FDA Branch Chief and, later, Heinrichand Fred recruited me to work for them in the EnvironmentalMutagenesis Branch at NIEHS. I was also convinced to join the EMSduring that first year of its existence.

2. The start of U.S. government genetic toxicity testing programs

In 1971 Marvin succeeded in persuading FDA to award twocontracts for testing direct food additives for mutagenicity in vitroand in vivo. At the time, the Genetic Toxicology Branch that heheaded was part of the Bureau of Foods, which later became theCenter for Food Safety and Applied Nutrition (CFSAN). I wasresponsible for the test protocols for the bacterial host-mediatedassay and Salmonella plate spot tests and yeast recombinationtests, and for reviewing the data, while Sidney Green wasresponsible for the in vitro cytogenetics and rat dominant lethalstudies. This contract turned out to be not very effective becausethe in vitro tests were without metabolic activation, the protocolsused were not very well developed, and the testing laboratories,like other labs at the time, had no experience in mutagenicitytesting. As a consequence, the test results were never published orused in any way. About 3 years later, when Gary Flamm was myFDA Branch Chief, I was asked to award a new contract for the invitro bacterial mutagenicity and host-mediated assays. This wasjust after the first Ames plate test publications appeared [6], andthe plate test and HMA protocols were specified in the contract.This subsequent contract was awarded to Litton Bionetics, whichhad hired David Brusick for this award as their director of genetictoxicology.

In 1975, the NCI formed an interagency ad hoc advisorycommittee on which I served with Gary Flamm (FDA), HeinrichMalling, and Fred de Serres (NIEHS). The committee was chaired byVirginia Dunkel (NCI), and its aim was to develop a program tovalidate in vitro genetic toxicity tests for identifying carcinogens,and to also examine the inter-lab reproducibility of the tests.1 Thetests were being evaluated as adjuncts to support the NCI’s cancerbioassay program that was initiated in 1971 in response to theNational Cancer Act of 1971 which tripled the NCI budget and gotthem into the carcinogen bioassay business. At the time NCIformed this gene-tox committee, Ames had not yet published theresults of the study that showed that his new plate test procedurewas effective for identifying and distinguishing carcinogens andnoncarcinogens [7,8], and there were no available studies on theeffectiveness of the other genetic toxicity tests we were consider-ing. Data generated under a previous NCI contract programinitiated in 1971 to evaluate a number of mutagenicity and DNAdamage tests as predictors of carcinogenicity were not useful for anumber of reasons, including protocol inconsistencies among labsdoing the same procedure, changes in protocols in mid-contractafter Ames’ publications describing the plate test with metabolicactivation, testing too few noncarcinogens, and the fact that thechemicals were not tested blind [10]. I had become involved in thatearlier program after the contract awards were made, and I hadbeen asked to monitor the in vitro microbial and yeast studies, andthe HMA, provide guidance to the labs, and evaluate the testresults.

This NCI interagency ad hoc advisory committee was interestingin several ways. Its organizer and Chair, Virginia Dunkel, was not ageneticist, but a cell biologist with primary interest in celltransformation systems. She saw the potential of genetox testingand enthusiastically advocated its use in the context of carcinogen

1 The background and genesis of this NCI program, and the reasons for theselection of the Salmonella and mouse lymphoma assays, were describedpreviously [9].

E. Zeiger / Mutation Research 773 (2017) 282–292 283

identification. At that time there were three in vitro mammaliancell gene mutation assays that were well described and that hadthe highest exposure at conferences and in publications. Thesewere the Chinese hamster cell mutation assay that had beendeveloped by Heinrich Malling and Ernie Chu at Oak Ridge NationalLaboratory [11], the L5178Y mouse lymphoma cell assay (MLA) thatwas developed by Don Clive [12], who was Gary’s post-doc atNIEHS, and a human lymphoblast assay [13] developed by BillThilly at the Massachusetts Institute of Technology (MIT) andwhich had been patented. Although we initially favored thelymphoblast assay because it was in a human cell, we agreed that itwould not be appropriate for the U.S. Government to finance thevalidation of an assay that would be licensed for profit. After muchdiscussion, the committee decided that this testing programshould concentrate on the Salmonella plate test [6], which wasthen all the rage, and the L5178Y mouse lymphoma assay [12],which would provide gene mutation information from a mamma-lian cell. As a junior person in the field, I was much impressed bythe ability of Heinrich and Gary, developers of the CHO and MLAassays, respectively, to objectively evaluate the merits of the twotests in the context of programmatic needs. Because the traditionalmammalian-cell chromosome aberration tests were consideredwell-established, a decision was made to not include these tests inthe validation program.

The publication from Ames’ lab showing a high concordance(85%) between Salmonella mutagenicity and rodent carcinogenicity[7], which came out during our discussions, provided support for ourselection of the Salmonella test. Other studies coming soonthereafter, reporting 87% [14] and 93% [15] concordance, reinforcedour hopes that the right combination of bacterial mutation andmammalian cell tests could eliminate the need for many of therodent cancer tests and allow resources to be directed towards in-depth carcinogenicity studies of chemicals of particular interest.

Virginia and I designed the Salmonella test protocols for thisstudy, and the mouse lymphoma assay protocols were designed byVirginia and Don Clive, the co-developer of the assay [12]. Requestsfor proposals from laboratories to perform Salmonella and mouselymphoma assays were advertised in 1975, and the contracts wereawarded in mid-1976. Four laboratories were awarded theSalmonella contracts, and two were awarded the mouse

lymphoma contracts (Table 1a). Virginia and I were the ProjectOfficers for the Salmonella contracts, and she and Don Clive for themouse lymphoma contracts.

The chemicals to be tested were selected from those that wereon test, or were selected for testing, in the NCI carcinogen bioassayprogram which started in the early 1970’s. Wherever possible, thesame batch of chemical would be used for the cancer andmutagenicity tests, which would allow a comparison of resultsfrom the two standardized protocols.

In June 1976, Virginia and I had just completed a pre-awardnegotiation at NCI in Bethesda, MD, with one of the contractfinalists when she was informed that there were a couple ofvisitors who would like to talk with her. They introducedthemselves as Ian Purchase and John Ashby of ICI CentralToxicology Laboratory in England. They described a project thatthey had recently completed and was being prepared forpublication on the effectiveness of 6 different in vitro and in vivotest systems to identify potential carcinogens [16]. Only one ofthese tests (the Salmonella assay) measured mutation [15]. Themost important thing to come out of this ICI project and itspublication was that it introduced John Ashby, an organic chemist,to the world of genetic toxicity testing, and vice versa. It alsomarked the beginning of a long, intellectually fruitful, and alwaysinteresting collaboration with John. John provided extraordinarycontributions to the field over the ensuing years throughpublication of a large number of interesting, insightful, andprovocative articles about the use and interpretation of variousgenetic toxicity tests and test combinations, as well as introducingmany novel ideas to the field.2

3. The development of the Salmonella and mouse lymphomatest protocols

Because of a lack of experience with the Ames test protocoland questions about the sensitivity and effectiveness of the

Table 1Initial NTP mutagenicity testing contracts.

Laboratory Principal Investigator

a. NCI contractsSalmonella + E. coli New York Medical College Herb Rosenkranz

EG&G Mason Research Institute/MicrobiologicalAssociatesa

Steve Haworth

Inveresk Research International Douglas McGregorSRI International Vince Simmonb and Kristien Mortelmans

Mouse lymphoma cell Litton Bionetics Brian MyhrSRI International Ann Mitchell

b. NIEHS contractsSalmonella Case Western Reserve University William Speck

EG&G Mason Research Institute/MicrobiologicalAssociatesa

Steve Haworth

SRI International Kristien MortelmansIn vitro chromosome aberrations & SCE Columbia University Art Bloom

Litton Bionetics Sheila GallowayMouse lymphoma cell Inveresk Research International Douglas McGregorRodent bone marrow chromosome aberrations & SCE Brookhaven National Laboratory Ray Tice

Oak Ridge Associated Universities Al McFeeDrosophila sex-linked recessive lethal and heritabletranslocation

Bowling Green State University Ron WoodruffBrown University Stan ZimmeringUniversity of Wisconsin Seymour Abrahamson and Ruby

Valencia

a During the course of the contract, EG&G Mason left the genetox testing business and the work and personnel (Steve and Tim Lawlor) moved to Microbiological Associates.b Vince left SRI shortly after the contract started and was replaced by Kristien Mortelmans, who had been hired as co-Principal Investigator.

2 In the mid-late 1990’s, as endocrine disruption and the identification ofendocrine-disrupting chemicals became the newest scientific concern, John shiftedhis interests to this area where he was just as provocative and productive.

284 E. Zeiger / Mutation Research 773 (2017) 282–292

different strains and metabolic activation preparations, Virginiaand I decided to test in strains TA98, TA100, TA1535, TA1537,TA1538, and E. coli WP2. Metabolic activation would be by S9preparations from Aroclor-induced and uninduced mouse, rat, andhamster livers. We used S9 from multiple species to determinewhich species was the most effective at metabolizing promuta-gens, and also to determine whether Aroclor induction was ofbenefit for all classes of promutagens or just those originallyreported by Ames. Repeat testing was not included in thecontracts, and all chemicals were tested under code to avoid biasby the testing laboratories or by Virginia and me when weevaluated the data and assigned our conclusions of positive,negative, or equivocal to the outcomes.

Because we suspected that some mutagenic chemicals couldhave very narrow response ranges between their nonmutagenicconcentrations and concentrations that were toxic, we decided touse half-log dose intervals. This spacing provided an additionaladvantage; it was prior to the availability of computer graphingprograms and half-log doses would allow us to plot the data onsemi-log graph paper (half-log intervals are equidistant in thisformat). Also, because we did not want to use resources inperforming preliminary toxicity assays, we used 7 test chemicalconcentrations and set the limit concentration at 333 mg/plate.While this is low by current standards, the majority of knownSalmonella mutagens at that time would have been detected in thisrange. We believed that such a three-order-of-magnitude rangewould be sufficient to test both nontoxic and highly toxicsubstances. We also decided to use 3 plates per test concentrationto provide a more ‘stable’ response than a 2-plate protocol and abetter estimate of plate-to-plate variation.

The results were evaluated during the testing of an initial 59chemicals, and it was obvious that there were a number ofchemicals that were not toxic or mutagenic at 333 mg, includingsome known or anticipated bacterial mutagens that possiblywould have shown up as mutagenic at higher concentrations.Therefore, for the second batch of 60 test chemicals, the labs wereinstructed to use the same protocol and number of testconcentrations, but raise the limit dose to 10,000 mg/plate. Thisvalue, like the previous one, was completely arbitrary. The resultsof these studies, and the plate count data, were published in twosupplemental issues of Environmental Mutagenesis [17,18].

Because of the anticipated volume of data (i.e., 6 bacterialstrains; 7 metabolic activation conditions; triplicate plates; 7 testchemical concentrations plus solvent and positive controls; and119 chemicals) multiplied by 4 labs, it was obvious that computersupport would be needed to manage the test data. This was beforedesktop computers, so data forms were designed and the data fromthe forms were keyed in by NCI computer support personnel,stored on computer tapes, and made available to us in printed formwith the plate counts, plate count means, and standard errors ofthe means. For one reason or another, and partly because ofoversight on my part, those data tapes were never transferred tothe NIEHS to be transformed into the database format being usedfor the NIEHS/NTP contracts. Because of this oversight, the NCISalmonella test data are not available through the NTP websitewith the other NTP genetic toxicity test data. Their only existence isin the two publications from these contracts [17,18].

The protocols that were developed for these NCI Salmonellacontracts became the basis for the protocols in the subsequent NTPSalmonella testing contracts that were awarded a few years later(below). They also served as the basis for OECD Test Guideline 471on Salmonella mutation tests [19].

Few laboratories were performing the mouse lymphoma TK+/�

assay in 1976, and those that did had only minimal experience withit when this contract was advertised, so the Principal Investigatorsand their staffs were initially trained and supervised by Don Clive’s

laboratory. Sixty-three chemicals were tested in both contractlaboratories under code. The initial protocol included preliminaryrange-finding studies with concentrations up to 5000 mg/ml forsolids and 5000 nl/ml for liquids. A 4-h exposure time was usedwith and without 5–10% Aroclor-induced rat liver S9 (it wasalready known that higher concentrations of S9 and longerexposure times were toxic to the mammalian cells, in contrastto the bacteria which could tolerate much higher concentrations).A number of protocol changes were made during the course ofthese contracts [20] which reflected the developing experiences ofthese and other laboratories, including the Clive laboratory, thatwere also performing the assay. Similar to the Ames test, theprotocols designed for these MLA tests influenced the protocolsdeveloped for subsequent NTP contracts, and also influenced OECDTest Guideline 476 on mammalian cell mutagenesis [21]. The testdata and results from the two-laboratory NCI studies werepublished in a series of articles in Environmental Mutagenesis[22–26].

4. At the NIEHS

In late 1975, I was approached by Heinrich Malling, whom I hadknown since starting work at FDA, who asked if I would beinterested in moving to NIEHS to be the head of the MicrobialGenetics Group of the Laboratory of Environmental Mutagenesis.At that time Fred de Serres was the Laboratory Director, andHeinrich was a Group Leader and, for want of a better term, theassistant Laboratory Director. It sounded like a great opportunity tome and I accepted, although I was a bit reluctant to leave theWashington, DC, area. I was offered the position in early 1976 andwas asked to make a number of trips to NIEHS for discussions inadvance of my formal start in July. It was during the first of thesediscussions that Heinrich asked me to design a large-volumemutagenicity testing program to be headquartered at NIEHS, andhe told me that I would be responsible for getting it operational.His offer was partly based on my involvement and experience withthe FDA and NCI testing contracts.

Why was NIEHS asked to establish its own, separate genetictoxicology testing program? At about the same time as the NCIcontracts were being designed and competed in 1975, Bruce Amespublished a paper in PNAS titled “Hair dyes are mutagenic: . . . ”

[27]. This got the attention of the FDA, the public, and also the U.S.Congress. In their 1976 NIH budget hearings, the House Committeeon Appropriations was aware of Ames’ finding that hair dyes, andcomponents of the dyes, were mutagenic and therefore, possiblycarcinogenic. With uncharacteristic haste, the Committee issuedthe following recommendation:

“Exploratory testing of environmental chemicals and othercompounds is turning up new examples of genetic effects(mutagenicity) in products in widespread use. For example,almost all commercially available hair dyes were recentlydiscovered to be genetically active in some of the newly-developed short-term tests.. . .The Committee believes that it is very important to thiscountry’s future well-being that NIEHS launch a program oftesting for mutagenicity all compounds produced in commer-cial quantities.” [emphasis added] [28]3

At that time, the most promising, popular, and best-definedtests were the Salmonella test (which was not yet being called the

3 The term “produced in commercial quantities” was not defined, so it wasinterpreted it as any chemical that the testing program personnel, or the NIEHS orother agencies, was interested in testing.

Ames test), in vitro mammalian cell chromosome aberration andsister chromatid exchange (SCE) tests, and the Drosophila sex-linked recessive lethal (SLRL) test. I proposed that the NIEHScontract testing program start out with these tests, with theability to make changes in the future as new tests emerged, or ifthe proposed tests were not performing as anticipated. Therodent dominant lethal assay was also very prominent at thattime, but it identified germ cell mutagens, and the NIEHS programwas directed at carcinogens. I also decided that the programshould have more than one contract lab for each test, as a back-upin the event of a problem in one lab, and also as a control for theother lab(s). I had not included the MLA in my original planbecause it was not well characterized at that time, and themajority, if not all, of the publicly available test results were froma single lab, Don Clive’s at Burroughs-Wellcome [12,29]. I thoughtthat we should wait for the results from the two NCI contractsthat Virginia Dunkel had just awarded before deciding on amammalian cell mutation test system. However, those two NCIcontracts were transferred to the NIEHS when the NTP wasformed, which provided a mammalian cell mutation componentto the testing program, and an additional contract for the assaywas awarded to Inveresk Research International (DouglasMcGregor) to test additional NTP chemicals.

My original plan, which reflected my enthusiasm for theprogram, and which was approved by Heinrich, Fred, and theNIEHS director, David Rall, called for testing 1000 chemicals/year inSalmonella, which was the fastest and least expensive test, andunspecified fewer chemicals in the other tests. However, afterthinking it through, I remembered that the Government’s workyear for contracting purposes is 2000 h. If I was responsible formanaging the testing and reviewing the test data for 1000chemicals/year, I, or somebody else, would have to review theresults of an average of one chemical every two hrs throughout theyear. Also, since the data would be keyed into the NIEHS computerby hand from pen-and-ink forms, the data entry people wouldhave the same work schedule in addition to the data from the othertests. There was also the added factor of sufficient, experiencedlaboratory personnel and facilities for this volume of testing. Giventhis information, Dr. Rall agreed to my proposal to lower the effortto 300 chemicals/year in Salmonella, with the possibility of futureexpansion.

Based on my experience with the FDA testing contracts, Irecommended that a chemist be hired into the program to direct acontract for a chemical repository to locate, purchase, code, anddistribute the test chemicals, and to oversee chemical analyses ofselected chemicals for purity and/or identity, if necessary. BecauseI anticipated a large volume of data from the different test systems,we would need procedures for their storage, retrieval, and analysis.Therefore, the program would also need somebody to set up andoversee the data processing and development of an electronicdatabase in the new area of digital computers, and a statistician todevelop analytical approaches to the data.

I requested 12 positions (with the hope of getting 9 or 10) tooversee the technical aspects of the contracts and providemanagement support for the program, including a chemist,database specialist, statistician, contracts coordinator, and secre-tary. At that time, the NCI’s cancer bioassay program testingcontracts were being managed by a ‘prime contractor’, which hadresponsibility for the management and oversight of all the testinglabs. This meant that the responsible NCI toxicologists andpathologists had no direct contact with the testing lab personneland needed to go through the prime contractor to communicatewith the bioassay labs, or to get information from those labs. WhenVirginia and I were designing the NCI genetox contracts theprevious year, we became aware of a number of problems with theNCI bioassay contracts that centered around poor supervision by

the prime contractor and a possible lack of awareness of scientificissues by the prime contractor’s staff and, therefore, also by the NCIpersonnel responsible for the individual chemicals. I insisted thatthe NIEHS testing program personnel be the responsible ProjectOfficers and have current knowledge and expertise in the areas ofmutagenesis and genetics. This meant hiring individuals whowould have their own research programs in genetics andmutagenesis. They would spend approximately 50% of their timeas contract Project Officers and the remainder of their timeperforming intramural research. This approach was designed toensure that the Project Officers overseeing the testing contractswould have current knowledge of the science to provide guidanceto the contractors. They would also be responsible for trouble-shooting problems with the assays and not have to depend on anintermediary contractor with possibly less expertise or knowledge.With the help of Larry Valcovic, who was working with Heinrich atthe time to develop rodent germ cell mutation tests, we christenedthis new testing program, the Environmental Mutagenesis TestDevelopment Program (EMTDP) [9,30,31]. A similar approach, i.e.,foregoing the prime contractor and having the intramuralscientists responsible for the scientific management and directionof the testing contracts, was adopted by the NIEHS/NTP when itassumed responsibility for the carcinogen bioassay testingprogram.

Much to my (and others’) surprise, all 12 requested positionswere granted in the 1978 budget. Three of these were immediatelytaken from me by the NIEHS upper management to hire threeindividuals for an anticipated new toxicology effort that I wasunaware of (and which subsequently became the NationalToxicology Program). The remaining positions were used to hiretwo lab researchers (Mike Resnick, a yeast geneticist, and JamesMason, a Drosophila geneticist) and their technicians, a contractcoordinator (Beth Anderson), chemist (Doug Walters), computerdatabase manager (Mike Rowley), statistician (Barry Margolin),and a secretary (Joyce Wilder). Beth Anderson’s scientificbackground and lab experience made her the ideal person to bethe Contracts Coordinator. She was responsible for tracking thecontracts, including budgets, shipment of chemicals, receipt of testresults, serving as a contact with the contract Principal Inves-tigators, the data processing group, and the NIEHS contracts officeon both technical and administrative matters, assisting with theinterpretation of data and preparation of manuscripts, and “otherduties as assigned.”

One position was reserved for the director of this new testingprogram because my intention was to be responsible for theSalmonella contracts and run my in-house research program,and find somebody else to run the testing program. However, Iwas informed by the NIEHS and NTP Director, Dave Rall, that heexpected to be in charge of the entire program. An expectationlike that, especially from Dr. Rall, was impossible to ignore, so Ireluctantly agreed. It turned out to be a good decision. The NTPformally came into being during the time that the NIEHScontracts were being awarded, and the following year the NCIcontracts were transferred to NIEHS and incorporated into theNTP testing program.4 Rather than move to North Carolina withher genetox contracts, Virginia Dunkel left the NCI and acceptedthe position of Director of the Genetic Toxicity Branch at FDA,the position previously held by Marvin Legator, Gary Flamm, andSidney Green. As a result, I was the Project Officer for thoseseven Salmonella contracts, and Bill Caspary, who had trans-

4 The NCI was initially a component of the NTP, along with the the NIEHS, FDA andNIOSH, but decided to transfer its Salmonella and mouse lymphoma testingcontracts, and its cancer bioassay testing program, to NIEHS and withdraw from theNTP.

ferred from NCI to NIEHS, was given responsibility for the twoMLA contracts.

5. The National Toxicology Program testing contracts

The NIEHS/NTP Salmonella testing contract protocol I selectedwas based on my experience with the NCI contract: the testerstrains (TA98, TA100, TA1535, TA1537), with no metabolicactivation, and 10% S9 preparations from rats and hamsters, threeplates per dose, and a limit dose of 10,000 mg/plate. If the initialtests were negative, the full protocol was to be repeated. If any ofthe tester strains/S9 conditions were positive, only the positivecondition(s) needed to be repeated [32]. Although the initial Amestest publications described the standard plate test, and it was usedfor the NCI contracts, I selected the preincubation protocol becauseof information that it was more sensitive for some chemicalclasses, such as linear nitrosamines [33] and some volatilechemicals tested in my lab at NIEHS [E. Zeiger, unpublishedobservations].

At the same time the Salmonella contracts were awarded, twocontracts each were awarded for chromosome aberrations and SCEin CHO cells, and for the sex-linked recessive lethal test inDrosophila (Table 1b). Mike Resnick and I were the Project Officersfor the initial CHO contracts, and Jim Mason was Project Officer forthe Drosophila contracts. Contracts for in vivo rodent bone marrowchromosome damage (aberrations; SCE) were awarded by MikeShelby in the early 1980’s to Ray Tice at Brookhaven NationalLaboratory and Al McFee at Oak Ridge Associated Universities.Additional in vivo contracts followed for micronuclei (MN) in bonemarrow and/or peripheral blood (to Jim MacGregor at USDA andsubsequently, John Mirsalis at SRI International, as PrincipalInvestigators). Some of the in vivo MN tests were performed instand-alone studies, but most of the peripheral blood testing wasintegrated into 14-day or 90-day subchronic toxicity tests in micethat were preliminary to cancer bioassays. At this time, we did notrequest peripheral blood erythrocyte micronucleus (MN) tests inrats because it was difficult to accurately differentiate immaturefrom mature erythrocytes in the peripheral blood slide prepara-tions before flow cytometry analysis was available in mostlaboratories. This integration of the MN test in mice used fortoxicity studies allowed us to get blood samples at the time ofsacrifice so that the MN test data would be obtained from the sameanimals as the other short-term toxicity endpoints, thus providingenhanced context for the genotoxicity endpoint and expandedtoxicity profiling. In addition to making the MN data moremeaningful, this also reduced animal use and the cost of obtainingthe data. Adding MN testing to the toxicology tests did not allow forthe inclusion of a positive control group, but we decided to goahead anyway and rely on the laboratory’s expertise and thevehicle-control values which were required to fall within in thelaboratory’s historical control range.

There was an interesting irony to the in vitro and in vivochromosome aberration, SCE, and MN contracts. In my design ofthe testing program, I was insistent that the NIEHS scientistssupervising the contracts be experienced in the procedures so as tobe able to adequately supervise the work, troubleshoot anyproblems, and analyze the data. Despite this, the Project Officersfor these cytogenetics contracts were me, Mike Resnick, and MikeShelby, with help from Beth Anderson. None of us had experiencewith performing cytogenetics assays in vitro or in vivo, but wewere responsible for evaluating the procedures and the test data.We were comfortable with this role because the PrincipalInvestigators of these contracts (Table 1) were excellent teachers.

Before the testing contracts were awarded, the chemist whohad been hired to support the EMTDP (Doug Walters) and I wentthrough the lists of chemicals being tested by the NCI, the IARC

carcinogen classification lists, and the Aldrich chemical catalog toselect the initial 300 hundred chemicals to be ordered by thechemical repository contractor (Radian Corporation) so that theywould be ready for shipment as soon as the testing contracts wereawarded. We also included chemicals that we were personallyinterested in and some that we felt had interesting structures. Itturned out that we had good foresight because many of thechemicals that we chose because of our interests were laternominated by one or another agency or organization for cancertesting.

In order to monitor laboratory performance and obtain data onintra- and inter-laboratory reproducibility, the repository wasdirected to include, at random, one known positive or negativechemical (from a list supplied to them) with each batch of 15 codedchemicals sent to each testing laboratory, and/or a repeat of achemical selected at random from one of the chemicals previouslytested by the same or different lab. The testing laboratories knew ofthis practice but had no idea as to which chemicals were thecontrols or duplicates because the chemicals were coded. Also,chemicals that were judged equivocal or weakly positive wereoften sent to the same or different lab without that lab’s knowledgefor a repeat test. Because of this practice, there were sufficient datain the NTP database to allow an analysis of the reproducibility ofthe assay when performed using the standardized protocol andevaluation criteria.

The first NIEHS contracts were awarded in late 1978 and early1979. The contractors agreed to follow the specified protocols anddata-reporting procedures, and test data began coming into NIEHSshortly after the contract awards. As can be seen from the list ofPrincipal Investigators for this first round of contracts (Table 1b),the program attracted individuals of the highest caliber, some earlyin their careers, to do the testing. During the first few months of thecontracts the test data were submitted on paper forms, which weregiven to data entry clerks who typed the information into acomputer-readable format.

When the NTP testing contracts started, the EMTDP bought intoan existing NCI computer software development contract (withBolt, Beranek, and Newman, Inc.) to develop a data capture,evaluation, and reporting system for the Salmonella and other testdata. Because this was pre-desktop computer days, the system thatwas developed used an in-lab computer (MINC; made by DataGeneral) connected to a small blue-screen terminal, and used 9-inch floppy disks, one per chemical [34]. The MINC was big (about3 ft. � 3 ft. � 2 ft.), noisy, and slow, but it allowed the lab techniciansto directly enter the plate counts, specific experiment protocolinformation and comments, technician name, and dates, and theprogram was compatible with Good Laboratory Practices (GLP).The lab would make a copy of the disk and mail the original to thesoftware development contractor who would enter the data intothe mainframe VAX computer at their location in Cambridge, MA,perform simple calculations, make the data available for me to readand receive reports on my office terminal, and provide dataprintouts for specific chemicals on request. At the time it wascutting-edge computer technology. Soon afterward, the softwaredevelopment and maintenance program was transferred from thecontractor to NIEHS because in-house Government-supportedcontractor personnel cost much less than outside computersoftware company personnel. The NIEHS in-house databasesupport contractors subsequently developed the software forthe in vitro chromosome aberration and SCE, in vivo MN,Drosophila, and MLA tests.

As soon as desktop PC’s became commercially available, thesupport contractor was tasked with reworking the data captureprograms so that they could be used with the testing laboratoryand NIEHS desktop computers. They were also tasked with revisingthe program so that the Salmonella plate counts could be captured

standard strains were negative and the chemical had a structurethat suggested that it would induce oxygen radicals or was analdehyde.

Because our Salmonella test protocol was not identifying a largeproportion of the known rodent carcinogens (many of which weresubsequently categorized as “non-genotoxic”), we modified theprotocol so that the repeat test of a negative response that had used10% S9, would be done using 30% S9. As with the limit doseselection,10 and 30% S9 were arbitrary values and not based on anyin situ physiological standard. Although Ames’ original methodspaper [43] recommended 4–10% Aroclor-induced rat liver S9 forroutine screening, we found that the sensitivity of the assay andthe level of the mutagenic response tended to increase withincreasing levels of S9, and that levels under 10% may not besufficient for many chemicals [51; unpublished studies]. Otherthan increasing the cost of the test, I assumed that we would notlose sensitivity by using the higher (30%) S9 concentration. A laterreview of the data showed that a number of chemicals that werenegative or equivocal with 10% were positive with 30% S9. As aconsequence, the protocol was changed so that the initial test used30% S9 and, if negative, the repeat test used 10%. However, wenever saw any chemicals that were negative with 30% but positivewith 10%.

When an analysis of the Salmonella database was performed in1991, there were 239 chemicals that were tested from 2 to 12 timesusing the same protocol in the same or different labs, not includingthe required repeat tests on the same sample. A few of these wereknown positives and negatives that were included to monitorlaboratory performance, but the majority were chemicals thatwere randomly sent to more than one lab, or chemicals thatproduced an equivocal or weak response in their initial test. Thisanalysis showed that the reproducibility of the test when equivocalresults were not included and weak positives were considered aspositive was 86.89% (206 chemicals; pairwise concordance;positive vs. negative) [52]. There was no difference in thereproducibility within labs compared to among labs.

During the first few years of the program, the repository wasasked to analyze all chemicals that produced positive or equivocalresults in any of the genetic toxicity tests for identity and purity, inaddition to other chemicals that were analyzed prior to testing forvarious reasons. Two things became clear – the chemical supplier’sstated purity was occasionally higher than the NTP-analyzedpurity, and that there was occasional mislabeling of chemicals, e.g.,the wrong salt or water of hydration or, of more concern, the wrongchemical name was on the label.

The frequency of mislabeling with the wrong chemical namewas 4/633 (0.63%) when differences in the salt or water ofhydration were not considered, and it was not confined to a singlesupplier. This was not a large value, but definitely worthy of note.We prepared a brief manuscript describing this to inform thescientific community, and I sent it to an Associate Editor of a well-respected toxicology journal (that shall remain nameless here). Itwas promptly returned to me with the comment that such apublication was unnecessary because good toxicologists alwaysconfirm the identity of their test chemicals and reagents beforeusing them. This was news to me and to the toxicologists I sharedthis with. However, a journal targeting the Quality Assurance andGood Laboratory Practices (GLP) community was interested, andour findings were eventually published [53].

One basic tenet of the testing program was that the perfor-mance of the tests would be continuously evaluated, and tests thatwere not performing to expectations or provided results that wereredundant to other, more facile tests, would be dropped fromroutine use. Because the genetic toxicity and carcinogenicity testswere performed using standardized protocols and evaluationprocedures, we were able to evaluate the effectiveness of the

genetox tests for predicting the outcome of the cancer assays. Anumber of such comparisons were made [17,18,54], but the onewith the biggest impact was the evaluation of 73 chemicalspublished by Ray Tennant and colleagues in 1987 [55]. A follow-upstudy with an additional 42 chemicals supported the 1987 results[56]. These studies clearly showed that the standard in vitromammalian cell tests (chromosome aberrations and SCE in CHOcells, and the MLA) were not as effective for distinguishingcarcinogens from noncarcinogens as was originally believed basedon earlier publications from individual laboratories and the EPAGene-tox program, which included primarily carcinogens [57–59].All the NTP studies [55,56,60] showed that results from the in vitrochromosome aberrations, SCE, and mouse lymphoma cell tests,when used in addition to Salmonella, did not add to the carcinogenpredictive value of the Salmonella test alone, that is, the assays, asrun, were not complementary. A Salmonella-negative chemicalthat was positive in a mammalian cell assay was equally likely to bea carcinogen or a non-carcinogen.

Because of the performance characteristics of the in vitromammalian cell tests [55,56], a decision was made to no longerperform the MLA, SCE, or chromosome aberration tests routinely,but to use them only if specifically requested by the nominatingagency or if other factors indicated that those test results would beuseful. Testing with the Drosophila SLRL assay was also discon-tinued at that time. Although a positive response in this assay washighly predictive of rodent carcinogenicity, it was considered tooinsensitive for routine use because only 20–30% of the carcinogenstested were positive [61]. The test was also relatively expensive,and there were concerns expressed that we should not be usinginsects to test for potential human health effects (curiously, thesesame voices had no objections to using bacteria and yeast).

The Tennant et al. publication [55] generated much discussion,and publications denouncing our conclusions as flawed for onereason or another, such as inadequate protocol or lab performanceor incorrect data evaluation procedures. The main concern was thehigh proportion of false positive results reported for the MLA tests(i.e., MLA positive/cancer negative). It is interesting to note that thein vitro SCE test had the same proportion of false positives, and formany of the same chemicals, but there was no outpouring ofconcern, possibly because no advocates stepped forward tochallenge those results. Subsequent publications of NTP [56,60],and later data [62] supported the findings of the Tennant paper, i.e.,that adding in vitro mammalian cell tests to the Salmonella test inthe basic test battery did not improve the overall prediction ofrodent carcinogenicity.

Shortly after the Tennant et al. publication [55] appeared, acolleague who was the director of a large industry genetictoxicology laboratory, accosted me at an Environmental MutagenSociety meeting and insisted that we should never have publishedthat article. As he explained it, he had spent much of his careerconvincing upper management that the in vitro mammalian celltests were necessary and critical for screening chemicals, and ourpublication, which concluded that these mammalian cell tests didnot add significantly to the Ames test’s prediction of carcinogenic-ity, undermined his position.

My response to him was the same as to the other critics of ourconclusions then and now as to the effectiveness of the variousgenetic toxicity screening tests for predicting rodent carcinoge-nicity – I am constrained by the available data. If anyone has datasupporting a different conclusion, let’s examine it and see whereand how it differs from the NTP data. Also, to comments that theNTP data were not relevant because they were based on chemicalsthat were not from the same chemical classes as the drug andpesticide producers work with, my same question applies – whereare the data showing different outcomes with non-NTP chemicals?Nobody provided independent test data that contradicted or called

directly from electronic plate readers. Another advantage to thePC’s was the reduction in size from 9-inch floppy discs to 3-inchnon-floppy discs. It wasn’t long afterward that the laboratory PC’swould send the data electronically to the NIEHS computerbypassing the need for any type of removable disc.

A part of my original plan for the testing program, in addition totesting chemicals of interest to the various government agenciesand other interested groups and individuals, was to develop adatabase that could be used to study the effectiveness of thevarious tests and test combinations. For this end, the selection oftest substances included many chemicals and their structuralanalogs, and chemicals previously tested for carcinogenicity fromcurated sources, e.g., the early NCI bioassay program and theextensive IARC list of human and rodent carcinogens.

At the time the testing program started, it was decided that allthe testing information would be publicly available, similar to theinitial NCI bioassay testing reports. Also, because this was beforethe development of the Internet, we decided to publish the testdata and results in peer-reviewed journals. NIEHS was convincedto support the publication of the Salmonella (and later in vitrocytogenetic and MLA data) in peer-reviewed supplemental issuesof Environmental Mutagenesis. Prior to their availability on the web,the results of approximately 1500 chemicals tested in Salmonella[17,18,32,36–39],108 in chromosome aberration and SCE tests [40],and 63 in the mouse lymphoma assay [20] were published in thesesupplements. In addition to these supplements, Salmonella,chromosome aberration, SCE, MLA, Drosophila, and in vivocytogenetics test results were also published in regular peer-reviewed journal issues. The data from all testing contracts arecurrently accessible from the NTP website’s genetic toxicitydatabase [35].

It wasn’t long after we started that we began receiving requestsfor results and data on specific chemicals or groups of chemicalsfrom other government agencies, industries, law firms, and privateindividuals. If the request came in by phone, we would ask for aletter (later, an email) for our files. The data would then be sent tothe requester along with a cover sheet describing the tests and theresults. If the results had already been published, the citation(s) tothe publications were also provided. This practice was reduced, butnot stopped, when the database became publicly available throughthe NTP web site.

The NTP cancer bioassay reports (the Blue Books) beganincluding the NTP genetox test data as an appendix to the reportbeginning with Technical Report No. 250 in 1986 [41], althoughthe test results were summarized in some of the earlier reports.David DeMarini, and later Kristine Witt, took responsibility forsummarizing the genetox test results, writing the genetoxsections of the reports, and providing data printouts in publica-tion format.

6. Data evaluation

One of my primary concerns when we started the NCI contracts,and later in the NTP program, was how the data were to beevaluated, and the criteria for judging a response positive ornegative. With the exception of the Drosophila sex-linkedrecessive lethal test [42], there were no generally acceptedstatistical analysis procedures that were designed to accommodatethe performance characteristics of the different tests. This was thereason for my request for a statistician (Barry Margolin) to developstatistical analysis procedures that were based on the testperformance, rather than just selecting a standard statistic.

In their initial methods publication, Ames recommended that apositive response be at least 2-fold the spontaneous frequency forthe strain and that reproducible dose-responses be present forsubstances with “low mutagenicity” [43]. Virginia and I did not

agree with such a fold-increase rule for strains that had lowbackground mutation frequencies (e.g., TA1535, TA1537, andTA1538). We refined the rule and required a dose-related increase,regardless of the level of the response, with at least two dosesgreater than, or equal to, a two-fold increase over background,unless the background was less than 10, in which case a three-foldincrease was required [17,18]. However, we recognized that despitetheir convenience and ease of use, these criteria for a positiveresponse (two- or three-fold, or any other �fold) were arbitrary,with no biological or statistical basis, but they served the purposeof providing a quick and easy approach to comparing the inter-laboratory performance.

Although two- and three-fold rules were used for the NCISalmonella contracts, I was not comfortable with drawing such arigid line (i.e., 2� or 3� increase) separating a positive from anegative response, regardless of the data pattern, e.g., narrow orwide SEM, non-monotonic increase, etc. Therefore, when theNIEHS Salmonella contracts started, I did not use the fold-rule, butrequired a reproducible, dose-related increase in revertants,regardless of whether or not it reached two- or three-fold overthe background [32]. Because the contract protocol required repeattests, information on the reproducibility of the responses wasalways available. This approach had a high degree of subjectivitybecause it also allowed consideration of inter-plate variability. As aresult, a number of chemicals were judged to be positive or weaklypositive in the absence of a two- or three-fold increase. Thisapproach was vindicated years later when statistical analysesshowed, using the NTP database, that the two-fold rule was tooconservative for strains with high spontaneous frequencies, andled to negative results which a subjective or statistical approachwould consider to be positive [44,45]. Off-the-shelf statisticalapproaches were also not sufficiently conservative, because thehigh spontaneous strains exhibited a hyper-Poisson distribution[46].

We similarly used nonstatistical approaches to evaluate the SCEresults. Although only one flask per concentration was used,statistical analyses of these responses were based on anexamination of the inter-flask variability using the variance from20-flask solvent controls that were run at Barry Margolin’s request.His analysis confirmed that standard, Poisson statistics with a pvalue of 0.05, were not sufficiently conservative. This is why weused the standard of a minimum 20% increase in the in vitro SCEtest, even though, statistically, this was equivalent to a p value of0.001 [47,48]. Our approach to the use and value of formal statisticshas been recently confirmed and supported [49].

7. Database exercises

One of the great things about developing a good qualitydatabase is that you can ask questions of it to determine itseffectiveness, identify ways to improve the protocol, and generallydata-mine it. When the first Salmonella testing contracts wereready to be re-competed, Barry Margolin, Ken Risko, and I did ananalysis of the performance of the ability of the four Salmonellatester strains/S9 combinations to identify mutagens. Approxi-mately 40% of all the chemicals tested in the program weremutagenic in at least one Salmonella strain. Testing with TA100,alone, detected 80% of the carcinogens identified by using all 4strains, and the combination of TA100 + TA98 detected 89% [50].Based on this analysis, the labs were directed to test initially inTA100 and TA98 and, if negative in both strains, to then test in TA97(which would replace TA1537) and TA1535. Given the proportionof all chemicals tested that were positive in TA98 and/or TA100,this new testing strategy would result in a significant saving oftime and resources. The newly developed strains TA102 and TA104would be used only if I requested them, and that would be if the

[9] E. Zeiger, The history and rationale of genetic toxicity testing – an impersonaland sometimes personal, view, Environ. Mol. Mutagen. 44 (2004) 363–371.

[10] L.A. Poirier, F.J. de Serres, Initial National Cancer Institute studies onmutagenesis as a prescreen for chemical carcinogens: an appraisal, J. Natl.Cancer Inst. 62 (1979) 9109–9926.

[11] E.H.Y. Chu, H.V. Malling, Mammalian cell genetics: II, chemical induction ofspecific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci.U. S. A. 61 (1968) 1306–1312.

[12] D. Clive, W.G. Flamm, M.R. Machesko, N.J. Bernheim, A mutational assaysystem using the thymidine kinase locus in mouse lymphoma cells, Mutat. Res.16 (1972) 77–87.

[13] W.G. Thilly, J.G. DeLuca, H. Hoppe 4th, H.L. Liber, B.W. Penman, Mutation assayin diploid human lymphoblasts: methodological aspects, J. Environ. Pathol.Toxicol. 1 (1977) 91–99.

[14] T. Sugimura, S. Sato, M. Nagao, T. Yahagi, T. Matsushima, Y. Seino, M. Takeuchi,T. Kawachi, Overlapping of carcinogens and mutagens, in: P.N. Magee, S.Takayama, T. Sugimura, T. Matsushima (Eds.), Fundamentals of CancerPrevention, University Park Press, Baltimore, 1976, pp. 191–215.

[15] D. Anderson, J.A. Styles, Appendix II. The bacterial mutation test, Brit. J. Cancer37 (1978) 924–930.

[16] I.F.H. Purchase, E. Longstaff, J. Ashby, J.A. Styles, D. Anderson, P.A. Lefevre, F.R.Westwood, An evaluation of 6 short-term tests for detecting organic chemicalcarcinogens, Br. J. Cancer 37 (1978) 873–959.

[17] V.C. Dunkel, E. Zeiger, D. Brusick, E. McCoy, D. McGregor, K. Mortelmans, H.S.Rosenkranz, V.F. Simmon, Reproducibility of microbial mutagenicity assays. I.Tests with Salmonella typhimurium and Escherichia coli using a standardizedprotocol, Environ. Mutagen. 6 (Suppl. 2) (1984) 1–251.

[18] V.C. Dunkel, E. Zeiger, D. Brusick, E. McCoy, D. McGregor, K. Mortelmans, H.S.Rosenkranz, V.F. Simmon, Reproducibility of microbial mutagenicity assays: II.Testing of carcinogens and noncarcinogens in Salmonella typhimurium andEscherichia coli, Environ. Mutagen. 7 (Suppl. 5) (1985) 1–248.

[19] OECD (Organisation for Economic Co-operation and Development), TestGuideline 471. OECD Guideline for Testing of Chemicals. Genetic Toxicology:Salmonella typhimurium Reverse Mutation Assay. Adopted 26 May 1983, OECD/Paris.

[20] EMM (Environmental and Molecular Mutagenesis) Alan R. Liss/New York. Vol.12, Suppl. 13, (1988) pp. 1–229.

[21] OECD (Organisation for Economic Co-operation and Development), TestGuideline 476. OECD Guideline for Testing of Chemicals. Genetic Toxicology: Invitro Mammalian Cell Gene Mutation Tests. Adopted 4 April 1984, OECD/Paris.

[22] A.D. Mitchell, C.J. Rudd, W.J. Caspary, Evaluation of the L5178Y mouselymphoma cell mutagenesis assay: intralaboratory results for sixty-threecoded chemicals tested at SRI International, Environ. Mol. Mutagen. 12 (Suppl.13) (1988) 37–101.

[23] A.D. Mitchell, C.J. Rudd, W.J. Caspary, Evaluation of the L5178Y mouselymphoma cell mutagenesis assay: intralaboratory results for sixty-threecoded chemicals tested at SRI International, Environ. Mol. Mutagen. 12 (Suppl.13) (1988) 37–101.

[24] W.J. Caspary, Y.J. Lee, S. Poulton, B.C. Myhr, A.D. Mitchell, C.J. Rudd, Evaluationof the L5178Y mouse lymphoma cell mutagenesis assay: quality-controlguidelines and response categories, Environ. Mol. Mutagen. 12 (Suppl. 13)(1988) 19–36.

[25] W.J. Caspary, D.S. Daston, B.C. Myhr, A.D. Mitchell, C.J. Rudd, P.S. Lee, Evaluationof the L5178Y mouse lymphoma cell mutagenesis assay: interlaboratoryreproducibility and assessment, Environ. Mol. Mutagen. 12 (Suppl. 13) (1988)195–229.

[26] B.C. Myhr, W.J. Caspary, Evaluation of the L5178Y mouse lymphoma cellmutagenesis assay: intralaboratory results for sixty-three coded chemicalstested at Litton Bionetics, Inc, Environ. Mol. Mutagen. 12 (Suppl. 13) (1988)103–194.

[27] B.N. Ames, H.O. Kamen, E. Yamasaki, Hair dyes are mutagenic: identification ofa variety of mutagenic ingredients, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 2423–2427.

[28] U.S. Congress, DHEW [Departments of Labor and Health, Education, andWelfare], and Related Agencies Appropriations Bill, 1977. Committee onAppropriations. National Institute of Environmental Health Sciences hearings.House of Representatives, 94th Congress, 2nd session. Report No. 94–1219,June 8, 1976.

[29] D. Clive, A linear relationship between tumorigenic potency in vivo andmutagenic potency at the heterozygous thymidine kinase (TK+/�) locus ofL5178Y mouse lymphoma cells coupled with mammalian metabolism, in: D.Scott, B.A. Bridges, F.H. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier,North Holland, Amsterdam, 1977, pp. 241–247.

[30] E. Zeiger, J.W. Drake, An environmental mutagenesis test developmentprogramme, in: R. Montesano, H. Bartsch, L. Tomatis (Eds.), Molecular andCellular Aspects of Carcinogen Screening Tests, IARC Scientific Publications,Lyon, 1980, pp. 303–313 No. 27.

[31] E. Zeiger, An overview of genetic toxicity testing in the National ToxicologyProgram, Ann. N.Y. Acad. Sci. 407 (1983) 387–394.

[32] S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, W. Zeiger, Salmonellamutagenicity test results for 250 chemicals, Environ. Mutagen. 5 (Suppl. 1)(1983) 3–142.

[33] T. Yahagi, M. Nagao, Y. Seino, T. Matsushima, T. Sugimura, M. Okada,Mutagenicities of N-nitrosamines on Salmonella, Mutat. Res. 48 (1977) 121–130.

[34] R.M. Rowley, E. Zeiger, C. Russell, A PROPHET-based laboratory data entry anddata-base management system for the Environmental Mutagenesis TestDevelopment Program, Comput. Biomed. Res. 15 (1982) 544–554.

[35] http://tools.niehs.nih.gov/cebs3/ui/ [searched Dec. 2016].[36] K. Mortelmans, S. Haworth, T. Lawlor, W. Speck, B. Tainer, E. Zeiger, Salmonella

mutagenicity tests. II. Results from the testing of 270 chemicals, Environ.Mutagen. 8 (Suppl. 7) (1986) 1–119.

[37] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonellamutagenicity tests. III. Results from the testing of 255 chemicals, Environ.Mutagen. 9 (Suppl. 9) (1987) 1–109.

[38] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonellamutagenicity tests. IV. Results from the testing of 300 chemicals, Environ. Mol.Mutagen. 11 (Suppl. 12) (1988) 1–157.

[39] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonellamutagenicity tests. V. Results from the testing of 311 chemicals, Environ. Mol.Mutagen. 19 (Suppl. 21) (1992) 2–141.

[40] S.M. Galloway, M.A. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D.Bloom, F. Nakamura, M. Ahmad, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick,B. Anderson, E. Zeiger, Chromosome aberrations and sister chromatidexchanges in Chinese hamster ovary cells: evaluations of 108 chemicals,Environ. Mol. Mutagen. 10 (Suppl 10) (1987) 1–175.

[41] NTP (National Toxicology Program), Toxicology and Carcinogenesis Studies ofBenzyl Acetate (CAS No. 140-11-4) in F344/N Rats and B6C3F1 mice (GavageStudies), National Toxicology Program Technical Report Series No. 250, U.S.DHHS, August, 1986.

[42] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statisticalsignificance of mutation frequencies, Mutat. Res. 9 (1970) 527–549.

[43] B.N. Ames, J. McCann, E. Yamasaki, Methods for detecting carcinogens andmutagens with the Salmonella/mammalian-microsome mutagenicity test,Mutat. Res. 31 (1975) 347–364.

[44] B.H. Margolin, Statistical studies in genetic toxicology: a perspective from theU.S. National Toxicology Program, Environ. Health Perspect. 63 (1985) 187–194.

[45] N.F. Cariello, W.W. Piegorsch, The Ames test: the two-fold rule revisited, Mutat.Res. 369 (1996) 23–31.

[46] B.H. Margolin, N. Kaplan, E. Zeiger, Statistical analysis of the Ames Salmonella/microsome test, Proc. Natl. Acad. Sci. U. S. A. 78 (1981) 3779–3783.

[47] S.M. Galloway, A.D. Bloom, M. Resnick, B.H. Margolin, F. Nakamura, P. Archer, E.Zeiger, Development of a standard protocol for in vitro cytogenetic testingwith Chinese hamster ovary cells. Comparison of results for 22 compounds intwo laboratories, Environ. Mutagen. 7 (1985) 1–51.

[48] B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E.Zeiger, Statistical analyses for in vitro cytogenetic assays using Chinesehamster ovary cells, Environ. Mutagen. 8 (1986) 183–204.

[49] R.L. Wasserman, N.A. Lazar, Editorial. The ASA’s statement on p-values:context, process, and purpose, Am. Stat. 70 (2016) 129–133.

[50] E. Zeiger, K.J. Risko, B.H. Margolin, Strategies to reduce the cost of mutagenicityscreening using the Salmonella/microsome assay, Environ. Mutagen. 7 (1985)901–911.

[51] E. Zeiger, R.S. Chhabra, B.H. Margolin, The effects of the hepatic S9 fractionfrom Aroclor 1254 treated rats on the mutagenicity of benzo(a)pyrene and 2-aminoanthracene in the Salmonella/microsome assay, Mutat. Res. 64 (1979)378–389.

[52] W.W. Piegorsch, E. Zeiger, Measuring intra-assay agreement for the AmesSalmonella assay, in: L. Hothorn (Ed.), Statistical Methods in Toxicology,Springer-Verlag, Heidelberg, 1991, pp. 35–41.

[53] E. Zeiger, A. Prokopetz, D.B. Walters, The frequency of mislabeled chemicalsfrom commercial suppliers, Account. Rsch. 3 (1993) 45–46.

[54] E. Zeiger, Carcinogenicity of mutagens: the predictive capability of theSalmonella mutagenesis assay for rodent carcinogenicity, Cancer Res. 47(1987) 1287–1296 (1987).

[55] R.W. Tennant, B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding,W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson, R. Minor, Prediction ofchemical carcinogenicity in rodents from in vitro genetic toxicity assays,Science 236 (1987) 933–941.

[56] E. Zeiger, J.K. Haseman, M.D. Shelby, B.H. Margolin, R.W. Tennant, Evaluation offour in vitro genetic toxicity tests for predicting rodent carcinogenicity:confirmation of earlier results with 41 additional chemicals, Environ. Mol.Mutagen. 16 (Suppl. 18) (1990) 1–14.

[57] S.A. Latt, J. Allen, S.E. Bloom, A. Carrano, E. Falke, D. Kram, E. Schneider, R.Schreck, R. Tice, B. Whitfield, S. Wolff, Sister-chromatid exchanges: a report ofthe Gene-Tox program, Mutat. Res. 87 (1981) 17–62.

[58] R.J. Preston, W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F.McFee, S. Wolff, J.S. Wassom, Mammalian in vivo and in vitro cytogeneticassays: a report of the U.S. EPA’s Gene-Tox program, Mutat. Res. 87 (1981) 143–188.

[59] D. Clive, R. McCuen, J.F.S. Spector, C. Piper, K.H. Mavournin, Specific genemutations in L5178Y cells in culture. A report of the U.S. EnvironmentalProtection Agency Gene-Tox program, Mutat. Res. 115 (1983) 225–251.

[60] E. Zeiger, Identification of rodent carcinogens and noncarcinogens usinggenetic toxicity tests: premises, promises, and performance, Regul. Toxicol.Pharmacol. 28 (1998) 85–95.

[61] P. Foureman, J.M. Mason, R. Valencia, S. Zimmering, Chemical mutagenesistesting in Drosophila. X. Results of 70 coded compounds tested for the NationalToxicology Program, Environ. Mol. Mutagen. 23 (1994) 208–227.

into question the NTP-data-based conclusions. Subsequent pub-lications [62,63] that used the NTP database, purged of what wasconsidered questionable or invalid MLA test results and supple-mented by industry test data, came to the same conclusions as theearlier NTP publications.

One example of the recognized value of the NTP database is thatits Salmonella and other genetic toxicity test data were used as acurated learning set by developers of commercial QSAR systems,including CASE, Leadscope, and VITIC. The NTP data still form thecore knowledge bases of these, and presumably other, QSARprograms.

The volume of chemicals tested in Salmonella, and the numberof testing contracts, decreased following the initial 3-contract, 100chemicals/laboratory start for a number of reasons. The chemicalnominations we were receiving for mutagenicity and/or carcino-genicity testing from government agencies and other sources werefewer than anticipated, and I could not justify selecting additionalchemicals from the Aldrich catalogue. Perhaps more critical, thebudget for mutagenicity testing contracts was decreasing andbeing spread out over more tests (e.g., in vivo bone marrowcytogenetics). However, because we were using the modifiedSalmonella test protocol (above), the majority of mutagenicchemicals were tested in only two strains, which reduced theaverage cost per test.

My experience with the inter- and intra-laboratory validation ofgenetic toxicity tests became very valuable in the mid 1990’s whenI became involved in the validation of alternative, i.e., in vitro, testsystems and, later, endocrine disruptor assays. I began workingwith the NIEHS alternatives program and the DHHS InteragencyCommittee to Coordinate and Validate Alternative Methods(ICCVAM) [64] which, among other efforts, identified and provideddirection for the validation of in vitro and in vivo toxicology andendocrine disruption tests as replacements or animal reductionalternatives for the currently used in vivo tests. The recommen-dations in the ICCVAM report were heavily influenced by ourextensive experience with the validation of genetic toxicity tests.

8. Summary

I spent the year before retiring from NIEHS (1999–2000) at theOECD in Paris writing and editing (non-genetox) Test Guidelinesand Guidance Documents, and overseeing validation studies of invivo endocrine disruption assays. Through the wonders of the faxmachine, emails, and with Kristine Witt’s assistance back at NIEHS,I was able to continue to review and evaluate the Salmonella andbone marrow MN data submissions during that time. A fewmonths after returning to the U.S., I retired from NIEHS at the endof December 2000 to the life of an independent consultant.Kristine, who was very familiar with the testing program, tookcharge and is still responsible for its guidance and the dataevaluation. In keeping with the evolving nature of the testingprogram and the genetox test methods, she made a number ofchanges to the Salmonella protocol and in the tests used. Currently,approximately 30 chemicals are being tested per year inSalmonella mutagenicity assays, and 15–20 chemicals undergotesting for MN and/or DNA damage (comet assay) in vivo. Anincreased effort focusing on in vitro tests for MN and DNA damageinduction in mammalian cells in vitro has also been initiated. TheSalmonella protocol has been modified to routinely include onlystrains TA100 and TA98, along with E. coli WP2 uvrA�/�, with andwithout 10% phenobarbital/5,6-benzoflavone-induced rat liver S9mix (in place of Aroclor) [65]. However, the testing contract retainsthe ability to use additional strains of Salmonella and additionalversions or modifications of S9 mix if needed. The subchronic orchronic animal studies use the same lot of the chemical as is usedfor the mutagenicity tests so that results from the in vivo genetic

toxicity tests can be routinely integrated into the NTP subchronictoxicity studies.

I was very lucky to be hired to do mutagenicity testing at thedawn of the mutagenicity-as-a-surrogate-for-carcinogenicity test-ing era. In addition to working for, and with, the scientistsresponsible for genetic toxicity’s current status as a scientificdiscipline and as an international regulatory requirement, I alsohad the opportunity to meet and get to know many of the leaders inthese efforts at the time I started work at FDA, and AlexanderHollaender, a principal founder of the Environmental MutagenSociety, shortly afterward. It is tempting to say that my careertrajectory was planned but, as I noted, it came about strictly bychance. Although I had originally viewed the FDA job as an interimposition that would allow me to complete my Ph.D. while gettingpaid, and then move on to my original interest which was clinicalmicrobiology and infectious diseases, by the time I finished my Ph.D. I was so thoroughly enjoying the work, the challenges, and mymany colleagues at the FDA and other organizations, that I did notconsider changing directions. There was also the additionalsatisfaction of being a part of the genesis of the field of genetictoxicology and of the development of genetic toxicity testingapproaches and data interpretations that are required, or beingused, internationally.

On reflection, it is fascinating how small, seemingly inconse-quential, independent events can control one’s life – a decision togrow a beard (because it seemed like a good idea at the time), beingoverheard in a hallway by somebody whom I did not know at thetime, a UC Berkeley student deciding to test his girlfriend’s hair dyein a newly developed test, . . . . There is a lot to be said about beingin the right place at the right time and being open to all careerpossibilities.

Acknowledgements

I acknowledge and appreciate the many colleagues I have hadover the years (only some of whom have been mentioned here) andour many enjoyable, intellectually stimulating, funny, and some-times testy, interactions that have made my career in genetictoxicology so interesting. I am also grateful to George Hoffmannand Kristine Witt for their many insightful comments andsuggestions on an earlier version of this manuscript, and to thetwo anonymous reviewers for their comments and suggestions.

References

[1] E. Zeiger, Historical development of the genetic toxicity test battery in theUnited States, Environ. Mol. Mutagen. 51 (2010) 781–791.

[2] M.G. Gabridge, M.S. Legator, A host-mediated microbial assay for the detectionof mutagenic chemicals, Proc. Soc. Exp. Biol. Med. 130 (1969) 831–834.

[3] DHEW (U.S. Department of Health, Education, and Welfare). Report of theSecretary’s Commission on Pesticides and Their Relationship to EnvironmentalHealth, parts 1 and 2. (1969) Washington, DC/U.S. GPO.

[4] E. Zeiger, Some factors affecting the host mediated assay response, Environ.Health Perspect. 6 (1973) 101–109.

[5] V.F. Simmon, H.S. Rosenkranz, E. Zeiger, L.A. Poirier, Mutagenic activity ofchemical carcinogens and related compounds in the intraperitoneal host-mediated assay, J. Natl. Cancer Inst. 62 (1979) 911–918.

[6] B.N. Ames, W.E. Durston, E. Yamasaki, F.D. Lee, Carcinogens are mutagens: asimple test system combining liver homogenates for activation and bacteriafor detection, Proc. Natl. Acad. Sci. U. S. A. 70 (1973) 2281–2285.

[7] J. McCann, E. Yamasaki, B.N. Ames, Detection of carcinogens as mutagens in theSalmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. U. S.A. 72 (1975) 5135–5139.

[8] J. McCann, B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals: Discussion, Proc. Natl. Acad. Sci. U. S.A. 73 (1976) 950–954.

[62] D. Kirkland, M. Aardema, L. Henderson, L. Muller, Evaluation of the ability of abattery of three in vitro genotoxicity tests to discriminate rodent carcinogensand noncarcinogens I. Sensitivity, specificity, and relative predictivity, Mutat.Res. 584 (2005) 1–256.

[63] D. Kirkland, E. Zeiger, F. Madia, R. Corvi, Can in vitro mammalian cellgenotoxicity test results be used to complement positive results in the Amestest and help predict carcinogenic or in vivo genotoxic activity? II.Construction and analysis of a consolidated database, Mutat. Res. 775–776(2014) 69–80.

[64] NIEHS (National Institute of Environmental Health Sciences), Validation andRegulatory Acceptance of Toxicological Test Methods: A Report of the Ad hocInteragency Coordinating Committee on the Validation of AlternativeMethods. NIH Publication No. 97–3981, March, 1997.

[65] T.-M. Ong, H. Mukhtar, C.R. Wolf, E. Zeiger, Differential effects of cytochromeP450-inducers on promutagen activation capabilities and enzymatic activitiesof S-9 from rat liver, J. Environ. Pathol. Toxicol. 4 (1980) 55–65.

Scientific feuds, polemics, and ad hominem arguments in basic and special-interest genetics ☆Elof Axel Carlson

Institute for Advanced Study, Indiana University, PO Box 8638, Bloomington, IN 47407-8638, USA

Scientific feuds, polemics, and ad hominem arguments in basic andspecial-interest genetics$

Elof Axel CarlsonInstitute for Advanced Study, Indiana University, PO Box 8638, Bloomington, IN 47407-8638, USA

Article history:Accepted 19 January 2017Available online 3 February 2017

Keywords:MullerCalabreseAd hominem argumentsConflict of interestPeer reviewLow-dose radiation exposureJournal policy to prevent ad hominemarguments

A B S T R A C T

Scientific disputes are commonly presented and settled in journal publications. Most are resolved by aweighing of evidence and new findings. In some cases the arguments are personal and in the form of adhominem attacks on the personality or integrity of an author of a journal article. Many famous scientists(e.g., Galileo, Newton, and Hooke) used ad hominem arguments in responding to their critics. WilliamBateson, W.F.R. Weldon, William Castle, and H.J. Muller used ad hominem arguments in theirpublications until the end of World War I, when editorial policy of journals changed. Motivating some ofthe attacks are philosophic differences (such as holistic or reductionist approaches to science),ideological differences (such as Marxist or Capitalist outlooks), politics (such as Cold War depictions byEast and West on fallout from nuclear testing), or conflicts of interest (which can be professional orfinancial such as the debates over nontraditional and orthodox medicine or over tobacco smoking andhealth). Most of the time, the disputes are motivated by honest disagreements over the interpretation ofthe data. A recent surge (2009–2016) of ad hominem attacks by Edward Calabrese has appeareddisparaging H. J. Muller, E. B. Lewis, other twentieth-century contributors to radiation genetics, and theNational Academy of Sciences. They address the mutational effects of low-dose radiation exposure.Calabrese’s attacks have led to responses by geneticists in the field of mutagenesis, by agencies criticizedby Calabrese, and by students and colleagues of those who have been accused of deception by Calabrese.This article reviews some of the history of ad hominem arguments in science and the background to theattacks by Calabrese. I argue that Calabrese’s characterization of Muller and his supporters is unjust,misleading, and hurtful. I also propose some methods for dealing with or preventing ad hominem attacksin professional journals.

1. Introduction

Scientists learn that their fields change not only by the additionof new data, new technology, new experiments, and new ideas, butalso by the necessary conflict of contending ideas that abound eachgeneration. I first became aware of the extensive presence ofcontroversy in the progress of science when, in 1965, I wrote thefirst draft of The Gene: A Critical History [1]. I was using the reprintlibrary at Woods Hole, Massachusetts, at the Marine BiologyLaboratory. As I took notes on my 5 � 8 cards and sorted these outfor each chapter, I was struck by the intensity of debates amongrespected geneticists. From the mid nineteenth century to the

1960s that I was covering for a history of how the idea of the genearose and developed, I found that disputes were far more likelythan a simple discovery and acceptance of new knowledge. Someof the disputes were quickly settled by new rounds of experiments.Some lingered for decades. Until the end of World War I thedisputes were often in print in professional journals. For thejournal Nature the disputes were carried out in the original articlessetting off the dispute and moved to the letters to the editor. Whenthe dispute got repetitious (usually after about three exchanges ofcorrespondence) the editors cut off the debate in print. My secondsurprise was that the name-calling and nastiness of the argumentsdisappeared not only in Nature but in the major professionaljournals where geneticists sent their manuscripts. I have askedlibrarians for help in tracking down how this policy arose and whohad the authority to engage other editors in shifting the debatesaway from ad hominem attacks, but I still don’t know how thishappened.

$ This article is part of the Reflections in Mutation Research series. To suggesttopics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann (ghoffmann@holycross.edu).

E-mail address: ecarlson31@netzero.com (E.A. Carlson).

journal homepage: www.else vie r .com/ locate / rev iewsmrCommunity address : www.elsevier .com/ locate /mutre s

the Lenin All-Union Academy of Agricultural Sciences meetings inMoscow in December 1936. A movement led by T.D. Lysenko(1898–1976) was challenging what they called “Weismannism-Mendelism-Morganism.” In its place, Lysenko promoted what hecalled Michurinism honoring the Russian equivalent of LutherBurbank (1849–1926) [13].

Like Burbank, I.V. Michurin (1855–1935) believed he could“train” plants by environmental modification and that these traitswere inherited [14] . Michurin also favored grafting one species’twigs on another species’ branch where the altered sap wouldimprove the quality of grafted fruit. Lysenko’s target was N.I.Vavilov (1887–1943), the head of agriculture in the USSR. The 1936meeting had Lysenko and his followers debating Vavilov and hisfollowers. Muller was a visiting investigator who had beenrecruited by Vavilov to the USSR. Each side presented evidencefor and against western genetics, characterized by Lysenkoists asbourgeois, capitalistic, and racist. When Muller presented hispaper, he attacked the philosophic failings of Lysenkoism andclaimed, in his defense of the findings of classical genetics that “Inchoosing between their acceptance and the adoption of the anti-genetic views of our opponents, we are confronted with a choicequite analogous to that between medicine and shamanism,between astronomy and astrology, between chemistry andalchemy” [15]. Muller was accused in turn by Lysenko’s supportersof being a racist because he claimed that if Lamarckism was correct,oppressed people and races would have become less healthy andless intelligent each generation. It took unusual courage for Mullerto attack Lysenko as a charlatan or pseudo-scientist to a large

audience especially when his two Russian students, Solomon Levit(1894–1938) and Isador Agol (1891–1937) had disappeared andMuller was told it would be “inconvenient” to inquire about them.They were both executed during the Stalin purges taking place atthat time.

Ad hominem and polemic writing (published and unpub-lished) continues in the molecular era of genetics. When ErwinChargaff (1905–2002) visited J.D. Watson and F.H.C. Crick(1916–2004) at Cambridge to discuss DNA, he wrote home thathe “had visited two clowns at Cambridge.” Chargaff’s essays in hisbook Heraclitean Fire, especially “Gullible’s Troubles,” are filledwith bon mots and digs reflecting his contempt for Watson andCrick and their supporters. He describes Francis Crick as having“the looks of a fading racing tout” and Jim Watson as “the other,undeveloped at twenty-three, a grin more sly than sheepish” [16].He called molecular biology “the practice of biochemistry withouta license.” At a meeting I attended at Cold Spring HarborLaboratory about 1962, someone in the audience asked SydneyBrenner why he did not name messenger RNA, “hermetic RNA”after Hermes because it was so swiftly turned over and hidden solong from the attention of molecular biologists, Chargaff stood upand said, “Gentlemen, may I remind you that Hermes was also thegod of thieves.”

For a more extended discussion of these controversies andmany others in genetics, I recommend reading my own book TheGene: A Critical History [17] and James Schwartz’s In Pursuit of theGene [18]. Fig. 1 shows some of the historic scientists discussed inthis commentary.

Fig. 1. Historic scientists discussed in this commentary.

130 E.A. Carlson / Mutation Research 771 (2017) 128–133

As I immersed myself in the history of science, I learned thatscientific disputes go back to the earliest efforts to understand theuniverse. Platonic dialogues show Socrates’ (470-399 BCE) greatskill in “leading the witness” to contradictory consequences ofideas that sound good but are not well thought out. His success inmaking his opponents look foolish led to his downfall, with falseaccusations of impiety, corrupting the youth of Athens, and leadingAthens to defeat by Sparta. Galileo (1564–1642) ridiculed hisscientific opponents in his debates with them and in print with hisfirst books introducing the Copernican solar system. This invited atorrent of criticisms of him and a run-in with the authority of theChurch, which his critics claimed was challenged by his presenta-tion of Copernicus’ model as factual. Galileo’s continued ridicule ofthese opposing arguments in print led to his trial and convictionresulting in his house arrest for the rest of his life.

Isaac Newton (1642–1727) and Robert Hooke (1635–1703)despised each other, each accusing the other of stealing ideas intheir working out of the laws of optics and moving bodies. Theywere both members of the newly formed Royal Society. It led toNewton avoiding the Society while Hooke was alive. Hooke’spersonality was combative. He had many disputes with otherscientists on priorities for discovery and the interpretation of theirwork. He served as the Society’s experimentalist and tested eachnew instrument or finding submitted to the society. He also wroteand published the Society’s first best-seller, The Micrographia, thatestablished the cell (which he named) as a unit composing the corktissue he examined under his microscope [2]. After Hooke died andNewton became President of the Royal Society, he had all ofHooke’s portraits removed and they disappeared or weredestroyed. Newton also accused Gottfried Leibniz (1646–1716)of stealing the idea of differential and integral calculus from him.He claimed it was not coincidental independent discovery butinformation Leibniz got from Newton’s correspondence with otherscientists known to Leibniz.

Charles Darwin (1809–1882) tried to avoid controversy bydelaying publication of his theory of evolution by natural selectionfor some twenty years and by having his friends, especiallybotanist Joseph Hooker (1817–1911) and zoologist Thomas Huxley(1825–1895), respond to critics. But books and articles appearedcriticizing him as a sloppy thinker or a thief who merely enlargedideas of his predecessors. When Darwin’s The Origin of Speciesappeared it was extensively reviewed. Adam Sedgwick (1785–1873), a geologist Darwin respected, after reading The Origin ofSpecies, wrote to Darwin on November 24, 1859:

“If I did not think you a good tempered & truth loving man Ishould not tell you that . . . I have read your book with more painthan pleasure. Parts of it I admired greatly; parts I laughed at till mysides were almost sore; other parts I read with absolute sorrow;because I think them utterly false & grievously mischievous– Youhave deserted– after a start in that tram-road of all solid physicaltruth– the true method of induction” [3]. Richard Owen (1804–1892) and Samuel Butler (1835–1902) were two of his moreprominent critics. Owen was a respected naturalist and Butler,while known for his fiction and numerous writings, opposed thereductionism in Darwin’s approach and favored a holistic view oflife and the universe verging on pantheism. After Darwin’s deaththe ad hominem arguments became more extensive and theattacks were inspired by religious belief, as evolution becameanathema to the newly developing fundamentalist movements inGreat Britain and especially in the United States.

2. Disputes and personal attacks in the history of genetics

We owe to William Bateson (1861–1926) the terms genetics,genotype, phenotype, heterozygous, homozygous, and allele. Hestudied embryologyatCambridge and went in 1883 to Johns Hopkins

University to work with William Keith Brooks (1848–1908). Brooksconvinced Bateson that the future field to study was heredity.Bateson returned and devoted himself to studying variation andpublished in 1894 a book on what was known about it [4]. Heintroduced the idea of homeotic mutations and meristic mutations.The homeotic mutations misplaced the location of organs. Themeristic mutationsduplicatedparts like extradigits on limbs orextravertebrae. Bateson argued that these were discontinuous events andcould lead to new species characteristics. He fought bitterly with theprevailing late nineteenth century Darwinian school led by FrancisGalton (1822–1911), Karl Pearson (1857–1934), and W. F. R. Weldon(1860–1906). That group founded a field of biometrics (Gaussiancurves and their departures) and had their own journal, Biometrika,that served as a vehicle for articles and editorial commentdenouncing Bateson. When Biometrika did not publish Bateson’sarticles, he wrote and subsidized the publication of his own book in1902, Mendel’s Principles of Heredity: a Defense [5]. It was thediscontinuity of hereditary traits studied by Mendel that appealedto Bateson, and we owe to Bateson the many years of battle hedevoted to promoting Mendelism against a far greater and acceptedstatistical approach to studying speciation and heredity used byWeldon and Pearson. It took another 20 years before R. A. Fishercombined Mendelism with the new field of population genetics tocreate a mathematical approach to evolution [6]. When Weldoncollapsed and died of a heart attack, his widow blamed Bateson forcausing his death. Bateson had written a scathing response toWeldon’s criticism and faint praise of Mendelism and Batesonclaimed, “I am disposed to think that unaided he is – to borrowHorace Walpole’s phrase – about as likely to light a fire with a wetdish clout (sic) as to kindle interest in Mendel’s discoveries by histempered appreciation” [7].

Bateson’s enthusiasm for Mendelism caught fire in the US. Oneof the first converts to Mendelism was William Castle (1867–1962).He began studying contrasting traits in mammals. He showed thatalbinism was a recessive trait. He worked on coat color in mice,guinea pigs, and rabbits. He believed in “genic contamination”when traits were heterozygous and that this accounted for thevariable expression of many traits that otherwise behaved asdominant or recessive characteristics. Opposing Castle’s viewswere the members of T.H. Morgan’s (1866–1945) Fly Lab, especiallyH.J. Muller (1890–1967). In 1914 Muller published a paper arguingthat the variability of Mendelian traits was not due to geniccontamination but to “residual inheritance” or what could also becalled modifier genes [8] . Castle and J.C. Phillips had presentedtheir evidence that piebald rats owed their variation to geniccontamination [9]. Muller presented his evidence that theextracted recessive was not contaminated and commented “It isdifficult to believe that this suggestion of Castle and Phillips wasnot made in a spirit of mysticism, when we consider also theirsuggestion that the gene may undergo contamination” [10]. Castlewas equally dismissive of Muller in his reply claiming that Mullerhad to invoke modifier genes (residual inheritance) or quantitativefactors for which he offered no evidence. “What a slender basis andwhat an absurd one from which to derive the ‘fundamentalprinciples’ that Mendelian factors are constant! Do biologists takethemselves seriously when they reason thus? Certainly no one elsewill long take them seriously” [11]. Castle took five years before heconceded that residual inheritance did account for the variation hestudied in both spotted rabbits and hooded rats [12]. For Muller theslap in the face given to Castle was costly. Muller told me thatMorgan had advised him not to publish the attack on Castle andthis led to a rupture in their relation. It was also the beginning ofMuller having a reputation as having a difficult personality. Whilemost geneticists do not engage in or approve of polemical or adhominem responses to critics, there are situations where suchresponses are applauded. An example of this was at the meetings of

E.A. Carlson / Mutation Research 771 (2017) 128–133 129

support the way Muller and Stern handled the discussion of thework and eventual publication of the low-dose experiments ofCaspari, Stern, and Spencer. I have never seen papers that includeunpublished research (still in a state of analysis) to reject longsupported theories. It is also puzzling that the Spencer paper wasconsistent with the LNT model and it was only the Stern- Casparipaper that seemed to contradict it. In Muller’s quick reading beforeleaving for Stockholm, he said it would require a statistical analysisthat he did not have time to provide. Calabrese chose instead tointerpret Muller not as disagreeing with him, but lying to him, tothe Nobel committee, and to the public.

When two scientists disagree on interpretation, most of thetime one is proven right and the other wrong as in the disputes ofBateson and Weldon or Muller and Castle but it would take verypersuasive evidence to claim that one position is based on a lie. Itshifts science away from experimentation and laboratory work tosettle issues and invites conspiracies, cover ups, and other illmotivations heaped on an opponent instead. I do not know ifCalabrese is aware of how painful it is for the Muller family,Muller’s students, Muller’s colleagues, and those who knewMuller to be told that he deliberately lied and bullied hiscontemporaries into adhering to the LNT model that he knew tobe false. When I worked on the Muller biography [32] I read anexchange of letters by Muller and Linus Pauling (1901–1994)during the debates over radioactive fallout from weapons testing.In that debate Edward Teller (1908–2003) considered Muller as analarmist and Pauling as duped by a left-wing outlook. Muller sawTeller as a Cold War apologist who rejected radiation exposure asa significant risk if it did not manifest immediate symptoms.Muller also objected to Pauling’s position as too extreme inclaiming significant damage from fallout because Muller felt thatit was necessary during the Cold War to have a strong nucleardeterrent. Pauling told Muller he would sue him if he did notretract that claim. Muller wrote him an apology that he had notmeant to criticize his personality, only his political position.

Calabrese has urged the Nuclear Regulatory commission to dropthe LNT model and replace it with a radiation hormesis responsemodel. He believes it will benefit nuclear industries and otherscientists and engineers applying radiation to society who will notbe encumbered by concerns over low doses of radiation. Calabresewas not unique in his disregard for low-dose exposure to radiation.Teller dismissed it and felt there was no individual risk to anyoneanywhere from worldwide fallout. When I interviewed WillardLibby (1908–1980) for the Muller biography, he downplayed theconcerns about fallout from weapons testing and felt Muller andPauling were using it for political purposes. He claimed Muller wasa double agent used by the USSR to sabotage the development ofnuclear weapons by the US [33]. Ironically, a few years earlierduring the Cold War, Muller was accused by Lysenkoists of being adouble agent sent to the USSR to sabotage its agriculturalprograms.

4. How should conflict be handled in interpreting geneticfindings?

Scientific debate is both healthy and necessary for science.When several scientists are working on a common topic,interpretations will differ and in most instances the debate isresolved by more data and more experiments. The use of adhominem arguments arises when one of the participants feels anattack on his or her interpretation is a personal attack that merits aresponse in kind. This is where editors play a role. The use of adhominem arguments does not add to the validity of aninterpretation. Tearing down an opponent does not prove thevalidity of one’s favored findings or diminish the validity of anopponent’s interpretation.

The first barrier to inappropriate character assassination is therole of the editor of a journal. Editors should require the deletion ofwords and phrases that reflect the author’s emotional feelings orthat cast doubt on another scientist’s integrity. Attacking acolleague as dishonest, a cheat, a liar, or a bully, should not gounchallenged. The editor should either reject the manuscript orsuggest a revision. The accuser then has to modify the manuscriptand, ideally, argue the case on scientific evidence or try anotherjournal. The second barrier is the neutrality of the referees whoread the manuscript. It is important that the editor check, whenpossible, if they have a close association or known hostility to theauthor of a manuscript.

The third barrier is in the instructions to authors submittingmanuscripts. It should be stated that conflicts of interest should benoted (many journals especially in health-related fields nowstipulate such disclaimers) and that manuscripts may disagreewith the interpretations of other colleagues but should not use adhominem arguments. A scientific finding should be judged by thevalidity of the evidence for it and the care with which it isexamined for its scientific merits. Those who submit manuscriptsmay be disappointed by the amount of time they have to spendtightening their arguments or delaying publication to do follow upexperiments to support their conclusions. The back and forthexchange results in better papers and allows readers to follow theevidence for the claimed results. It also reduces opportunities forcritics and competitors to resort to ad hominem arguments.

A fourth barrier would be finding a proper venue for discussionsof a scientist’s personality or mental habits as a scientist. Thesemight more properly be submitted to journals of the history,sociology, or philosophy of science where both editors and refereesare more familiar with this aspect of science. Even here, adhominem arguments should be rejected or carefully edited so thatclaims of cheating, plagiarism, conflicts of interest or deception aredocumented and peer reviewed. Many universities have used legaladvice to investigate and render verdicts on staff accused ofprofessional misconduct. While the personality flaws or differ-ences of scientists might be useful for studies of how scienceworks, editors should have the authority to publish such peer-reviewed articles or to reject them. I personally prefer theobjectivity of science journals that refrain from commenting onthe psychological aspects of the scientist and focus on thearguments associated with evidence, experimentation, data, andreasoning associated with a submitted manuscript. Ad hominemattacks on a competitor’s interpretations are distractions. If ascientific area of study is contentious, the solution is moreexperiments and continued peer review of the results.

While scientists like Newton and Hooke could be nasty to otherscientists it is their valid scientific findings that enter the stream ofknowledge that enhances a field of knowledge. If there are errorsother scientists will find them, and science continues a necessarypruning process in which inadequate or false findings are weededout. Science is an immense enterprise. Good work can come fromnasty people. Some good scientists may be neurotic or cranks.Some respected scientists may publish an article flawed with anerror later shown to be wrong. And many good natured scientistswho are wonderful colleagues may be modest contributors to newknowledge, lack deeper insights, or do not provide the rigor neededto justify their views. Scientific journals are places where newknowledge and ideas should be found. Contested models orfindings in submitted articles should be discussed on theirscientific merits. Referees of submitted articles who suspect fraudor bias or find errors should point that out for the editors toevaluate. We benefit more from research journals, I believe, byexcluding sensational interpretations of scientific personalitiesand focusing on the accuracy and strength of findings andarguments that enrich our knowledge.

132 E.A. Carlson / Mutation Research 771 (2017) 128–133

3. The vilification campaign of Edward Calabrese against H.J.Muller

I have provided the evidence for a long history of polemicalwriting in the sciences to illustrate that the personalities ofscientists vary and if given the opportunity criticism can be nastyand wounding. Most scientists today do not encounter thistradition of verbal trade-offs that are sprinkled in the literatureof the past, especially in published journal articles or in letters tothe editor of journals that permit such ripostes. I will devote therest of this article to a series of attacks by Edward J. Calabrese on H.J. Muller, Ed Lewis (1918–2004), Curt Stern (1902–1981), JamesCrow (1916–2012) and others. At least 14 papers by Calabrese haveappeared on this issue [19]. What distinguishes this type of attackis that most of these attacks are on geneticists who are dead andwho cannot reply. Also, Calabrese attacks the characters of those hecriticizes, assigning unproved motivations to them by conjectureand, in Muller’s case, questioning his honesty. The characters ofMuller, Lewis, Stern, and Crow by those who knew them are verypositive and appear in the Biographical Memoirs of the NationalAcademy of Sciences [20].

Calabrese is a Professor of Environmental Health Sciences at theUniversity of Massachusetts in Amherst. He received most of hiseducation at the University of Massachusetts including a Ph.D. inthe Department of Entomology in 1973 and an Ed.D. in education in1974. He shifted to research in a neglected field called hormesis.This field argues that there is a biphasic response to chemicals andradiations that is usually contradictory. Thus, small doses of toxicchemicals (or ionizing radiation) are usually harmless or may evenbe beneficial. Calabrese claims there is, for most toxic agents, athreshold that must be reached before a toxic agent causes harm.Most of his publications that criticize the “Linear no thresholdmodel” (LNT) for ionizing radiation, are not based on experimentsthat he or his laboratory have carried out with ionizing radiation.They are based on data he has obtained from reading the literatureon low-dose effects of ionizing radiation.

About 2010, Calabrese called me to ask if I had a copy or if I hadread the original written manuscript of Muller’s Nobel Prizeacceptance speech. I told him I didn’t recall if Muller had saved theoriginal speech notes or manuscript but he should check the LillyLibrary. He called a second time and presented his charge thatMuller deliberately withheld information from an experimentcarried out by Curt Stern (1902–1981). We disagreed. I did not hearfrom him again, but I learned about a year later that he hadpublished an article accusing Muller of dishonesty (the pressrelease for U Mass used the term “liar”) [21]. This was Calabrese’sclaim: Just before Muller went to Stockholm to receive his Nobelaward, he received a manuscript from Curt Stern who had done alow-dose experiment with Ernst Caspari (1909–1984) using fruitflies. It was similar to an earlier experiment that Stern worked onwith Warren P. Spencer. The earlier work was consistent with theno threshold model for the dose used. The work with Casparishowed a less-than-expected radiation-induced mutation ratealthough Stern mentioned to Muller that this might be a statisticalor procedural problem because of the small number of mutationsin both the controls and the induced flies. Muller said he was toobusy working on his other papers and his preparations for theNobel Award to give it attention in detail but he would do so on hisreturn. In Stockholm, Muller gave his talk and in it he claimed therewas no threshold dose for mutations induced by ionizing radiation,and he pleaded for caution in human radiation exposure. This was1946 so a lot of concern about radiation existed after the atomicbombs were used on Hiroshima and Nagasaki in 1945. Thereference to linearity is found in only one paragraph of Muller’sacceptance speech of 11 pages. In a letter December 9, 1946 inStockholm, Muller wrote his wife Thea “I have so far written only

half of my Nobel speech for Wednesday (quite illegibly) & that istwice as long as it should be, & nothing at all yet of the after dinnerspeech I must give at the city banquet tomorrow evening. But I stillhave tomorrow morning to work” [22].

Calabrese argues that Muller should have mentioned the thenunpublished work of Caspari and Stern and that by claiming therewas no threshold dose, he was deliberately withholding evidenceto the contrary and thus lying. What Calabrese does not mention isthat Muller had been in the field 19 years since he initiated it in1927. During that time numerous laboratories had studied theinduction of gene mutations and demonstrated linearity for thedoses they compared. These articles (several dozen) cover a rangeof doses and organisms used to establish the LNT model ofradiation. All were published between 1927 and 1946. Thus,virtually none were motivated about worldwide fallout (unknownat the time to geneticists) or atomic war (unknown before 1945)[23]. It also included efforts to use attenuated vs. acute doses of thesame dosage of radiation. In 1939 Muller’s doctoral student S.P.Ray-Chaudhuri (1907–1994) at the University of Edinburghcompared a dose of 400 roentgens given to fruit flies over 30 dayscompared to the same dose given in 30 min. Both groups gave thesame mutation rate [24]. Muller argued that the attenuated dosewas being administered at a rate of about one chest x-ray every10 min. That was about 0.01 to 0.1 roentgens per chest x-raydepending on the dose used by medical practitioners. Calabresesuggested that Muller’s “overreaction” to low dose effects wasfrom his concern over nuclear weapons. Muller’s concern actuallybegan in 1928. His student, H. Bentley Glass (1906–2005) told methat he attended a public lecture Muller gave at Baylor Universityon his findings of x-ray induced mutations. When Muller toldmedical students and their teachers that they should protectthemselves and their patients, several got up and walked out inprotest [25].

Those familiar with the field of mutation research are familiarwith other factors that make this a difficult field to interpret.Mutation rates may vary, as Muller and Altenburg discovered in1919 when they made the first estimate of the spontaneousmutation rate in fruit flies [26]. When Muller used the moreaccurate ClB technique in 1928 for identifying X linked lethals, hefound the rate was lower by a factor of ten. The ClB stock wasdesigned to provide objective quantitative data (the number ofvials with a missing category of male flies carrying an induced orspontaneously arising X linked lethal) [27]. At the time he did notknow the cause, which he thought might reflect “mutator genes” ordifferences in responses by different strains. This also applies todayto differences in factors such as repair enzymes present, catalaseand other enzymes that may or may not be in identical amounts inthe cells of different strains of a species or among different species.For an overview of these issues, see my book Mutation [28].

Calabrese added Ed Lewis and James Crow as co-conspirators inthe effort to maintain the LNT rule. Lewis claimed that theinduction of leukemias among survivors of the atomic bombings inJapan followed a linear plot. He assumed that an inducedchromosome break or gene mutation may lead to the initialleukemic cell [29] . Crow worked out the mathematics of geneticloads in populations. Calabrese also criticized (with ad hominemarguments about their integrity) the National Research Council(NRC) for writing the BEAR [Biological Effects of Atomic Radiation]reports and not dropping the LNT argument for radiation safety[30]. This prompted a published rebuke from the NRC. The authorsof that rebuke, R.J. Cicerone (Chair of the NRC at the NAS) and K.D.Crowley (Chair of the Nuclear and Radiation Studies Board of theNRC) reviewed Calabrese’s charges and dismissed them asunfounded and inappropriate: “Calabrese produces no evidencethat Muller inappropriately influenced the BEAR committee or thatthe NAS or the BEAR committee misled anyone” [31]. They also

References

[1] E.A. Carlson, The Gene: A Critical History, W.B. Saunders, Philadelphia, 1966.[2] R. Hooke, 1665. Micrographia Royal Society, London Available as a free eBook,

Project Gutenberg, www.gutenburg,org/ebook/15491.[3] Sedgwick revisited and repeated his views in The Spectator 1860. See David L.

Hull, Darwin and His Critics. The Reception of Darwin’s Theory of Evolution,University of Chicago Press, 1973.

[4] W. Bateson, Materials for the Study of Evolution, Macmillan and Co., London,1894.

[5] W. Bateson, Mendel’s Principles of Heredity: a Defence, Cambridge UniversityPress, London, 1902.

[6] R.A. Fisher, The Genetical Theory of Natural Selection, Clarendon Press,London, 1930.

[7] W. Bateson, Mendel’s Principles of Heredity: A Defence, Cambridge UniversityPress, London, 1902; cited in Carlson, 1966, p. 12.

[8] H.J. Muller, The bearing of the selection experiments of Castle and Phillips onthe variability of genes, Am. Nat. 48 (1914) 567–576.

[9] W. Castle, J.C. Phillips, Piebald Rats and Selection, Carnegie Institution ofWashington, 1914 (Publication no. 195).

[10] H.J. Muller, The bearing of the selection experiments of Castle and Phillips onthe variability of genes, Am. Nat. 48 (1914) 567–576.

[11] W.E. Castle, Mr. Muller on the constancy of Mendelian factors, Am. Nat. 49(1915) 37–42.

[12] W.E. Castle, Piebald rats and selection, a correction, Am. Nat. 53 (1919) 370–375.

[13] T.D. Lysenko, Heredity and Its Variability (Translated from the Russian byTheodosius Dobzhansky), Columbia University Press, New York, 1948.

[14] James Crow, Plant breeding giants: Burbank the artist; Vavilov the scientist,Genetics 158 (4) (2001) 1391–1395.

[15] H.J. Muller, The Present Status of the Experimental Evidence. UnpublishedAddress Presented December 1936, Lenin All-Union Academy of AgriculturalSciences, Lilly Library, Indiana University, 1936, pp. 32.

[16] Chargaff Erwin, Heraclitean Fire – Sketches from a Life Before Nature, Essay:Gullible’s Troubles, Rockefeller University Press, New York, 1978, pp. 100–103.

[17] See Reference [1].[18] Schwartz James, In Pursuit of the Gene – From Darwin to DNA, Harvard

University Press, Cambridge, 2008.[19] E.J. Calabrese, The road to linearity: why linearity at low doses became the

basis for carcinogenic risk assessment, Archive of Toxicology 83 (2009) 203–225 Calabrese E.J. 2011, Key studies used to support cancer risk assessmentquestioned. Environmental and Molecular Mutagenesis 52: 595–606.Calabrese, E. 2012 Muller’s Nobel Prize lecture: When ideology prevailed overscience Toxicological Science 2012 March 1126 (1) pp 1–4 doi 10 1093/toxsci/kffr338. E pub 2011 Dec 13; Calabrese, E. J. 2015 On the origins of the linear nothreshold (LNT) dogma by means of untruths, artful dodges and blind faith.

Environmental Research 432–442. These are a sample of Calabrese’s articles onthe LNT debate.

[20] The Biographical Memoir for Muller is by E.A. Carlson(2009), for Lewis byHoward Lipschitz (2014), for Crow by Daniel Hartl and Rayla Greenberg Temin(2014), and for Stern by James Neel (1987). All are accessible free on-linefromwww.nasonline.org/memoirs..

[21] Crok Marcel, Attack on Radiation Geneticists Triggers Furor, (2017) (www.sciencemag.org/news/2011/10/attack-radiation-triggers-furor, 18 October2011).

[22] H.J. Muller, The production of mutations, J. Hered. 38 (1946) 259–270 (The twocontradictory low dose papers were eventually published two years later:Spencer, W. P. and C. Stern 1948, Experiment to test the validity of the linear rdose mutation rate relationship at low doses. Genetics 33: 43–74; and E.Caspari and C. Stern 1948, The influence of chronic radiation with gamma raysat low doses on the mutation rate in Drosophila melanogaster. Genetics 33: 75–95. The letter from Muller to his wife is from a collection of family letters thathis daughter, Helen J. Muller, hopes to publish and I thank her for permission toquote from this letter).

[23] D.E.Lea, Actions ofRadiation onLiving Cells, Cambridge UniversityPress,London,1946 (see also the English translation of N.V. Timofeev-Ressovsky, M. Delbrückand K.G. Zimmer 1935, On the nature of gene mutation and gene structure, In:Sloan, P.R. and B. Fogel 2011, Creating a Physical Biology: The Three-Man Paperand Early Molecular Biology. University of Chicago Press Chicago).

[24] S.P. Ray Chaudhury, The validity of the Bunsen-Roscoe Law in the production ofmutations by radiation of extremely low intensity, Proc. R. Soc. Edinburgh 62(1994) 55–72.

[25] H. Bentley Glass, Introduction, in: E.A. Carlson (Ed.), Man’s Future Birthright –

Essays on Science and Humanity by H.J. Muller, State University of New YorkPress, Albany, 1973.

[26] H.J. Muller, E. Altenburg, The rate of change of hereditary factors in Drosophila,Proc. Soc. Exp. Med. 17 (1919) 10–14.

[27] H.J. Muller, The measure of gene mutation rate in Drosophila, its highvariability, and its dependence on temperature, Genetics 13 (1928) 279–357.

[28] E.A. Carlson, Mutation: The History of an Idea from Darwin to Genomics, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 2009.

[29] E.B. Lewis, Leukemia and ionizing radiation, Science 125 (1957) 963–972.[30] E. Calabrese, How the US National Academy of Sciences misled the world

community on cancer risk assessment, New Findings Challenge HistoricalFoundations of the Linear Dose Response, (2013), pp. 1105–1106, doi:http://dx.doi.org/10.1007/s00 204-013-1105-6 (Review Article).

[31] R.J. Cicerone, K.D. Crowley, Letter from Ralph J. Cicerone regarding EdwardCalabrese’s post published online first on August 4th. How the US NationalAcademy of Sciences misled the world community on cancer risk assessment.New findings challenge historical foundations of the linear dose response,Arch. Toxicol. 88 (2014) 171–172, doi:http://dx.doi.org/10.1007/s 00 204-013-1175-4 (DOI 10.1007/s00 204-013-1105-6, Review Article).

[32] E. A. Carlson, Genes, Radiation, and Society: the Life and Work of H. J. Muller,Cornell University Press, Ithaca, 1982. See Chapter 24, Human mutations andthe radiation danger (pp 336–351), and Chapter 25, The fallout controversy (pp352–357).

[33] E.A. Carlson, Interview with Willard Libby April 8 at UCLA. Manuscript Copy atLilly Library, Indiana University and diary at Cold Spring Harbor LaboratoryArchives, 1971.

Mutation Research-Reviews in Mutation Research 776 (2018) 78–81

Mutagenesis: Interactions with a parallel universe ☆Jeffrey H. Miller

Department of Microbiology, Immunology, and Molecular Genetics, The Molecular, Biology Institute, and The David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA

Mutation Research-Reviews in Mutation Research

journal homepage: www.elsevier.com/locate/mutrev

Mutagenesis: Interactions with a parallel universe☆

Jeffrey H. MillerDepartment of Microbiology, Immunology, and Molecular Genetics, The Molecular, Biology Institute, and The David Geffen School of Medicine, University of California, LosAngeles, CA 90095, USA

Keywords:Mutagen synergyAlternate modes of mutagenesisIndirect mutagenesisBase analogsMutation spectraReflections

A B S T R A C T

Unexpected observations in mutagenesis research have led to a new perspective in this personal reflection based onyears of studying mutagenesis. Many mutagens have been thought to operate via a single principal mechanism,with secondary effects usually resulting in only minor changes in the observed mutation frequencies and spectra.For example, we conceive of base analogs as resulting in direct mispairing as their main mechanism of mutagenesis.Recent studies now show that in fact even these simple mutagens can cause very large and unanticipated effectsboth in mutation frequencies and in the mutational spectra when used in certain pair-wise combinations. Here wecharacterize this leap in mutation frequencies as a transport to an alternate universe of mutagenesis.

1. Introduction

The concept of parallel universes (see Fig. 1) has intrigued science andscience fiction writers, e.g. [1–3], as has the amusing “Hitchhiker’s Guideto the Galaxy” description of someone’s words falling through a “worm-hole in the fabric of the space-time continuum” and being instantlytransported to a distant galaxy in a completely different time frame [4].

When we reflect on what we currently understand about mutagen-esis, we can in fact envision different modes of mutagenesis involvingthe same mutagenic agent as being in parallel universes. This can beseen with recent published revelations concerning base-analog muta-gens [5], once thought to be the simplest of mutagens, yet seen incombination their actions are more complex. When I published my firstpaper on mutagenesis 40 years ago [6], I thought mutagens such as 2-aminopurine (2AP) acted only by straightforward mispairing, as ori-ginally conceived by pioneers such as Benzer, Freese, Brenner, Crick,and others [7–10], and as reproduced in reviews and standard text-books from that point until the present day, e.g. [11–13]. Only now do Iappreciate the importance of the alternate pathways by which theseagents can act and even interact with one another.

2. Base analog mutagenesis in the standard universe

Base analogs are derivatives of the normal bases, adenine (A),guanine (G), cytosine (C), and thymine (T). Normally, when in a DNAdouble helix, these four bases pair according to the Watson-Crickpairing, A with T, and G with C. Rare tautomers can mispair, leading toincorrect bases being inserted during replication that will result inmutagenesis if not corrected. Fig. 2 shows the structures of a set of baseand nucleoside analogs.

Compounds such as 2-aminopurine (2AP; recall that adenine issimply 6-aminopurine) can mispair with cytidine more frequently thanadenine does, because the mispairing tautomers are more frequent.Often the actual tautomers are different than those originally envi-sioned (see Fig. 3) [14,15], but the concept is the same. Likewise,

Fig. 1. A representation of the different parallel “worlds” that might exist in other pocketsof the multiverse. Image credit: public domain, retrieved from https://pixabay.com/en/globe-earth-country-continents-73397/; see [1].

https://doi.org/10.1016/j.mrrev.2018.01.002Accepted 10 January 2018

☆ This article is part of the Reflections in Mutation Research series. To suggest topics and authors for Reflections, readers should contact the series editor, G.R. Hoffmann (ghoffmann@holycross.edu).

E-mail address: jhmiller@microbio.ucla.edu.

analogs of cytidine, such as zebularine (ZEB; cytidine lacking the aminogroup) [16] can make mispairs apparently more frequently than cyti-dine itself.

Each base or nucleoside analog generates mutations by its specificmispairing. We can ascertain this by comparing the mutagenic spectraof each mutagen at an array of sites. The Escherichia coli rpoB gene is anexcellent target for this, as it has as many as 92 base-substitution

mutation possibilities detected so far [17–19] that lead to the ri-fampicin resistant phenotype (Rifr). There are 28 transitions amongthese possible mutations (i.e. A:T→G:C or G:C→A:T). Seymour Ben-zer’s elegant studies of the rII system in phage T4 first demonstratedthat different mutagens have different favored sites (hotspots) [8], andwe can see that reflected here in the rpoB gene for several mutagens ormutators (Fig. 4). This shows that rather than each activating the sameprocess, instead, they each have an individual “fingerprint.” There areindirect mechanisms of mutagenesis that base analogs could in prin-ciple activate (see below), but they do not seem to do this when thesecompounds act alone to any degree that changes the fingerprints seen inFig. 4. These other mechanisms of mutagenesis are part of a differentuniverse.

3. The universe of dNTP ratios

There is another form of mutagenesis, totally apart from that causedby direct acting mutagens, that emanates from changing the deoxy-ribonucleoside triphosphate (dNTP) ratios, the building blocks of DNAthat fuel replication. Microbial and higher cells maintain a precisebalance of dNTP levels and ratios, and replication speed e.g. [20,21].Changes in dNTP pools affect mutation rates [22–33]. Relatively smallchanges in the ratios of the 4 different dNTPs can result in large in-creases in replication errors and subsequent mutation rates, e.g.[23,24]. We know this because of the analysis of different mutants withaltered dNTP ratios. Mutants defective in DCD (deoxycytidine deami-nase) or NDK (nucleotide diphosphate kinase) have increased dCTP anddGTP [21,23,25,26], and decreased dATP [23,25] and higher rates ofcertain base substitutions [23,25–27,34]. The double mutant deficientin both DCD and NDK has a larger imbalance and a more extrememutation rate increase [24]. Moreover, mutants carrying engineeredalterations of RNR (ribonucleotide reductase), which controls the ratiosof dNTPs through a set of allosteric sites, have high mutation rates andspecific alterations in dNTP levels [35] (see below). Changes in theabsolute levels of dNTP also affect mutation rates. Thus, when the levelsof all dNTPs are increased, mutation rates increase [31,32], and whenthey are all decreased, mutation rates are lowered [33].

Base analogs may alter dNTP ratios, and this was appreciatedalready during earlier studies of mutagens such as 2AP and 5-

Fig. 2. The structure of four base or nucleoside analogs discussed here (Figure adaptedfrom [5]).

Fig. 3. Structures of the possible AP-C mispair hydrogen-bonded configurations. Left,neutral wobble; right-upper, ionized Watson-Crick configuration, protonated on the APresidue; right-lower, ionized Watson-Crick configuration, protonated on the C residue.Figure reproduced from [14] with permission of the publisher (American Chemical So-ciety).

Fig. 4. The transition mutation spectrum in rpoB seen after treatment of a wild-type strainwith 2AP, or ethyl methanesulfonate (EMS), or after growth of a mismatch repair-defi-cient (MMR−, mutS) strain without treatment. (Data from [5,17]).

J.H. Miller Mutation Research-Reviews in Mutation Research 776 (2018) 78–81

bromodeoxyuridine (5BrdU) [36–38]. Interestingly, increasing theconcentration of single base analogs in mutagenesis studies does notchange the ratios past the tipping point needed to stimulate this lattertype of mutagenesis. Thus, both of these modes of mutagenesis arestuck in their own universe.

4. Transition from one universe to another

We recently began a study of the effects of combinations of muta-gens, beginning with base analogs [5]. Although there have been stu-dies of compounds that act as mutagen enhancers by inhibiting specificrepair enzymes [39,40] or inhibiting enzymes that inactivate certainmutagenic compounds [41–44], and also studies showing that inter-calating agents can increase the mutagenicity of bleomycin by perhapsincreasing their access to the DNA [45,46], the topic of mutagensynergy still remains a vastly unexplored field. We were surprised tofind that certain pairwise combinations of base analogs gave strongsynergistic or even antagonistic or suppressive effects. The combinationof ZEB and 2AP gave the largest effect, increasing mutation frequencies35-fold over that from the addition of frequencies from both mutagensused alone (Fig. 5).

The combination of 2AP and 5-azacytidine (5AZ) generated a muchsmaller increase. Also, the combination of 2AP and 5-bromodeoxyur-idine (5BrdU) actually gave a 7-fold decrease in the frequency expectedfrom adding the two individual frequencies (Fig. 6).

What can cause the dramatic synergistic effect in mutation fre-quencies seen in the 2AP+ZEB pairwise combination? We can

examine the “fingerprint” revealed by looking at the mutational spec-trum in the rpoB gene and try to compare it with that from other mu-tagens or processes. Fig. 7 shows that the mutational spectrum of thecombination is very different from the spectra of either of the twomutagens alone. Specifically, the main hotspots seen in 2AP and in ZEBspectra are not prominent in the combination spectra, but two newhotspots are: the A:T→G:C changes at positions 1547 and 1598. Al-though the hotspot at 1547 appears in a number of situations, parti-cularly that of a mismatch repair deficient strain [17,18], the hotspot at1598 is noteworthy in that it is very rare. In fact, it appears as a majorsite in only one mutational profile among all the observed rpoB spectrainduced by mutagens or generated by repair deficiencies.

Schaaper and coworkers have reported that a specific engineeredmutation in the gene encoding RNR results in a high mutation rate, anda spectrum in rpoB that has hotspots at four A:T→G:C sites, these being1532, 1538, 1547, and 1598 [35]; for related work in yeast see also[47]. Moreover, the altered RNR generates a dNTP imbalance, withincreased dCTP and dGTP, and reduced dATP that thus favors themisincorporation of dGTP across from a template T. These four sites areamong the A:T→G:C mutational sites (that lead to Rifr) where the nextnucleotide incorporated is either dGTP or dCTP [17]. The combinationof the specific nucleotide imbalance and the next nucleotide results inthese sites being more mutagenic. It is likely that the simple addition oftwo base analogs, ZEB and 2AP, results in a related situation, althoughmore pronounced, yielding even higher mutation frequencies. In fact,ZEB has been reported to be a direct inhibitor of thymidylate synthase[48], and we have shown that exogenous thymidine counteracts thebactericidal effect of ZEB [5]. Thus dTTP levels are clearly reduced.2AP might compete with adenine and result in an ultimate reduction ofdATP levels. It is possible that the suppressive effect of the5BrdU+2AP combination results from opposite effects on dNTP ratiosultimately generated by each mutagen.

We have recently detected two additional strong synergies (un-published), one involving the antibiotic trimethoprim, an inhibitor ofdihydrofolate reductase that results in reduction of thymidine levels,and a second involving the intercalating agent ICR191. These findingsshow that mutagenic synergy is not restricted to the interactions of twobase analogs (see also [45,46]).

5. Conclusions

We see here that the addition of two different mutagens cansometimes trigger a process that neither mutagen alone can activate,allowing the transition from one parallel universe to another, sort oflike the uttered sentences in “The Hitchhikers Guide to the Galaxy”,that fell through a crack in the Space-Time Continuum and weretransported to another world and time. It is of great interest to see how

Fig. 5. The frequencies of Rifr mutants found in cultures grown with either no mutagen(LB), or with 5 μg/ml ZEB, or 500 μg/ml 2AP, or 5 μg/ml ZEB+500 μg/ml 2AP. (Figurefrom [5]).

Fig. 6. The frequencies of Rifr mutants found in cultures grown with either no mutagen(LB), or with 20 μg/ml 5AZ, or 500 μg/ml 2AP, or 5 μg/ml 5BrdU, or 20 μg/ml5AZ+500 μg/ml 2AP, or 5 μg/ml BrdU+500 μg/ml 2AP. (Figure from [5]).

Fig. 7. The mutation spectrum in rpoB of mutations caused by 2AP or ZEB (bottom), orthe combination of 2AP+ZEB (top). The transitions (G:C→A:T, A:T→G:C) are shownas a percentage of the total mutations in each sample (Figure from [5]).

Mutation Research-Reviews in Mutation Research

journal homepage: www.elsevier.com/locate/mutrev

Risks of aneuploidy induction from chemical exposure: Twenty years ofcollaborative research in Europe from basic science to regulatoryimplications☆

Micheline Kirsch-Voldersa,⁎, Francesca Pacchierottib,⁎, Elizabeth M. Parryc, Antonella Russod,Ursula Eichenlaub-Rittere, Ilse-Dore Adlerfa Laboratory for Cell Genetics, Faculty of Sciences and Bioengineering, Vrije Universiteit Brussel, Brussels, Belgiumb Laboratory of Biosafety and Risk Assessment, ENEA CR Casaccia, Roma, Italyc Institute for Life Science, Swansea University Medical School, UKdDepartment of Molecular Medicine, University of Padova, Padova, Italye Institute of Gene Technology/Microbiology, Faculty of Biology, University of Bielefeld, Bielefeld, GermanyfNeufahrn, Germany

This "Reflections" paper on aneuploidy isdedicated to J. M. Parry who was the inspirer,promoter and coordinator of the EU-aneuploidy projects.

Keywords:AneuploidyMicronucleusNon-disjunctionChromosome segregationMitosis/meiosisThresholds

A B S T R A C T

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferationand diseases is still lacking. This review reflects on the efforts and results of a large European research networkthat, from the 1980′s until 2004, focused on protection against aneuploidy-inducing factors and tackled thefollowing problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2)aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays toidentify aneugenic factors and classify them according to their modes of action; 5) the need for reliable,quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for me-chanistic insight into the consequences of aneuploidy for human health. This activity brought together a con-sortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelinesand to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EUresearch programmes on aneuploidy were having a valuable scientific approach based on well-selected com-pounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibilityand inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascinationwith the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at theright time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprisebenefited from a solid partnership under the lead of an inspired and stimulating coordinator. The researchprogramme elucidated the major modes of action of aneugens, developed scientifically sound assays to assessaneugens in different tissues, and achieved the international validation of relevant assays with the goal ofprotecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will requireadditional research, and the study of effects of exposure to multiple agents should become a priority. It is hopedthat these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

1. Introduction

The consequences of aneuploidy for human health is not a new areaof interest. More than a century ago Theodor Boveri already linked theincorrect chromosome number in sea urchin embryos with abnormal

development; moreover, he hypothesized that having the wrongnumber of chromosomes might cause cells to grow in an uncontrollableway and become the seeds of cancerous tumors [1,2]. With the far-reaching discoveries of the last century on the cytological and mole-cular structure and function of the genome, it became apparent that a

https://doi.org/10.1016/j.mrrev.2018.11.002

☆ This article is part of the Reflections in Mutation Research series. To suggest topics and authors for Reflections, readers should contact the series editor, G.R.Hoffmann (ghoffmann@holycross.edu).

⁎ Corresponding authors.E-mail addresses: mkirschv@vub.ac.be (M. Kirsch-Volders), francesca.pacchierotti@enea.it (F. Pacchierotti).

Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications ☆Micheline Kirsch-Volders a *, Francesca Pacchierotti b *, Elizabeth M. Parry c,Antonella Russo d, Ursula Eichenlaub-Ritter e, Ilse-Dore Adler f

a Laboratory for Cell Genetics, Faculty of Sciences and Bioengineering, Vrije Universiteit Brussel, Brussels, Belgiumb Laboratory of Biosafety and Risk Assessment, ENEA CR Casaccia, Roma, Italyc Institute for Life Science, Swansea University Medical School, UKd Department of Molecular Medicine, University of Padova, Padova, Italye Institute of Gene Technology/Microbiology, Faculty of Biology, University of Bielefeld, Bielefeld, Germanyf Neufahrn, Germany

widespread these phenomena are, as many different combinations ofcompounds with mutagenic properties are used in chemotherapy andalso in antibiotic treatments. Moreover, compounds used in the en-vironment, such as pesticides, can act as mutagen enhancers[41,42,44], underlying the importance of more extensive testing forsynergies involving a range of products used in the environment, asnoted previously; see [44].

Funding

This research was funded by a faculty research grant from theUniversity of California.

Acknowledgements

This research was funded by a faculty research grant from theUniversity of California.

References

[1] E. Siegel, Is There Another ‘You’ Out There In A Parallel Universe? (2018)Forbes.com, Nov. 18, 2016 (https://www.forbes.com/sites/startswithabang/2016/11/18/is-there-another-you-out-there-in-a-parallel-universe/ ); Image credit: publicdomain, retrieved from https://pixabay.com/en/globe-earth-country-continents-73397/.

[2] M. Kaku, Parallel Worlds, New York, Doubleday, 2004.[3] M.L. Franks, The Universe and Multiple Reality, iUniverse, Inc., Lincoln, NE, 2003.[4] E.C. Adams, The Hitchhiker’s Guide to the Galaxy, Chapter 31, Pan Publishing,

1979.[5] J. Ang, L.Y. Song, S. D’Souza, I.L. Hong, R. Luhar, M. Yung, J.H. Miller, Mutagen

synergy: hypermutability generated by specific pairs of base analogs, J. Bacteriol.198 (2016) 2776–2783.

[6] C. Coulondre, J.H. Miller, Genetic studies of the lac repressor IV. Mutagenic spe-cificity in the lacI gene of Escherichia coli, J. Mol. Biol. 117 (1977) 577–606.

[7] S. Benzer, E. Freese, Induction of specific mutations with 5-bromouracil, Proc. Natl.Acad. Sci. U. S. A. 44 (1958) 112–119.

[8] S. Benzer, On the topography of the genetic fine structure, Proc. Natl. Acad. Sci. U.S. A. 47 (1961) 403–415.

[9] F.H. Crick, L. Barnett, S. Brenner, R.J. Watts-Tobin, General nature of the geneticcode for proteins, Nature 192 (1961) 1227–1232.

[10] E. Freese, The specific mutagenic effect of base analogs on phage T4, J. Mol. Biol. 1(1959) 87–105.

[11] J.W. Drake, R.H. Baltz, The biochemistry of mutagenesis, Annu. Rev. Biochem. 45(1976) 11–37.

[12] J.H. Miller, Perspective on mutagenesis and repair: the standard model and alter-nate modes of mutagenesis, Crit. Rev. Biochem. Mol. Biol. 40 (2005) 155–179.

[13] E.C. Friedberg, G.C. Walker, W. Seide, R.D. Wood, R.A. Schultz, T. Ellenberger, DNARepair and Mutagenesis, American Society for Microbiology, Washington, DC,2006.

[14] L.C. Sowers, Y. Boulard, G.V. Fazakerly, Multiple structures for the 2-aminopurine-cytosine mispair, Biochemistry 39 (2000) 7613–7620.

[15] I.J. Kimsey, K. Perzold, B. Sathyamoorthy, Z.W. Stein, H.M. Al-Hashimi, Visualizingtransient Watson-Crick-like mispairs in DNA and RNA duplexes, Nature 519 (2015)315–320.

[16] V.E. Marquez, A.J. Kelley, R. Agbaria, T. Ben-Kasus, J.C. Cheng, C.B. Yoo,P.A. Jones, Zebularine: a unique molecule for an epigenetically based strategy incancer chemotherapy, Ann. N. Y. Acad. Sci. 1058 (2005) 246–254.

[17] L. Garibyan, T. Huang, T.M. Kim, E. Wolff, A. Nguyen, T. Nguyen, A. Diep, K. Hu,A. Iverson, H. Yang, J.H. Miller, Use of the rpoB gene to determine the specificity ofbase substitution mutations on the Escherichia coli chromosome, DNA Repair 2(2003) 593–608.

[18] E. Wolff, M. Kim, K. Hu, H. Yang, J.H. Miller, Polymerases leave fingerprints:analysis of the mutational spectrum in Escherichia coli rpoB to assess the role ofpolymerase IV in spontaneous mutation, J. Bacteriol. 186 (2004) 2900–2905.

[19] C.H. Corzette, M.F. Goodman, S.E. Finkel, Competitive fitness during feast andfamine: how SOS DNA polymerases influence physiology and evolution inEscherichia coli, Genetics 194 (2013) 409–420.

[20] S. Gon, J.E. Camara, H.K. Klungsøyr, E. Crooke, K. Skarstad, J. Beckwith, A novelregulatory mechanism couples deoxyribonucleotide synthesis and DNA replicationin Escherichia coli, EMBO J. 25 (2006) 1137–1147.

[21] B.A. Nordenskjold, L. Skoog, N.C. Brown, P. Reichard, Deoxyribonucleotide poolsand deoxyribonucleic acid synthesis in cultured mouse embryo cells, J. Biol. Chem.245 (1970) 5360–5368.

[22] B.A. Kunz, S.E. Kohalmi, T.A. Kunkel, T.A. Mathews, E.M. McIntosh, J.A. Reidy,Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance ofgenetic stability, Mutat. Res. 318 (1994) 1–64.

[23] R. Schaaper, C.K. Mathews, Mutational consequences of dNTP pool imbalances in E.coli, DNA Repair 12 (2013) 73–79.

[24] L. Tse, T.M. Kang, J. Yuan, D. Mihora, E. Becket, K.H. Maslowska, R.M. Schaaper,J.H. Miller, Extreme dNTP pool changes and hypermutability in dcd ndk strains,Mutat. Res. 784 (2016) 16–24.

[25] Q. Lu, X. Zhang, N. Almaula, C.K. Mathews, M. Inouye, The gene for nucleosidediphosphate kinase functions as a mutator gene in Escherichia coli, J. Mol. Biol. 254(1995) 337–341.

[26] A. Sanchez, S. Sharma, S. Rozenzhak, A. Roguev, N.J. Krogan, A. Chabes, P. Russell,Replication fork collapse and genome instability in a deoxycytidylate deaminasemutant, Mol. Cell. Biol. 32 (2012) 4445–4454.

[27] G.L. Weinberg, B. Ullman, D.W. Martin Jr., Mutator phenotypes in mammalian cellmutants with distinct biochemical defects and abnormal deoxyribonucleoside tri-phosphate pools, Proc. Natl. Acad. Sci. U. S. A. 78 (1981) 2447–2451.

[28] G.L. Weinberg, B. Ullman, C.M. Wright, D.W. Martin Jr., The effects of exogenousthymidine on endogenous deoxynucleotides and mutagenesis in mammalian cells,Somat. Cell Mol. Genet. 11 (1985) 413–419.

[29] M. Meuth, The molecular basis of mutations induced by deoxyribonucleoside tri-phosphate pool imbalances in mammalian cells, Exp. Cell Res. 181 (1989) 305–316.

[30] M. Meuth, E. Aufreiter, P. Reichard, Deoxyribonucleotide pools in mouse-fibroblastcell lines with altered ribonucleotide reductase, Eur. J. Biochem. 71 (1976) 39–43.

[31] L.J. Wheeler, I. Rajagopal, C.K. Mathews, Stimulation of mutagenesis by propor-tional deoxynucleoside triphosphate accumulation in Escherichia coli, DNA Repair 4(2005) 1450–1456.

[32] S. Gon, R. Napolitano, W. Rocha, S. Coulon, R.P. Fuchs, Increase in dNTP pool sizeduring the DNA damage response plays a key role in spontaneous and induced-mutagenesis in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 108 (2011)19311–19316.

[33] E. Becket, L. Tse, M. Yung, A. Cosico, J.H. Miller, Polynucleotide phosphorylaseplays an important role in the generation of spontaneous mutations in Escherichiacoli, J. Bacteriol. 194 (2012) 5613–5620.

[34] J.H. Miller, P. Funchain, W. Clendenin, T. Huang, A. Nguyen, E. Wolff, A. Yeung,J. Chiang, L. Garibyan, M.M. Slupska, H. Yang, Escherichia coli strains (ndk) lackingnucleoside diphosphate kinase are powerful mutators for base substitutions andframeshifts in mismatch repair deficient strains, Genetics 162 (2002) 5–13.

[35] D. Ahluwalia, R.M. Schaaper, Hypermutability and error catastrophe due to defectsin ribonucleotide reductase, Proc. Natl. Acad. Sci. U. S. A. 110 (2013)18596–18601.

[36] D.H. Persing, L. McGinty, C.W. Adams, R.G. Fowler, Mutational specificity of thebase analogue 2-aminopurine in Escherichia coli, Mutat. Res. 83 (1981) 25–37.

[37] M.F. Goodman, R.L. Hopkins, R. Lasken, D.N. Mhaskar, The biochemical basis of 5-bromouracil- and 2-aminopurine-induced mutagenesis, Basic Life Sci. 31 (1985)409–423.

[38] R.L. Hopkins, M.F. Goodman, Deoxyribonucleotide pools base pairing, and se-quence configuration affecting bromodeoxyuridine- and 2-aminopurine-inducedmutagenesis, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 1801–1805.

[39] R.C. Grafstrom, I.-C. Hsu, C.C. Hsu, Mutagenicity of formaldehyde in Chinesehampster lung fibroblasts: synergy with ionizing radiation and N-nitroso-N-me-thylurea, Chem.-Biol Interact. 86 (1993) 41–49.

[40] H. Danaee, H.H. Nelson, H. Liber, J.B. Little, K.T. Kelsey, Low dose exposure tosodium arsenite synergistically interacts with UV irradiation to induce mutationsand alter DNA repair in human cells, Mutagenesis 19 (2004) 143–148.

[41] E.D. Wagner, K. Repetny, J.S. Tan, T. Gichner, M.J. Plewa, Mutagenic synergybetween paraoxon and mammalian or plant-activated aromatic amines, Environ.Mol. Mutagen. 30 (1997) 312–320.

[42] T. Gichner, E.D. Wagner, M.J. Plewa, Pentachlorophenol-mediated mutagenic sy-nergy with aromatic amines in Salmonella typhimurium, Mutat. Res. 420 (1998)115–124.

[43] F. Catterall, E. Copeland, M.N. Clifford, C. Ionnides, Effects of black tea theafulvinson aflatoxin B1 mutagenesis in the Ames test, Mutagenesis 18 (2003) 145–150.

[44] E.D. Wagner, M.S. Marengo, M.J. Plewa, Modulation of the mutagenicity of het-erocyclic amines by organophosphate insecticides and their metabolites, Mutat.Res. 536 (2003) 103–115.

[45] G.R. Hoffmann, M.V. Ronan, K.E. Sylvia, J.P. Tartaglione, Enhancement of the re-combinagenic and mutagenic activities of bleomycin in yeast by intercalation ofacridine compounds into DNA, Mutagenesis 24 (2009) 317–329.

[46] G.R. Hoffmann, A.M. Laterza, K.E. Sylvia, J.P. Tartaglione, Potentiation of themutagenicity and recombinagenicity of bleomycin in yeast by unconventional in-tercalating agents, Environ. Mol. Mutagen. 52 (2011) 130–144.

[47] D. Kumar, A.L. Abdulovic, J. Viberg, A.K. Nilsson, T.A. Kunkel, A. Chabes,Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools, Nucl. Acids Res.39 (2013) 1360–1371.

[48] M. Herranz, J. Martin-Caballero, M.F. Fraga, J. Ruiz-Cabello, J.M. Flores, M. Desco,V.E. Marquez, M. Esteller, The novel DNA methylation inhibitor zebularine is ef-fective against the development of T-cell lymphoma, Blood 107 (2006) 1174–1177.

insights coming from the application of Next Generation Sequencing(NGS) provided an independent validation of the conventional cyto-genetic information and more data about the parental origin of aneu-ploidy, as recently reviewed [22–26].

Abnormalities of chromosome number in cancer cells have beenreported widely over the last century. In general, most cancers displaygenome instability [27] and high levels of chromosome instability fre-quently associate with the most aggressive tumours [28,29]. Currentlyit is hotly debated how changes in chromosomal instability (CIN) can beinduced by cancer therapy to drive cells over the edge of toleratedimbalance and aneuploidy (discussed by [30]). One recently discoveredmechanism of rapid changes in the genome involves micronuclei fromerrors in chromosome segregation generating extensive rearrangementsand segmental aneuploidies termed chromothripsis [31,32]. Cancercells generally show chromosome instability, and aneuploidy serves asan enabling factor in tumour evolution, e.g., by the loss of hetero-zygosity of tumour suppressor genes. Rapid and random changes indosage of multiple genes on multiple chromosomes has the potential ofgiving cancer cells the karyotype for better fitness. CIN provides avariable genetic tumour landscape that can contribute to patient- andtumour-specific characterization and treatment (e.g., [33]). It has re-cently been recognized that polyploidy, in particular tetraploidisation,may be a common event in tumour evolution [34]. Polyploid cells cangive rise to more genetic instability and aneuploidy, especially whenpolyploidisation involves loss of control of centrosome numbers atspindle poles [35]. Despite evidence for the implication of chromosomeimbalance in cancer, the exact role of aneuploidy as a cause and/or aconsequence of carcinogenesis remains controversial.

1.2. Chemical induction of aneuploidy attracts the attention of scientistsand regulators: the premises for a European research project

During the 1970s and early 80s, few research groups in Europe andelsewhere were studying aneuploidy. At that time, Saccharomyces cer-evisiae, Aspergillus nidulans, Neurospora crassa and Drosophila melano-gaster were widely used in genetic research. Thus, it was natural to usethese excellent experimental models to investigate the induction ofaneuploidy by environmental mutagens. As a consequence, there was aneed to assess the feasibility of extrapolating results from these modelsystems to higher eukaryotes, human beings included. These con-siderations prompted the National Institute of Environmental HealthSciences (NIEHS) to organize the Workshop on Systems to DetectInduction of Aneuploidy by Environmental Mutagens [36] in November1978, in Savannah, Georgia, USA, and the U.S. Environmental Protec-tion Agency (EPA) to establish in 1984 the Aneuploidy Data ReviewCommittee, largely composed by U.S. members. The Committee re-ports, published in 1986 [37], highlighted many gaps in the data thathampered test comparisons and pointed to further research needs. Al-ready at that time, the importance of understanding mechanisms ofmitotic and meiotic chromosome distribution were becoming apparent,as documented by the EPA/NIEHS sponsored symposium "Aneuploidy:Etiology and Mechanisms," held in May 1985, in Washington, DC [38].Interestingly, in 1976, the European Commission Research Programmein Radioprotection had initiated a specific programme to assess the riskof radiation-induced non-disjunction [39]. Along with these initiativesorganized by regulatory authorities, the scientific community felt theneed to exchange experience and results in this emerging field, andBaldev K. Vig organized the first of a successful series of meetings onChromosome Segregation and Aneuploidy in 1989 in Reno, Nevada,USA [40]. This group continued to meet in different countries at 3-yearintervals providing stimulating interactions between scientists andregulatory bodies (Table 1). Notably, after the first one, all othermeetings were held in Europe, testifying to how lively this researchfield has been in European Countries. The financial support granted toEuropean research groups by the Commission of European Commu-nities and the coordination of the effort by Swansea University led by

Jim Parry certainly played an instrumental role (Table 2).

1.3. The birth of a European research network on aneuploidy: comingtogether of the expert laboratories

We were brought together by our academic interest in aneuploidyand a delight and fascination in observing mitotic/meiotic figuresunder the microscope, in particular with fluorescent probes. At the timeof the initiation of the project, the use of immunofluorescence as astandard tool to analyse mitosis and meiosis was still in its infancy. Forinstance, the three dimensional shape of the spindle and dynamicchanges in chromosome alignment in large cells like mammalian oo-cytes had mainly been studied in spreads or by electron microscopy,thus severely limiting the extent of retrievable information. Analyses ofcell components like centrosomes were not commonly used to assess theinfluence of chemical exposures on somatic and germ cells, althoughthe relevance and chances to treat diseases like cancer by anti-micro-tubule drugs were recognized as an area of active research in phar-macology and cell biological research [41]. In contrast, the impact ofthe environment through the induction of structural or numericalchromosomal aberrations and micronuclei was just being tested usingnewly available probes like centromeric markers (e.g., sera from pa-tients affected by an autoimmune disease named calcinosis, Raynaud'sphenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia(CREST), which contain antikinetochore antibodies [42]).

Drawing the different stages of the mitotic and meiotic divisionswhen teaching was quite feasible and coherent, but answering students’essential mechanistic questions in genetics was still inadequate andlacking information and therefore was both frustrating and stimulatingat the time; for instance, the deeper knowledge of microtubule poly-merisation kinetics was not paralleled by a similar understanding ofpolymerisation dynamics in a real cellular time and space context,particularly during cell division. At that time the mechanisms re-sponsible for chromosome segregation/missegregation were still poorlyunderstood.

With respect to meiosis it was largely unknown how bivalentchromosomes segregate the parental univalents during the first meioticdivision, while their sister chromatids are held together until anaphaseII. The beauty and the mystery around this key event in biology pushedus to look more and more deeply at the mechanisms operating at thechromosomal, cellular, and molecular levels.

During the late 1980′s and early 90′s, the discovery of cell cyclecheckpoints opened a new field linking genomic instability andcheckpoint deficiency, in particular the discovery of the DNA damagecheckpoint in Saccharomyces cerevisiae [43], of the SAC in buddingyeast [44] and of the genes required for cell cycle arrest in response toloss of microtubule function [45], culminating in granting the Nobelprize in Physiology/Medicine to Leland H. Hartwell, R. Timothy (Tim)Hunt and Sir Paul M. Nurse in 2011.

In addition to our scientific curiosity as cell biologists we shared theprofound conviction/intuition that aneuploidy plays a major role in cell

Table 1Years and locations of the Conference Series on ChromosomeSegregation and Aneuploidy.

Year Location

1989 Reno, Nevada, USA1992 Aghia Pelagia, Crete, Greece1995 Sorrento, Italy1998 Porto, Portugal2001 Chartres, France2004 Cortona, Italy2007 Naantali, Finland2010 Edinburgh, UK2013 Breukelen, The Netherlands2016 Galway, Ireland

M. Kirsch-Volders, et al. Mutation Research-Reviews in Mutation Research 779 (2019) 126–147

wrong number of chromosomes can not only critically influence cu-mulative gene dosage in a cell but is associated in many cases withadverse health effects at the tissue and organ level, in relation to dys-functional regulation of the cell type. These findings in the past pro-vided the ground for our changing view of the “fixed” genome and therelevance of changes with respect to disease, aging, fertility and evo-lution of our species [3–7].

In light of the potential relevance of genome instability, it has beenimportant to consider different issues related to aneuploidy inductionand its consequences with respect to cell type and stage of development:1) aneuploidy origin and consequences when occurring in germ cellsand preimplantation embryos, 2) origin and consequences of chromo-some imbalance when occurring in somatic cells, 3) aneuploidy as aresult of environmental factors, 4) dose-effect relationships for ex-posures to aneugens in vitro and in vivo, 5) the need for validated assaysto identify and classify aneugenic factors according to mode of action,6) the need for reliable, quantitative data suitable for regulating ex-posure and preventing aneuploidy induction, 7) the need for mechan-istic insight to understand the consequences of aneuploidy for humanhealth.

From the 1980′s until 2004, a large consortium of European re-search teams joined to tackle these questions with existing and newmethodologies. This effort contributed to better understanding of themechanisms responsible for aneuploidy induction and more accuratedetection of aneuploidy. The new tools and discoveries encouragedregulatory activities within the European Union (EU) and theOrganization for Economic Co-operation and Development (OECD) toprotect the human population against induced aneuploidy and con-tributed to advances in cancer research and treatment of human dis-eases related to genome instability. The subject of aneuploidy, frommolecular mechanisms to consequences for human health, remainscritical and the number of scientific articles published each year in thisfield is huge. At the end of October 2018, by searching for the word"aneuploidy" in Titles and Abstracts, Pubmed identified over 5500 pa-pers published in the last 5 years. Overall, more than 42,000 papers (theoldest published in 1958) are available in the database. These paperscontinue to investigate the cellular targets assuring correct chromo-some segregation, the link between aneuploidy and cancer, and thedifferent role of chromosome imbalance in early and late stages ofcarcinogenesis. Aneuploidy in germ cells and embryos, implantationfailure and trisomy, effects related to age, environmentally- and life-style-associated chromosomal imbalance, and prenatal diagnosis re-main major fields of interest in reproduction. This review summarizesthe major motivations and steps that consolidated a long-lasting, EU-funded, successful research network on chemically induced aneuploidy;it offers reflections on the history, and it identifies gaps for present andfuture research in this field.

1.1. Aneuploidy and polyploidy: diverse mechanisms of origin andconsequences

Aneuploidy is defined as the alteration of chromosome number thatis not a multiple of the haploid complement. This condition is differentfrom polyploidy in which cells harbour a multiple of their haploidkaryotype.

Polyploidy may result from mitotic failure, endoreduplication orcell fusion. Polyploid cells arise most commonly as a result of in-complete cell division, such as failure of cytokinesis producing a singletetraploid binucleate cell with a double number of chromosomes.Failure of the Spindle Assembly Checkpoint (SAC) to maintain mitoticarrest in cells with disrupted mitotic spindles or severe DNA damageresults in cells passing from M phase to G1 without undergoing normalmitosis and cytokinesis. This abnormal cell cycle transition is some-times termed “mitotic slippage”. If cells exit mitosis with all chromo-somes retained in one nucleus, the reformed interphase cell is tetra-ploid. Endoreduplication is another form of polyploidisation where the

genome replicates multiple times without intervening entry into Mphase, such as the polytene chromosomes formed in cells of the salivaryglands of Drosophila larvae. Polyploidy may also arise from cell fusion.Several cancer-causing viruses, such as Hepatitis B and C, Epstein-Barr,and Human Papilloma Virus (HPV) are fusogenic [8]. Unlike polyploidythat stems from mitotic errors or endoreduplication, cells of differenttypes can fuse and produce hybrid polyploid cells.

In mammals polyploidy is rare. However, there are several instanceswhere the generation of polyploid cells is a normal feature of differ-entiation. One form of this polyploidisation is called endomitosis andoccurs for instance in megakaryocytes during their differentiation. Inthe mammalian liver, embryonic cells are primarily mononucleate but,depending on species, the adult liver contains a high percentage ofbinucleate cells, at least some of which are thought to occur as a resultof incomplete cytokinesis [9,10].

Aneuploidy results mainly from chromosome non-disjunction andlagging of chromosomes, with chromosome distribution errors occur-ring during mitosis or meiosis. From a mechanistic point of view, an-euploidy can result from errors in the many processes controlling thefidelity of chromosome replication, separation and segregation duringmitosis. This means that there are targets other than DNA for aneugenicactivity. In most normal cells, a surveillance system responds to thepresence of abnormal chromosome content to halt cell cycling, thuscausing stalling of cytokinesis, cell death or inducing senescence[11–13]. Mistakes during cell division, generating changes in chromo-some content and producing aneuploid or polyploid progeny cells, arereviewed by Kirsch-Volders et al. [14]. Novel mechanisms of errors inhuman oocytes have recently been discovered that lead to precociousseparation of sister chromatids in homologous chromosomes at meiosisI instead of meiosis II, predisposing to first- or second-division meioticerrors (for a recent review see [5]). Polyploid cells generated by mitoticslippage may undergo abnormal division to generate aneuploid cells. Amajor consequence of segregation defects is change in the relative do-sage of products from genes located on the missegregated chromo-somes. Abnormal expression of transcriptional regulators can also affectgenes on the properly segregated chromosomes. There is evidence thatepigenetic changes in chromatin, particularly at centromeres, maycontribute to errors in chromosome segregation and DNA hypomethy-lation has been linked to increased X chromosome loss in male germcells or zygotes [15]. The consequences of perturbations in gene ex-pression and epigenetic alterations depend, respectively, on the specificchromosomes and chromosomal domains affected, and on the interplayof the aneuploid phenotype with the environment. Most often, theseabnormal chromosome distributions are detrimental to the health andsurvival of the organism. However, in a changed environment, altera-tions in gene copy number may generate a more highly adapted phe-notype [16,17].

During mammalian embryogenesis genome doubling typically leadsto embryonic lethality. In humans, congenital triploidy and tetraploidymay account for up to 10% of spontaneous abortions [18]; triploidy ismostly derived from dispermic fertilization and not from a process ofendoreduplication [19–21]. Chromosome segregation errors duringgamete formation in meiosis and mitotic errors during preimplantationdevelopment are a primary cause of implantation failure, spontaneousabortion, human birth defects, and reduced fertility or infertility. Whileaneuploidy is detected in 0.3% of livebirths, estimates in gametes andembryos indicate that spontaneous numerical errors may be ratherfrequent, ranging from 5% to 60% depending on gender, parental age,the chromosome involved and the method of study [22,23]. Data ob-tained from spontaneous abortions suggest that these events are asso-ciated with aneuploidy in about 35% of cases [22,23], and thus supportthe existence of a strong selection filter for the aneuploid condition inpre- and early postimplantation stages. It is well-known from 50 yearsof cytogenetic surveys that female gametes are highly prone to aneu-ploidy when compared to male gametes or mitotic cells, and that agedwomen display the highest frequency of meiotic errors [22,23]. Novel

chemically induced aneuploidy, the group tested the application ofbone marrow [50,51] and spermatocyte [52] cytogenetic analyses tothe detection and characterization of aneugenic risks. The added valueof the international collaboration supported by European grants wasconfirmed by interlaboratory comparisons, joint papers and growingdatabases [53,54].

Ursula Eichenlaub-Ritter initially studied spindles and chromosomesegregation in protists [55] but then shifted her interest to chromosomesegregation and aneuploidy in oocytes. Working in collaboration withthe group of Ann Chandley at the MRC in Edinburgh (UK) and RogerGosden at the University of Edinburgh, maternal age-related changes inthe oocyte spindle and increases in unaligned chromosomes at meta-phase I were for the first time identified by employing spindle im-munofluorescence in a mouse model [56,57]. Later, immuno-fluorescence studies were extended to human oocytes in cooperationwith the group of Andre Stahl and Jean-Marie Luciani at the Laboratoryof Embryology and Cytogenetics of the University of Marseille (France),Faculty of Medicine [58]. After moving to the University of Bielefeld inGermany, the laboratory worked toward identifying the origins of an-euploidy in human oocytes, focusing on women of advanced maternalage and with a reduced follicle pool and exposures causing chromo-somal errors. In the 1980s, the in vitro maturation of mammalian oo-cytes was still in its infancy, and the Bielefeld group was among the firstto follow cell cycle progression throughout in vitro maturation usingmouse oocytes as model and employing spindle fluorescence and cy-togenetics to assess mechanisms and fidelity of chromosome segrega-tion in oocytes. This led to the recognition that disturbances in reg-ulation of the cell cycle and escape from meiotic arrest were importantetiological factors in predisposition to aneuploidy in mammalian oo-cytes. These factors were studied in relation to ageing, exposures toaneugenic chemicals and effects of the chromosomal constitution, suchas the presence of translocations [59,60]. The keen interest in aneu-ploidy in meiosis and its etiological factors [61,62] and in the effects ofexposures to aneugens was stimulated by collaborative efforts in EUprojects, led by Jim Parry.

In Roma, the laboratories of the National Committee for NuclearEnergy (CNEN, later ENEA) were focusing their research on germ cellchromosome aberration induction, using the cytogenetic analysis ofmouse spermatocytes and oocytes as the main tool. Initially, the pri-mary interest was on ionizing radiation effects, which fit with the CNENresearch agenda and the EURATOM Programme [39]. It was discoveredthat X rays and neutrons induced not only chromosome breaks but alsonon-disjunction in male germ cells [63]. This research was soon ex-tended to chemical agents with the main goal being comparison ofaneugenic effects in mammalian germ cells with those induced in As-pergillus nidulans [64] or in Drosophila melanogaster [65]. Later, thebattery of approaches was expanded to include the more difficult cy-togenetic analysis of oocytes [66] and the refinement of bone marrowmetaphase analysis by means of bromodeoxyuridine-based differentialstaining of first, second and third cell generations [67]. With some ofthese tools ENEA informally joined the European research network onaneuploidy through its collaboration with the Italian National Instituteof Health [68]. At the same time, Antonella Russo moved from ENEA tothe University of Padova, where she established micronucleus analysisin mouse spermatids as a tool faster than metaphase chromosomecounting for testing the induction of structural and numerical chro-mosome changes in male germ cells [69,70]. A very profitable colla-boration developed between the ENEA laboratory and John Mailhes,Louisiana State University Medical Center, Shreveport, USA, who wasan expert in mouse oocyte cytogenetic analyses. He came to Roma as avisiting scientist in 1990 and in the following 3 years FrancescoMarchetti, who had just completed his thesis work on the aneugeniceffects of griseofulvin in mouse zygotes [71] at the ENEA CasacciaResearch Center, was a fellow in Mailhes' laboratory.

From the beginning, the Italian research community had been activeon the topic of chemically induced aneuploidy. In December 1981,

CNEN organized a one-day workshop entitled “The contribution ofexperimental research to unraveling causes and mechanisms of aneu-ploidy and non-disjunction”, which gathered 7 different groupsworking with various models, including yeasts, other fungi, plants,mammalian cell cultures and mice. In 1986, the Italian GeneticAssociation convened the Symposium “Chemically InducedAneuploidy: Tests and Mechanisms,” in Spoleto, where 17 contributionswere presented either as lectures or posters. The database of EU-fundedprojects (http://cordis.europa.eu/projects/result_it?q='aneuploidy'%20AND%20(contenttype%3D'project'%20OR%20/result/relations/categories/resultCategory/code%3D'brief','report'), queried by thesearch term “aneuploidy”, returns 5 projects on chemically-inducedaneuploidy coordinated by various Italian laboratories between1982–1995.

Belgium has a long tradition of research on chromosome segrega-tion. Edouard Van Beneden (1846–1910) discovered in Ascaris howchromosomes are organized in meiosis [72]. In the middle of last cen-tury, studies on the antimitotic properties of tubulin inhibitors werestimulated by Pierre Dustin at the Faculty of Medicine of the UniversitéLibre de Bruxelles (ULB) for chemotherapeutic purposes [41] and byPaul Janssen, founder of Janssen Pharmaceutica, and his researchgroup [73]. One of the goals in the Brussels laboratory in the 80′s was toassess the role of chromosomal changes in vivo during chemically in-duced rat hepatocarcinogenesis [74–76] and mouse skin tumorigenesis[77]. Four Belgian Universities—ULB, Université Catholique de Lou-vain (UCL), Université de Liège (ULg), and Vrije Universiteit Brussel(VUB)—collaborated in this challenging project supported by the Na-tional Research Foundation (FNRS-FWO), showing that numericalchromosomal changes, including polyploidy, aneuploidy and cen-tromere-bearing micronuclei, are induced during some steps of thecarcinogenic treatments. At VUB the team focused on in vitro effects ofchemicals, including metals, on mitotic figures, using methods thatallowed us to combine mechanisms and effects, in casu by differentialstaining of chromosomes and spindle [78–80]. A parallel aim was todiscriminate aneugens from clastogens by C-banding [81], DNA contentand area measurement of micronuclei induced in vivo in mouse bonemarrow [82]. The results were encouraging but no research money wasmade available for aneuploidy studies in Belgium. In 1986 the grouphad the opportunity to organize at the Vrije Universiteit Brussel theXVIth annual meeting of the European Environmental Mutagen Societyand a special session on risks of aneuploidy. Jim Parry was present atthe meeting, and the Belgian team was invited to enter a large Europeanexercise.

Joining our efforts AND Jim Parry’s tenacity made the difference.Indeed Jim was the central figure open to our scientific thoughts andsuggestions, coordinating the projects, making contacts with EU sci-entific officers, stimulating many young scientists in the different labs,allowing exchanges of methodologies between labs and co-tutoring PhDstudents. He played an essential role in the education of young re-searchers, in making enough money available for research, in im-plementing our findings at the regulatory level, and last but not leastdeveloping an international team with friendly relationships amongcolleagues, of which the present paper is a result and a tribute to him(Fig. 1).

2. The EU research projects

The European Communities’ (EC) Environmental ResearchProgrammes were intended to provide a scientific basis for EC en-vironmental policy and support the development of anticipatory en-vironmental management.

The 3rd and 4th Environmental Research and DevelopmentProgrammes of the European Community (EC), later European Union(EU), 1981–1986 and 1986–1990, provided funding to 21 laboratoriesto investigate “Genetic Effects of Environmental Chemicals” (see Tables2 and 3). A wide range of studies were performed using many different

death and diseases like cancer. In a way, it was obvious that aneuploidycan modify the gene balance: having an irregular number of chromo-somes, almost by definition, leads to imbalances in transcript andprotein abundance in aneuploid cells and to secondary effects de-pending on the type of proteins involved (among others, DNA repairgenes, proto-oncogenes, tumour suppressor genes, cell cycle controlgenes, apoptosis genes, and chromatin remodelling genes). However, atthat time, assessing nucleotide changes in DNA sequence (i.e., pointmutations) was dominating mutagenesis and the regulation of chemi-cals, while numerical chromosomal aberrations were not much in focus;cell genetics was centered essentially on structural chromosome mu-tations rather than changes in chromosome copy number. Numericalchromosome mutations were known from aneuploidies in newborns butnot taken much into account for the evaluation of potential mutagens.Even today, the term mutation is often wrongly restricted to changes ofthe DNA sequence that can modify gene expression. In view of theknown dramatic effects of chromosomal imbalance in germ cells, zy-gotes/embryos and offspring, and the growing interest in the potentialinvolvement of aneuploidy/polyploidy in cancer, it is important toidentify the mechanisms and agents that may perturb the accuratesegregation of chromatids/chromosomes both in somatic and germcells.

The best opportunities to discuss these questions were the annualmeetings of the European Environmental Mutagen Society (EEMS). Thissociety, now renamed European Environmental Mutagenesis andGenomics Society (EEMGS), is a member of the InternationalAssociation of Environmental Mutagenesis and Genomics Societies(IAEMGS). EEMGS is a friendly society devoted to exchanging researchand concerns among academics, companies and regulators, and at thattime it alternated its meetings between Eastern and Western Europeancountries. This platform clearly favored contacts among geneticists, cellbiologists and scientists interested in studying aneuploidy, along withpharmaceutical and chemical industries and regulators. It catalyzed thestart of an EU-funded collaborative project on aneuploidy.

Inspiration, motivation, intuition and good partners have alwaysbeen essential for creative research but without funds such innovativeresearch in experimental sciences remains impossible. When researchprojects on aneuploidy were emerging in different European countries,they lacked significant local financial support.

Research in Swansea in the late 1960s and early 1970s involvedinvestigations into recombination, DNA repair and the tolerance ofaneuploidy in the yeast Saccharomyces cerevisiae. In the early 1970s thiswork used various chemical mutagens as well as radiation, and this ledthe group to the research area of environmental mutagenesis. Yeaststrains were constructed to detect all the genetic endpoints that couldbe modified by chemical and radiation exposure: point mutation, mi-totic crossing over, mitotic gene conversion, mitotic and meiotic an-euploidy [46]. These strains of yeast were used throughout the 1970sand early 1980s.

Although yeast is a good model organism for genetic studies, themain conclusion from the first collaborative study was that many ofthese assays were unsuitable for routine screening in the field of en-vironmental mutagenesis. Major problems with the yeast test systemswere the failure to detect reference aneugens like colcemid and thefailure of meiotic systems to take up molecules that are large. Thus, theSwansea group moved its environmental mutagenesis studies into invitro mammalian cell culture systems where there was the additionalbonus of actually observing chromosomes and cell division micro-scopically [47].

During the ‘80 s, at the GSF-Institut für Säugetiergenetik inNeuherberg, Germany, the research group led by Ilse-Dore Adler gainedknowledge and experience with male mouse germ cell cytogenetic as-says [48] and contributed to validating the mouse bone marrow mi-cronucleus test in comparison to the standard metaphase analysis ofchromosome aberrations [49]. Prompted by regulatory agencies’ en-couragement to develop and validate tests for the assessment ofTa

SynopsisofEU-funded

projectscoordinatedby

SwanseaUniversity.

Duration

Coordinator

Noofpartners

Countries

Acomparativestudyofthegenetic

toxicology

ofenvironm

entalm

utagensandcarcinogens

1982-1986

EC-scientificoffi

cer:HeinrichOtt

Over15

SeeTable3

Geneticeffectsofenvironm

entalchemicals

1987-1990

EC-scientificoffi

cer:AndrewSors

Over15

SeeTable3

Thedetectionandevaluationofaneugenicchemicals

1991-1994

SwanseaUniversity,UK(JMParry)

9Belgium,Greece,Italy,Germany,Sweden,The

Netherlands,UK

Chemicallyinducedaneuploidy

1994-1996

5Germany,Hungary,The

Netherlands,UK

Thedetectionandhazardevaluationofaneugenicchemicals

1997-2000

4Belgium,Germany,UK

ProtectionoftheEuropean

populationfrom

aneugenicchemicals(PEPFAC)

2001-2005

5Belgium,Germany,Italy,UK

cellular targets producing disturbances to chromosome segregationleading to aneuploid progeny. However, it was clear that we hadonly a poor understanding of the complete range of possible che-mical/target interactions and their relative importance in terms ofaneuploidy induction;

(2) the available test systems had substantial practical and theoreticallimitations that precluded their routine use for the screening andassessment of potential aneugens. For example, in vitro tubulinpolymerisation assays were of value only for a discrete range ofaneugens (i.e., the spindle inhibitors), and fungal test systemsshowed a low predictive value for mammalian cells and intact an-imals [54,84].

Overall, the comprehensive range of genotoxicity studies under-taken illustrates the strengths of European co-operation and the benefitsto participating laboratories in technological development and transfer.

After these pioneering efforts, a more coordinated phase followed,in which the Swansea lab, starting from its specific research with theyeast Saccharomyces cerevisiae and cultured mammalian cells [47], tookthe lead (Table 2). Between 1991 and 2000, within the framework of itsEnvironment Research and Development Programme, the EC Directo-rate General (DG) XII supported three successive collaborative researchprojects coordinated by Swansea University, mainly aimed at devel-oping and validating assay systems for the detection and evaluation ofchemicals capable of inducing numerical chromosome changes both insomatic and germ cells [84,85]. Through the years the research focusmoved from test validation to hazard characterization, culminating in2001 with the project "Protection of the European Population fromAneugenic Chemicals (PEPFAC)" that investigated the aneugenic ac-tivity of chemicals of high concern for human populations, such as bi-sphenol A, natural and synthetic hormones, industrial chemicals andpharmaceuticals. The project involved five laboratories with distinctand complementary expertise in Bielefeld, Brussels, Neuherberg, Romaand Swansea [85].

A broad published database from the EU 1997–2000 project called“The detection and evaluation of aneugenic chemicals” shows expandedknowledge of cellular interactions that might lead to aneuploidy andthe nature of the chemicals that induce numerical chromosome changesin somatic and germ cells. The project was subdivided into a series ofinter-related research activities:

(1) Investigations into the diverse mechanisms that might lead to an-euploidy induction, the nature of the chemicals that can modify thefidelity of mitotic and meiotic chromosome segregation and eval-uating their relative importance.

(2) The development of methods to investigate the metabolism of an-eugenic chemicals and to define the metabolic requirements ofpotential test systems for their study.

(3) The development of methods to detect chemical aneugens in mi-totic and meiotic cells with emphasis on the development of mo-lecular probes capable of identifying specific chromosomes in test

species and humans, including in interphase nuclei [86].(4) The development of methods to study the role of aneuploidy in

somatic and inherited diseases with emphasis on the analysis of thecarcinogenic potential of aneugenic chemicals.

(5) Identification of the physicochemical features of chemicals thatresult in aneugenic activity and the development of a suitablemodel capable of predicting aneugenic activity based upon thesephysicochemical characteristics.

(6) The application of the results of the project to the development of aregulatory framework for the assessment of aneugenic chemicals.(For review see [87]).

The EU 2001–2005 project “Protection of the European populationfrom aneugenic chemicals (PEPFAC)” was part of the EU FrameworkProgramme 5: Quality of Life, Management of Living Resources(2001–2004). A key aim of the PEPFAC project was to make use ofcomplementary methodologies developed by the collaborators tocharacterise the mechanisms of action of aneugenic chemicals. Thesemethods were used to produce detailed dose-response data for aneu-genic chemicals in both somatic and germ cells suitable for use inregulation and risk assessment. Data were generated for spindle-mod-ifying aneugens to test the hypothesis of a threshold effect as a con-sequence of multiple microtubules being disrupted for occurrence ofchromosome missegregation. This mechanism contrasted with non-threshold effects induced by clastogenic agents, with only a single ortwo DNA insults. Comparisons were also made with aneugens thatmodify the fidelity of chromosome segregation by mechanisms otherthan spindle inhibition. The project evaluated the contribution of fac-tors such as apoptosis, cell cycle checkpoint genes, target cell age andculture progression, type of cell division (i.e., mitosis or meiosis), andtarget tissue to the shapes of the dose-response curves and potentialthresholds of activity. The assessment of the potential genotoxicity ofchemicals was based upon the use of in vitro screening and detectionmethods supported by in vivo methods to confirm or not whether ac-tivity detected in simple test systems such as cultured mammalian cellsis reproduced in intact animals. PEPFAC collaborators (Fig. 2) de-termined the relative requirements for in vitro and in vivo methods inthe assessment of aneugenic chemicals, and parameters such as therelative sensitivities of somatic versus germ cells and males versus fe-males. The data from the project were evaluated for their ability togenerate a hazard and risk-estimation model for aneugenic chemicalsand to integrate these models into the regulatory process.

The coordination of research efforts, such as that supported by theEU, was providing an effective mechanism for the concentration ofresearch on specific problems, for the rapid dissemination of experi-mental data and the transfer of technology within the European Union.The groups were meeting regularly, stimulating early exchange of re-sults and a strong collaborative impulse (Table 5).

3. The scientific results

The EC/EU projects on aneuploidy achieved important results froma methodological standpoint, contributing to the development, stan-dardization and validation of reliable methods to measure the fre-quency of aneuploidy in various cell types (Fig. 3). Eventually, thisprogress led to the implementation of new international guidelines ongenotoxicity testing. At the same time, many chemicals, including somerelevant for potential human exposure, were extensively characterizedfor their aneugenic effects, and their mode of action, as well as the dose-effect relationships, were elucidated in somatic and germ cells.

3.1. Methodological achievements in somatic and germ cells

3.1.1. Adapted MN assay in vitro and in vivo in somatic cellsOwing largely to the ease of scoring and relevance for both clasto-

genic and aneugenic effects, micronucleus assays have been applied for

Table 4Chemicals selected for use in the EC/EU aneuploidy projects.

Chemical Selection criteria [84]

Colchicine Spindle poison, positive controlEconazole nitrate Membrane modifierChloral hydrate Spindle poisonHydroquinone Spindle poisonDiazepam Modifier of centriole functioningThiabendazole Spindle poisonCadmium chloride Inconclusive in somatic cells, possible germ cell aneugenThimerosal Representative mercury compound, possible aneugenPyrimethamine Inconclusive, possible aneugenVinblastine sulphate Modifier of spindle elongation

systems, and a group of laboratories investigated the “induction ofaneuploidy by environmental chemicals”. These studies ranged fromassessing the ability of a chemical to modify tubulin polymerisation tothe induction of aneuploid offspring in rodents, reflecting the fact thataneuploidy may be induced by interaction with a variety of targetmolecules and structures within the cell. The participants demonstratedthat a variety of chemicals were capable of inducing aneuploidy infungal cultures (for review see [83]). In the next stage the coordinatedresearch project involved the activities of European laboratories thatwere directly supported by the EC, as well as collaborators fromcountries outside EC (e.g., Sweden, Switzerland, United States, NewZealand) (Tables 2 and 3).

Their major research efforts aimed at:

(1) evaluating the significance to the human population of aneuploidyinduced by chemicals that had previously been shown to producepositive results in fungi,

(2) developing and validating assay systems for the detection of che-micals capable of inducing chromosome number changes and

(3) providing mechanistic understandings of the modes of action ofaneugenic chemicals.

Before starting the study, the participants in the EC project under-took a literature search and evaluation of those chemicals that had beentested either directly or indirectly for their ability to induce aneu-ploidy/polyploidy and/or those cell division modifications that may bepredicted to lead to numerical changes. Using these data, chemicalswere selected that could be used to evaluate various test systems forpotential use in the detection, assessment and the regulation of aneu-genic chemicals (Table 4).

These EC Research Projects (see [84]) demonstrated that:

(1) aneugenic chemicals were capable of interacting with a variety of

Fig. 1. Jim Parry in a photograph from the late 1990s.

Table 3Test systems and participants in EC Aneuploidy projects (3rd and 4th Environmental Research and Development Programmes, 1981–1990) [84].

Test system Participant

In vitro tubulin polymerisation 1. Institute of Toxicology, Zurich and Hoffmann-La Roche, Basel, Switzerland2. University of Goteborg and Kobi Pharmacia, Sweden

Mitotic aneuploidy in Saccharomyces cerevisiae 1. Biological Sciences, Swansea, UK2. Hoffmann-La Roche, Basel, Switzerland

Mitotic aneuploidy in Aspergillus nidulans 1. Istituto Superiore di Sanità, Roma, Italy2. "Democritus", Athens, Greece

Induction of aneuploidy sectors in plants 1. Environmental Protection Agency, Research Triangle Park, USAInduction of cell division aberrations in cultured mammalian cells 1. University of Pisa, Pisa, Italy

2. Biological Sciences, Swansea, UKChromosome counts during mitotic cell division in cultured mammalian cells 1. Biological Sciences, Swansea, UK

2. Sylvius Laboratory, Leiden, The Netherlands3. University of Pisa, Pisa, Italy

Induction of micronuclei and the use of antibody probes in cultured mammaliancells

1. Biological Sciences, Swansea, UK2. National Institute for Research on Cancer, Genova, Italy3. Vrije Universiteit, Brussels, Belgium4. Università "La Sapienza", Roma, Italy5. Cancer Research Laboratory, Auckland, New Zealand

Cell division abnormalities in rodent bone marrow 1. GSF-Institut für Säugetiergenetik, Neuherberg, Germany (renamed Helmholtz ZentrumMuenchen)2. ENEA CRE Casaccia and Istituto Superiore di Sanità, Roma, Italy

Induction of micronuclei in rodent bone marrow 1. GSF-Institut für Säugetiergenetik, Neuherberg, Germany (renamed Helmholtz ZentrumMuenchen)2. Vrije Universiteit, Brussels, Belgium3. University of Pisa, Pisa, Italy4. ENEA CRE Casaccia and Istituto Superiore di Sanità, Roma, Italy

Chromosome counts in rodent bone marrow 1. GSF-Institut für Säugetiergenetik, Neuherberg, Germany (renamed Helmholtz ZentrumMuenchen)2. ENEA CRE Casaccia and Istituto Superiore di Sanità, Roma, Italy3. University of Pisa, Pisa, Italy

Chromosome counts, division abnormalities and micronuclei in rodent germ cells 1. GSF-Institut für Säugetiergenetik, Neuherberg, Germany (renamed Helmholtz ZentrumMuenchen)2. ENEA CRE Casaccia and Istituto Superiore di Sanità, Roma, Italy

hybridization (FISH) depended upon the construction of DNA probesfor whole chromosomes or specific chromosome regions such as cen-tromeres or telomeres and the availability of optical detection systems(reviewed by [91]). In the 1980s, centromeric probes became availablefor the human and mouse genomes, initially by means of customizedproduction and later on through commercial channels. They were soon

applied to distinguish centromeric-positive micronuclei considered tobe a specific marker of whole chromosome loss from centromeric-ne-gative micronuclei produced by chromosome breaks. In addition toFISH methods using DNA probes, a primed in situ approach (PRINS)was applied to detect centromeric and telomeric DNA repeats [92]. Theuse of fluorescent antibodies against kinetochores was another

(caption on next page)

decades in vitro in cultured cells and in vivo in several tissue types.However, the mechanisms for the induction of micronuclei by aneugenswere not clear, and no validated assay was available. Moreover, therewas a major difficulty for in vitro assays, due to the fact that micro-nucleus formation requires cell division. It is therefore important todiscriminate cells that divided in vitro from those that did not divide. Adecisive breakthrough came from the work of Michael Fenech and hiscolleagues who developed and refined the cytokinesis block micro-nucleus assay (CBMN) [88] that permits the identification of cells thathave divided once as binucleates and cells that have not divided and aretherefore mononucleates. Our network validated this approach inhuman lymphocytes, which were selected as the model system becauseof their euploid karyotype and human origin. Interlaboratory compar-isons were run to standardize the method.

The mouse bone marrow micronucleus test was already the goldstandard for evaluating the in vivo genotoxicity of chemicals in somaticcells. For aneugens, a critical question was determining the most ef-fective protocol. Due to their specific mode of action on non-DNA tar-gets, exposure designs and timing for sacrifice were re-evaluated [49].However, there was concern that the bone marrow micronucleus testcould not be suitable to detect chemical aneugens after oral or

inhalation exposure, because under these conditions chemicals couldlikely bind proteins on the proximal tissue, such as gut or lung epithelia,and would not reach bone marrow at effective concentrations. Withinthe EU network, therefore, in vivo micronucleus assays for both epi-thelial gut and lung cells were developed [89,90].

3.1.2. Fluorescence in situ hybridization for aneuploidy detectionOur ability to identify and count chromosomes improved in the

1980s because of technological developments and molecular advancesin our understanding of DNA and chromosome structure. Prior to this,individual chromosomes could only be identified in metaphase cellpreparations by their particular shape and/or by chromosome bandingmethods. Aneuploidy could be estimated in such preparations by simplycounting chromosomes. Although cumbersome and not suitable as ascreening approach, chromosome counting in metaphase yielded soliddata on in vivo chemically induced aneuploidy, especially for the ana-lysis of unavoidably small numbers of oocytes and zygotes. For othercell types that could be collected in large quantities, like lymphocytes,in vitro cultured immortalized cells and sperm, chromosome countingwas successfully replaced by more practical approaches.

The development of methods such as fluorescence in situ

Fig. 2. The team of the PEPFAC project (left to right: M. Kirsch-Volders, I.-D. Adler, J.M. Parry, U. Eichenlaub-Ritter, F. Pacchierotti, E.M. Parry).

Table 5EC/EU Contact Group Meetingsa.

Year/Date Location Host

1982 1-2 April Lyon, France IARC, Lyon1983 4-7 January London, UK S. Neale: Courteau Institute, London1984 8-10 July Swansea, UK J.M. Parry: University College of Swansea1985 24-28 April Athens, Greece A. Kappas: Nuclear Research Center “Democritos” Athens1988 17-20 January Noordwijkerhout, NL F. Sobels: University of Leiden1989 7-12 March Cordoba, Spain R. Marcos: University Autonoma de Barcelona1990 5-10 June Helsinki, Finland M. Sorsa: Institute of Occupational Health, Helsinki1991 10-15 September Estoril, Portugal J. Rueff: University of Lisbon1993 15-16 May Croydon, UK D. Anderson: BIBRA and University of Bradford1993 27-29 April Barcelona, Spain J.M. Parry (during the EEMS Meeting)1994 21-28 May Corfu, Greece N.A. Demopoulos: University of Patras1995 9-13 April Noordwijkerhout, NL P. Lohman: University of Leiden1996 29 May-2 June Patras, Greece N.A. Demopoulos: University of Patras1998 21-23 March Athens, Greece S. Kyrtopoulos: National Hellenic Research Foundation, Athens2002 2-6 October Brussels, Belgium M. Kirsch-Volders: Vrije Universiteit Brussel, Brussels2003 10-11 February Swansea J.M. Parry: Mid-term report meeting

a These meetings gathered the participants to multiple EC/EU funded projects on genetic toxicology, including those more specifically devoted toaneuploidy. As such, they favored many productive discussions about a broad array of research topics.

them, aneugenic effects were shown, although the effects were oftensmall (especially in comparison with data in oocytes) and not alwaysconsistent between laboratories [52,54,68]. The sensitivity window ofprimary spermatocytes to aneuploidy induction was limited and, ac-cordingly, the harvest time after treatment was a critical variable. Thedemonstration of meiotic delay by some of the tested chemicals pro-vided supportive information about the mechanisms of aneuploidy in-duction. By the sperm FISH method, it was possible to compare theeffects induced by diazepam in mice and men finding that both pro-longed and acute exposure to this widely used drug induced an increaseof aneuploid sperm in both species, and that men appeared to be about10 and 100 times more sensitive than mice to chronic or acute ex-posure, respectively [115].

3.2.3. Mechanistic insights into chromosome mis-segregation in oocytesexposed to pesticides, drugs, industrial and endocrine disrupting chemicals

Studies on aneuploidy induction in oocytes exposed in vitro eitherafter isolation from follicles, or in preantral follicle culture were carriedout, and they were compared to in vivo exposure by various adminis-tration routes. Nocodazole, a reference aneugenic chemical that affectsmicrotubule dynamics and polymerization kinetics was initially testedto establish the validity of the approach [59,119,122]. The slightlylower dose of nocodazole inducing aneuploidy in mouse oocytes fromfollicle culture compared to denuded oocytes suggested a synergisticeffect of the cytostatic chemical on the somatic and germ cells of thefollicle. Indeed, gene expression studies demonstrate functional inter-actions between the granulosa cells of the follicle and the mammalianoocyte that might affect oocyte maturation, chromosome segregationand developmental potential [132]. Aneuploidy induction in oocytes bynocodazole was confirmed in vivo after intraperitoneal treatment, butonly at high doses, pointing to chemical bioavailability as a criticalfactor for aneugenic risks [122].

The project then moved to assessing the effects of more en-vironmentally relevant chemicals. Trichlorfon, an organophosphatepesticide, was tested because of a report suggesting an association be-tween consumption of trichlorfon-contaminated fish and a cluster ofDown syndrome cases in a Hungarian village [133]. Spindle aberrationswere indeed demonstrated in mouse oocytes exposed to low, physio-logically relevant concentrations of trichlorfon during in vitro growthand maturation [121,134].

Diazepam, a tranquilizer used worldwide, which had been shown toinduce chromosome loss in cultured rodent cells [135,136] and to in-crease the frequencies of aneuploidy in mouse and human sperm [115],was also found to hinder spindle assembly, cell cycle progression andchromosome alignment in mouse oocytes after in vitro exposure[118,126].

A publication suggesting aneugenic activity of inadvertent, chroniclow dose exposure of female mice to bisphenol A (BPA), a widely usedplasticizer with endocrine disrupting activity [137], had raised concernabout this chemical. While there were difficulties to reproduce theseobservations in vivo, possibly because of phytoestrogens in animal dietacting as a confounding factor [94,123,138], using the preantral follicleculture in vitromodel, in which dietary influences were excluded, it wasindeed shown that prolonged low BPA treatment induced spindle andmeiotic abnormalities in mouse oocytes that can predispose to chro-mosomal errors [139].

2-Methoxyestradiol (2-ME), a metabolite of 17beta-estradiol and anatural component of follicular fluid that binds to the colchicine do-main of tubulin [140], was also shown to induce multipolar spindlesand aneuploidy [141]. Since ovarian concentrations of 2-ME could bealso increased by exposure to environmental pollutants activating theexpression of enzymes in the metabolic pathway from 17beta-estradiolto 2-ME, these observations pointed to possible indirect mechanisms ofaneuploidy induction in oocytes through activation of metabolic en-zymes in hormonal pathways.

4. New concepts

4.1. Metabolic activation of aneugens

Many chemical carcinogens require metabolic transformation toproduce their active genotoxic metabolite(s). Metabolic activation anddetoxification take place via the phase I and phase II enzyme systemslocated primarily in the mammalian liver. Rodent liver microsomalpreparations (S9) have been used extensively for in vitro genotoxicitytesting to mimic these biotransformation systems [142], but S9 pre-parations are also toxic to mammalian cell cultures, so that their use islimited to short exposure times representing a fraction of the cell cycle.An alternative is the use of genetically engineered cells that expressmetabolising enzymes that generate ultimate metabolites en-dogenously. This allows exposure periods to be extended to last for thewhole cell cycle, which is particularly important for the detection ofcompounds whose effects are specific to a particular stage of the cellcycle like aneugens [84].

A number of cell lines have been developed that constitutively ex-press cDNAs encoding biotransformation enzymes [143]. Geneticallyengineered Chinese hamster and human lymphoblastoid cell lines havebeen developed for the detection of clastogenic and aneugenic meta-bolites produced from indirect acting compounds [144–146].

Doherty and colleagues [146] have described the use of humanlymphoblastoid cells, AHH-1, that have low native CYP1A1 activity andMCL-5 cells that contain cDNAs for the human cytochromes 1A2, 2A6,3A4, 2E1 and microsomal epoxide hydrolase in addition to the nativeCYP1A1 activity [147]. These cells were used to investigate the po-tential role of cytochrome P450 in the aneugenic activity of 15chlorinated hydrocarbons. The cytokinesis-block micronucleus assaywas employed to enumerate micronuclei in binucleate cells, which hadundergone a single nuclear division, and with the use of specific andnon-specific centromere probes the distribution and segregation ofchromosomes was assessed to measure non-disjunction in binucleatecells and chromosome loss in micronuclei. This methodology demon-strated the ability of metabolically competent cell lines to metabolizehalogenated hydrocarbons to genotoxic species, including both clasto-gens and aneugens. The biotransformation of chemicals to aneugenicspecies had not previously been demonstrated.

Trichlorfon failed to induce micronuclei in either cell line at pH 7.3,while at pH 5.5 both chromosome loss and non-disjunction were in-duced in both cell lines, indicating an activity independent of P450. Atconcentrations up to 20 μg/ml the activity at pH5.5 was predominantlychromosome loss and non-disjunction, but at higher concentrations itwas mostly the induction of centromere negative micronuclei, i.e.,chromosome breakage [148].

The inclusion of metabolically competent cells in the in vitro mi-cronucleus assay therefore creates a powerful system for detectinggenotoxicants and may be extended to elucidate both mechanisms ofbioactivation and modes of genotoxic insult.

4.2. DNA versus non-DNA targeting mutagens

Indirect mechanisms of genotoxicity can be defined as interactionswith non-DNA targets leading to genotoxic effects. This includes lipidperoxidation and protein adducts. As far as proteins are concerned,research focused on inhibition of repair enzymes, cell cycle controlproteins, apoptosis-related gene products, damage to nuclear laminae,protection mechanisms against oxidative damage, metabolizing en-zymes, and tubulins of the mitotic/meiotic spindle. Fig. 4 shows theoverview of the mechanisms differentiating directly versus indirectlyacting genotoxicants, as described by Kirsch-Volders et al. in 2003[124].

approach, complementary to FISH methods, for identifying the origin ofmicronuclei [93], while immunofluorescent staining of spindle andcentrosomes allowed the exploration of mechanisms of chromosomemis-segregation and targets of chemical aneugens in mitotic andmeiotic cells [94,95]. Finally, when chromosome-specific pericen-tromeric probes became available for the human genome (but un-fortunately not for the mouse genome), they could be applied to detectnon-disjunction events in the main nuclei of binucleate cells preparedfor the CBMN assay. Coupled with the analysis of micronuclei, thisapproach allowed the assessment that non-disjunction of chromosomesis generally the major mechanism of aneuploidy induction by chemicalsin lymphocytes [14,96–100].

The accurate assessment of chromosome instability and more spe-cifically aneuploidy depends upon the analysis of relatively large cellnumbers. Thus interphase cytogenetics is today considered a muchmore efficient molecular cytogenetic method to assess aneuploidy,compared to M-phase.

3.1.3. Aneuploidy detection in post-meiotic male germ cellsThe occurrence of meiotic aneuploidy is a major social and medical

issue [22,23], and early experimental approaches to verify the aneu-genic activity of chemicals were based on chromosome counting atmeiosis II in rodents. As an alternative to this laborious approach,methods for detecting aneuploidy in post-meiotic stages of male germcells were developed in the 1980s and '90s.

Early spermatids are cells immediately after the two consecutivemeiotic divisions, and their identification among the heterogeneoustesticular cell populations is based on morphological features. Twodifferent methodologies, known respectively as the suspension method[101] and the dissection method [102], were developed to perform amicronucleus test in early round spermatids of mice and rats. Later, theassay was improved by application of kinetochore immunofluorescentstaining or FISH/PRINS detection of centromeric and telomeric se-quences [92,103–106]. Application of a meiotic MN assay representedan opportunity for direct comparisons with the large databases alreadyavailable from in vivo and in vitro MN assays [107,108]. EU-sponsoredprojects led to important accomplishments in the standardization andvalidation of new methodologies for the assessment of aneugenic actionof chemicals in germ cells [109,110].

The methodology for detection of aneuploid sperm by multicolourFISH was developed in early 1990s in human samples, taking advantageof the availability of new chromosome-specific centromeric probes[111–113]. I.-D. Adler at the GSF-Institut für Säugetiergenetik in Neu-herberg, Germany, started a collaboration with A. Wyrobek, LawrenceLivermore National Laboratory (LLNL, USA), to standardize the assayfor detecting aneuploid or diploid mouse sperm [114,115]. The ap-proach was indeed very successful and FISH-based chromosomecounting in sperm became a cross-species bridging biomarker forcomparative studies in mouse, rat, and humans. Stringent criteria ofanalysis were defined and validated for the correct identification ofnormal and aneuploid cells. The EU PEPFAC project [85] further con-tributed to the development of a robust assay for estimation of malegerm cell aneuploidy in rodents and established a first database of male

germ cell chemical aneugens [115–117].

3.1.4. Ex vivo/in vitro assays in oocytesWithin our research network, novel in vitro tests were introduced to

assess the risks of aneugens to female germ cell genomic integrity. Thecooperation between the Bielefeld and Neuherberg laboratories led tothe first studies using high resolution three-dimensional confocal im-munofluorescence microscopy to study the acentriolar spindle ofmammalian oocytes in response to potentially aneugenic exposures[118]. This was followed by development of non-invasive polarizationmicroscopy of the oocyte spindle in living mouse and human oocytes asa novel tool to assess hazards by aneugenic chemicals [119]. Co-ordinated experiments in Bielefeld and Roma made it feasible to com-pare results after exposures of mouse oocytes in vivo and in vitro[94,120–122], while dose-response relationships in male vs femalegerm cells, and in mitotic vs meiotic cells could be compared thanks tocollaborations among the groups in Neuherberg, Brussels and Swansea[123–125]. The Bielefeld group, in cooperation with other members ofthe consortium, developed methods to analyze the relative suscept-ibility of individual chromosomes to congression failures and thealignment of autosomes and sex chromosomes on the oocyte spindle forthe first time, combining FISH technology and spindle immuno-fluorescence in oocytes [126].

3.2. Achievements in characterising aneugenic hazards posed by chemicalsin somatic and germ cells

3.2.1. Threshold dose-effect relationships and consequences of alteredchromosome segregation

After the cytokinesis block MN assay in lymphocytes was validated,dose-effect relationships were studied by comparing known clastogensversus aneugens: threshold dose-responses were found with aneugens.Depending on the dose, it was also found that tubulin inhibitors couldinduce delayed mitotic arrest and mitotic slippage leading to tetra-ploidy in the following cell cycle (for review see [127]).

To better understand the mechanisms influencing possible survivalor death of the cells with genomic imbalance, cell lines were used toassess the potential of aneuploidy and/or polyploidy to induce apop-tosis. In collaboration with Enrico Cundari (CNR, Roma, Italy), the VUBteam demonstrated that apoptosis can be induced by aneuploidy/tet-raploidy and that it might be dependent on p53 status [128–130].

Evidence also exists in mouse oocytes about thresholds for aneu-ploidy induced by tubulin-binding chemicals [66,119,131].

3.2.2. Small but significant aneugenic effects in male germ cellsWhen the European projects on chemically induced aneuploidy

started, it became clear that the database on germ cell aneugens wasscanty. Thus, the first effort was to collect data on different chemicals tobe tested by standardized methods. The cytogenetic analysis of mousesecondary spermatocytes was chosen at Neuherberg and Roma as themost suitable approach due to greater relative abundance of such cellscompared to oocytes. Chemicals with presumed different primary tar-gets or mechanisms of action were tested (Table 4) and, for some of

Fig. 3. Some original pictures from our EU-granted projects. (a) Applications of FISH in cytokinesis blocked human binucleated lymphocytes with pancentromericprobes (left image, yellow fluorescence) for identication of centromeres in micronuclei, or with chromosome specific centromeric probes (middle and right image,chromosome 1 is red; chromosome 17 is green) for discrimination between chromosome loss and non-disjunction events. Original images from VUB, courtesy of A.Elhajouji. (b) Left: a normal mouse oocyte showing the spindle and congressed chromosomes; middle and right: mouse oocytes containing aberrant spindles withprominent cytoplasmic microtubule asters (white arrows), as well as misaligned chromosomes (yellow arrow). Original image and more details in [141]; (c) from leftto right examples of: hyperploid mouse bone marrow metaphase with 42 chromosomes (original image and more details in [67]; secondary mouse oocyte withhyperploid chromosome set (n=22, upper triangle) and corresponding hypoploid polar body with n=18 chromosomes (bottom triangle) as originally shown in[122]; polyploid mouse zygote [120]. (d) left column, top to bottom, a mouse spermatid carrying a MN due to chromosome loss (green fluorescence, minor satellitecentromeric sequences; red fluorescence, major satellite pericentromeric sequences, both detected by PRINS as described in [203]; right panel showing the appli-cation of mouse sperm FISH assay as originally reported in [204] (yellow, green and red fluorescence represent respectively X, Y and chromosome 8). Left side of thepanel shows normal epididymal sperm, while in the right side two examples of aneuploid sperm are visible (top, XY8; bottom, X88). Published images are reproducedwith permission.

identifying in vivo the cells that divided after exposure to the mutagen.More recently, new developments in FISH combining chromosomeprobing and cell cycle specific markers showed the existence ofthresholds for aneuploidy induced by spindle poisons in peripheralblood of mice [155].

4.5. Induction of apoptosis by aneuploidy/polyploidy

When the project started, apoptosis was considered to be inducedprimarily by structural DNA damage rather than changes in chromo-some number. Collaboration with Enrico Cundari (CNR, Roma, Italy),an expert on apoptosis who came as visiting Professor at the VUB la-boratory, led the team in Brussels to assess the possibility of apoptosisinduction by aneuploidy and tetraploidy. It was demonstrated thatdepending on the concentration of tubulin inhibitors the cell can bepermissive leading to aneuploidy, or it can enter apoptosis under con-trol of p53 (for reviews see [156,157]). This question, considered novelat that time, is now a well-accepted concept that also plays an im-portant role in chemotherapy.

4.6. Narrow stage specific sensitivity of germ cells to aneugens

In germ cells (both male and female) the time of sensitivity isnarrow. Sperm FISH analysis in Hodgkin’s disease patients undergoinga combined chemotherapy including vincristine and vinblastine showedsignificantly increased aneuploidy frequencies that generally (but notalways) returned to baseline levels 1–2 years after the end of treatment[158,159]. In view of the increasing success of cancer therapies, theseand similar data in young women [160] are going to be increasinglyimportant when providing genetic counseling to those patients wishingto have babies after chemotherapy.

4.7. Do unique germ cell aneugens exist?

The EC funded project "Detection of Germ Cell Mutagens" offeredthe opportunity to test the activity of nine chemicals, most of themalready found to give heritable effects, using a panel of germ-cell assayscarried out in parallel with in vivo somatic tests [109]. This projectcould verify whether specific germ cell mutagens exist. At the end of theproject, the conclusion was that all germ-cell mutagens were also so-matic cell mutagens. The project was not focused on aneuploidy, al-though the spermatid micronucleus assay was among the new meth-odologies to be validated [109]. As far as aneuploidy is concerned, thequestion remains open, given the existence of specific differences re-lated to checkpoint activity and spindle organization in the mammaliangerm cell line, and especially in mammalian oocytes. Indeed, it hasbeen recently remarked that germ cell tests are necessary and cannot bereplaced by assays in somatic cells [161].

5. Translation of efforts from basic science to regulatoryimplications

The results obtained by the EU projects were a trigger to reconsiderhow to perform tests of mutagenicity and to improve the battery oftests. This Section gives an overview of progress in this area.

5.1. Guidelines for mutagenicity testing: UK-COM and UKEMS as anexample

During late 1970s and early 1980s there were very few publishedguidelines on how to conduct mutagenicity tests. Guidelines from theOrganization for Economic Co-operation and Development (OECD) didnot appear for the first time until 1983. The MRC/ICI/NIEHSInternational Collaborative Study [162,163], suggested that no singlemutagenicity test would detect all classes and examples of chemicalcarcinogens.

Under the leadership of Jim Parry, the UKEMS established a sub-committee in March 1982 to determine the minimal criteria that shouldbe achieved to comply with mutagenicity testing requirements in theUnited Kingdom.

The processes by which UKEMS achieved its testing recommenda-tions in the 1980s and early 1990s were employed in the InternationalWorkshops for Genotoxicity Testing (IWGT) and made a significantimpact on OECD guidelines and the International Conference onHarmonization (ICH) guidance [164].

At the same time the Committee on Mutagenicity of the UKDepartment of Health and Social Security (DHSS) was preparingguidelines on testing [165]. Whereas the UKEMS recommendationsconcentrated on method, the DHSS guidelines were concerned morewith strategy (i.e., which tests to conduct).

The UK Committee on Mutagenicity of Chemicals in Food,Consumer Products and the Environment (COM) is an independentexpert advisory committee reporting to the UK Chief Medical Officerwith a general remit to advise on important general principles or newscientific advances in connection with mutagenic hazard or risk and topresent recommendations for mutagenicity testing. In 1989, guidancefor testing of chemicals for mutagenic potential concentrated onmethods to detect point mutations or structural chromosome aberra-tions. In 2000 this advice was amended in view of rapid developmentsin technology and the development of many new methods [166]. TheCOM reaffirmed its general 1989 advice that screening for mutagenicityshould be based on a limited number of well validated and informativetests and also proposed that methods to detect the potential hazard ofchemicals that may induce aneuploidy be considered. This was to takeaccount of the association between aneuploidy and heritable effects ingerm cells, and potential carcinogenicity. Thus, the COM concludedthat the testing of chemicals for potential aneugenic activity should beincluded in genotoxicity testing strategies.

The COM recommended a three stage testing strategy for the de-tection of mutagenic hazard, related to the anticipated likelihood ofhuman exposure. Stage 1 testing involves screening for clastogenicityand indications of aneuploidy by either in vitro metaphase analysis orthe in vitro micronucleus test. It was recommended that, if a metaphasetest showed indications of potential aneugenicity by effects on themitotic index or polyploidy, then this should be investigated by pro-cedures such as FISH and chromosome painting. Alternatively, an invitro micronucleus test could be used employing methods to classify themicronuclei using kinetochore or centromere staining. Similar tests foraneuploidy can be used in in vivo studies where appropriate.

The COM acknowledged that in some cases there might be athreshold concentration effect for the induction of aneuploidy and thatin such cases the dose-response for the induction of non-disjunctionshould be determined as this effect may occur at a lower concentrationthan that inducing chromosome loss.

The convincing results obtained within our EU projects and by otherresearch teams internationally brought the UK Committee onMutagenicity of Chemicals in Food, Consumer Products and theEnvironment [166] to the following conclusions:

• From the theoretical point of view, given the association betweenaneuploidy and heritable effects in germ cells, and potential carci-nogenicity, testing of chemicals for potential aneugenic activityshould be included in genotoxicity testing strategies. Data fromstudies of induced aneuploidy had been used for the classification ofchemicals in the EU and thus the COM advice was timely.• From the practical point of view, major changes in the new strategyproposed were the consideration of the detection of the potentialhazard of chemicals which may induce aneuploidy (numericalchromosome aberrations) and the application of the in vivo assaysfor tissues other than the bone marrow. The objective was to set outa scientifically valid testing strategy comprising those methods be-lieved to be the most informative, best validated and

4.3. Dose-effect relationships for aneugens: a narrow window for testing andconsequences for classification

The relationship between a mutagen, its target(s), and the measuredendpoint is illustrated in Fig. 5 [149]. In general, no effect may be seenwith an aneugen at low dose because it is unlikely that enough targetsare hit to disrupt the fidelity of chromosome distribution to thedaughter cells. Exceptions may exist in the case of some clastogenicagents, like mitomycin C, that induce breaks in pericentric hetero-chromatin and can cause aneuploidy by centromere function impair-ment [150–152].

An intermediate dose may allow cells with imbalanced chromosomenumber to survive mitosis and aneuploidy can be detected. At highdose, the disturbances may be too strong to allow cell survival and/orcompletion of mitosis. For designing aneuploidy tests, one has to con-sider that exposures at the highest possible doses may not maximize theeffect. Exposure doses that cause only moderate cell cycle alterationsmay be more efficacious in inducing chromosome malsegregation andshould be a preferred approach to study design. There may be a verynarrow window of effect both in somatic cells and in germinal cells.Consequences of positive results in any of the tissues should be definedin terms of classification according to international categories, i.e.,possible carcinogens/mutagens/germ cell mutagens. Even in the case ofa demonstrated threshold in aneugenic response to a chemical, it will benearly impossible to define a safe dose for humans because thethreshold may vary by orders of magnitude between test species andhumans [115]. In conclusion, when testing an unknown chemical in anassay capable of detecting aneugens, a single positive concentration,even without a dose-response, should be regarded as subject to classi-fication.

4.4. Threshold versus non-threshold dose-effect relationships

Starting from the concept of indirect acting mutagens, it was theo-retically expected that in contrast to DNA-binding mutagens, mutagens

that induce their genotoxic effects through non-DNA binding wouldshow threshold concentration-effect response curves. This relationshipbetween a mutagen, its target(s), and the measured endpoint is illu-strated in Fig. 5 [149].

Applying the in vitro micronucleus assay in combination with FISHfor centromeric regions on flow-sorted micronuclei [96] and chromo-some specific probes on binucleated cells in the CBMN assay [99], athreshold for selected spindle inhibitors (colchicine, mebendazole,carbendazim, nocodazole) was demonstrated. In contrast to the aneu-gens, the clastogen MMS (methylmethanesulfonate) did not show athreshold dose-response. The data on aneugens were confirmed andextended later by different laboratories, as reviewed in a special issue ofMutagenesis on the micronucleus assay [153]). However, recent workalso showed the existence of experimental thresholds for some DNA-binding chemicals [154].

Comparison of thresholds for induction of non-disjunction by no-codazole in in vitro exposed human lymphocytes and in vitro maturing,nocodazole-exposed mouse oocytes suggests that extrapolation fromsomatic to germ cells for spindle interfering agents might be feasible -the values were in the same range [124].

Extrapolation from in vitro threshold values to the in vivo situation insomatic cells was technically difficult, due to the difficulty of

Fig. 4. Conceptual model of mechanisms by which directly or indirectly actinggenotoxicants, as well as other molecules, can play a role in carcinogenesis(grey arrows). Continuous white arrows indicate possible paths from cell da-mage to cell proliferation. Dashed white arrows represent possible compensa-tion mechanisms that, after extensive cell loss due to apoptosis or cytotoxicity,may lead to cell proliferation of adjacent cells in the same tissue. Originallyfrom [124].

Fig. 5. The relationship linking a mutagen (M), its cell targets (T) and themeasured endpoint (E) may produce different dose-effect responses; whenseveral specific interactions are required to induce a genotoxic endpoint, athreshold effect can be expected [149]. In the drawing, M1.1-1.n and T1.1-1.nindicate multiple molecules of the same mutagen or the same target. The threehypothetical relationships represented here may correspond to the interactionof a single molecule of a DNA-binding mutagen with the DNA, leading to genemutation or chromosome break (single hit-single target); the interaction ofseveral molecules of an aneugenic compound with β tubulins (multiple hits-single target) that can cause spindle deficiency and chromosome loss with athreshold effect; the interaction of a single molecule of an aneugen with mul-tiple molecules of the same target (single hit-multiple targets) that also maygive rise to chromosome segregation errors with a threshold effect.

extend some of them to the in vitro chromosome aberration test TG 473.The parameters concern the assessment of cytotoxicity, which is criticalin performing a scientifically relevant MN assay, since MN expression isdependent on cell division. They confirm the use of (1) relative popu-lation doubling (RPD) and the relative increase in cell counts (RICC) inthe absence of cytokinesis blocking agent; (2) the cytokinesis-blockproliferation index (CBPI) and the replication index (RI) when the cy-tokinesis block assay is used [168]. These parameters take into accountcell proliferation from the beginning of the treatment, instead of pre-viously acceptable parameters that only measure the cell counts at theend of the treatment. Moreover the TG 473 for in vitro chromosomeaberration testing was revised to adopt this approach as well, and RPDand RICC were recommended for cell lines.

5.3. In vivo micronucleus assay: bone marrow, gut and lung

During the 2nd IWGTP, held in Washington, DC, a working groupdiscussed procedures for in vivo MN assays, reaching consensus about anumber of issues. They considered the detection of aneuploidy andassays in tissues other than bone marrow (germ cells, other organs,neonatal tissue) [107]. A major conclusion was that CREST- or FISH-labelling approaches could be considered reliable methods to detectaneugens by the in vivo micronucleus assay in somatic cells. They alsoevaluated the available data from the spermatid MN assay. Most ofthese results were produced during the EC project “Detection of GermCell Mutagens” [109]. The conclusion was that clastogenic and aneu-genic compounds can be detected and distinguished by this assay, withvery good agreement between the two methods (see 3.1.3) and theresponse of rats and mice.

6. Mechanisms of chromosome segregation errors andaneuploidy: still a hot topic

As pointed out before, aneuploidy can occur when there is a failureamong a multitude of potential targets. Since the 1980s, the importanceof clarifying the pathways leading to aneuploidy in somatic and germcells was appreciated in designing and interpreting tests and protocols.However, only more recently, mechanisms of (geno)toxicity are beingintegrated into hazard characterization by, for instance, the AdverseOutcome Pathways approach (AOP) [176]. For aneuploidy, this is apowerful tool to assess the strength of evidence and identify gaps ofknowledge [140,177–179]).

There has been much progress in understanding mechanisms con-trolling genomic stability, and we will point out a few important re-views.

For studies of chromosome/chromatin structure, new microscopicapproaches, such as electron microscopic tomography (ChromEMT),that overcome previous limitations of resolution, enable the visualisa-tion of chromatin fibers in 3D in the nucleus [180,181].

Concerning chromosome segregation, a special issue of Biologypublished in 2017 was devoted to an update on the mechanisms in-volved and the consequences of chromosome segregation errors inmitosis and meiosis [34]. A detailed review concerning meiosis hasbeen recently proposed by Gorbski [182]. Mechanisms of aneuploidy inhuman eggs are extensively reviewed by Webster and Schuh [23], andillustrations of the surveillance mechanisms ensuring accurate chro-mosome segregation at the mitotic checkpoint can also be found in[183–185].

Since our early observations on apoptosis induced by micro-nucleation/aneuploidy/tetraploidy, the field evolved to a detailed andspecific discrimination between apoptosis, necrosis, and senescence, asa result of cytokinesis failure, mitotic catastrophe and mitotic slippage[184].

The destiny and impact on health of a micronucleus resulting fromchromosome lagging is now better understood [127]. It was shown thatdue to micronucleus membrane deficiency, the chromosome (or

chromosome fragment) isolated in the micronucleus is not able to bereplicated properly, which leads to chromosome fragmentation (chro-mothripsis). Furthermore, random recombination of the fragmentsleads to a massively rearranged mutant chromosome (chromoanagen-esis), which may also undergo rounds of breakage/fusion cycles if thetelomeres are not present at the ends of the mutated chromosome. Therecombined fragments may also be integrated or not in other chromo-somes. This process of chromothripsis and chromoanagenesis [31] leadsto enhanced genetic instability [186]. In addition, cyclic GMP-AMPsynthase (cGAS) surveillance of DNA from micronuclei was discoveredby combining live-cell laser microdissection and single cell tran-scriptomics [187]. This remarkable discovery, which links genomicinstability caused by micronucleus formation to innate immunity, in-dicates that self-DNA liberated from shattered chromosomes in micro-nuclei into the cytosol after breakdown of the micronucleated envelopetriggers a proinflammatory response. The authors concluded that asmicronuclei formed from lagging chromosomes activate interferon-sti-mulated gene expression, recognition of micronuclei by cGAS may actas a cell-intrinsic immune surveillance mechanism that detects a rangeof neoplasia-inducing processes [187]. This new knowledge has over-turned the traditional concept of micronuclei from that of passive in-dicators of DNA damage to active players in the formation of DNAstrand breaks, chromosomal rearrangements, inflammation and tu-morigenesis [29,32,188].

The impact of aneuploidy during gametogenesis and embryogenesisis an important cause of reproduction failure and human disease[22,23]. There are still several open questions concerning the specificsensitivity of the two gametogenesis processes, based on temporal,developmental, cell and hormonal differences between males and fe-males [125,189]. It was recently reported that at the first cleavage di-vision two separate spindles are formed where paternal and maternalchromosomes independently congress; this unpredicted finding mightexplain erroneous divisions into more than two blastomeric nuclei ob-served in mammalian zygotes. It also demonstrates how much we stillhave to discover about mechanisms of chromosome segregation in thegermline [190]. A more extensive use of genomic investigations, ima-ging approaches and time-lapse analyses is needed to clarify these is-sues and to identify the occurrence of chromosome segregation error,both in rodent systems [161,191] and directly on human samples[5,192–194].

The impact of losing chromosome balance and the impact of an-euploidy in cancer have been reviewed and discussed with sometimesdivergent conclusions [3,183,195,196]. Although chromosomal in-stability is a hallmark of cancer and a driver of tumour evolution itremains unclear whether it is a mere bystander or a driver of metastaticprogression; of note, recent studies suggest that micronucleus formationin cancers together with induction of the cGAS-STING inflammatorypathway promote the metastatic phenotype [197]. The role of aneu-ploidy and micronuclei in cancer induction, progression and metastasisrequires further investigation in in vivo experimental cancer models.

7. Reflections and recommendations on research strategies

Fig. 6 shows our group of collaborators at the time of completion ofthis historical review for Reflections in Mutation Research. In the fol-lowing paragraphs, we summarize the strengths/weaknesses of the EUresearch programmes on aneuploidy, and we offer some reflections andsuggestions for future research.

The varied projects and collaborations were highly successful at thefundamental, translational, applied and regulatory levels, especially forthe following reasons:

- Our starting strategy was to test a selected number of known/po-tential aneugens and clastogens in a battery of tests, both in vitro andin vivo, both in somatic and in germ cells, trying to combine me-chanisms with effects when possible, controlling rigorously the

complementary for endpoint. The Committee expressed the opinionthat routine screening for both clastogenicity and aneugenicity waspossible using the in vitro micronucleus test, with the use of cen-tromeric probes to identify the nature of any micronuclei induced.Alternatively, essentially equivalent information could be obtainedfrom an assay using metaphase analysis and appropriate stainingprocedures to highlight alterations in structure and number.

5.2. OECD in vitro micronucleus assay in mammalian cells (MNvit) todetect clastogens and aneugens

5.2.1. The 2nd International Workshop on Genotoxicity Testing ProceduresIn the 1990′s, the in vitroMN assay had become an attractive tool for

genotoxicity testing because of its capacity to detect not only clasto-genic and aneugenic events but also some epigenetic effects. Otherstrengths were its predictivity for cancer and its simplicity of scoring,accuracy, wide applicability in different cell types in vitro and in vivoand its amenability to automation. Implementation of in vitroMN assaysin the battery of tests for hazard and risk assessment of potential mu-tagens/carcinogens was therefore fully justified.

At the “2nd International Workshop on Genotoxicity TestingProcedures” (IWGTP), held in Washington, DC, 25–26 March 1999,current methodologies and data for the in vitro micronucleus test werereviewed. As a result, guidelines for the conduct of specific aspects ofthe protocol were developed. Agreement was achieved on the followingtopics: choice of cells, slide preparation, analysis of micronuclei, toxi-city, use of cytochalasin-B, number of doses, and treatment/harvesttimes [167]. Because there were several important in vitromicronucleusvalidation studies in progress, it was not possible to design a definitive,internationally harmonized protocol at that time. After completion ofthese studies, the data were reviewed at the “3rd International Work-shop on Genotoxicity Testing” in Plymouth, UK, 28–29 June 2002. Datafrom studies coordinated by the French Society of Genetic Toxicology,Japanese collaborative studies, European pharmaceutical industry va-lidation studies, along with data from Lilly Research Laboratories, wereused to make recommendations on the main aspects of the in vitromicronucleus protocol [168]. The impact of the results of EU projectswas fundamental. The major recommendations concerned:

(1) Demonstration of cell proliferation: both cell lines and lymphocytescan be used, but demonstration of cell proliferation in both controland treated cells is required for acceptance of the test.

(2) Assessment of toxicity and dose range finding: assessment of toxi-city should be performed by determining cell proliferation (e.g.,increased cell counts (CC) or population doubling (PD) withoutcytochalasin-B, or cytokinesis-block proliferation index with cyto-chalasin-B) and by determining other markers for cytotoxicity (e.g.,confluency, apoptosis, necrosis), which can provide valuable addi-tional information.

(3) Treatment schedules for cell lines and lymphocytes.(4) Choice of positive controls: without S9-mix both a clastogen (e.g.,

mitomycin C or bleomycin) and an aneugen (e.g., colchicine)should be included as positive controls, and a clastogen that re-quires S9 for activity (e.g., dimethylnitrosamine or cyclopho-sphamide) should be included when S9 is used in those cell typesthat cannot activate this agent directly.

(5) Duplicate cultures and number of cells to be scored.(6) Repeat experiments: for each experiment in lymphocytes, blood

from 2 healthy young and nonsmoking donors should be compared;in cell lines, the experiments need only to be repeated if the firstone is negative.

(7) Statistics: statistical significance should not be the sole factor fordetermining positive results. Biological meaning should serve as aguideline, taking into account historical control ranges and cyto-toxicity.

5.2.2. The retrospective validation by the European Centre for theValidation of Alternative Methods

The primary goal of this retrospective validation was evaluating thepotential of the MNvit as an alternative to the standard in vitro chro-mosome aberration test (CAT). Several studies comparing in vitro CATand MNvit had already been performed. A high correlation was ob-served in each of the studies (> 85%); however, no formal validationfor the micronucleus in vitro assay had been carried out. Therefore, in2004, a working group was established by the European Centre for theValidation of Alternative Methods (ECVAM) to perform a retrospectivevalidation of the existing data. To evaluate whether the test met all datarequirements requested by the ECVAM principles on test validity, amodular approach to validation was followed. This approach is definedby seven validity modules: (1) test definition, (2) within-laboratoryreproducibility, (3) transferability, (4) between-laboratory reproduci-bility, (5) predictive capacity, (6) applicability domain and (7)minimum performance. Module 7 was not considered, as this was aretrospective evaluation of data.

The working group first evaluated the available published data andcame to the conclusion that two studies [169,170] met the criteria for aretrospective validation according to the criteria previously defined bythe working group. These two studies were evaluated in depth (in-cluding reanalysis of raw data) and provided the information requiredto assess the reliability (reproducibility) of the test. For the assessmentof the concordance between MNvit and in vitro CAT, additional pub-lished data were considered.

Based on this retrospective validation, the ECVAM ValidationManagement Team concluded that MNvit was reliable, reproducible,transferable, predictive and relevant [171], and it can therefore be usedas an alternative method to in vitro CAT. Following peer review, theseconclusions were formally endorsed by the ECVAM Scientific AdvisoryCommittee.

5.2.3. OECD guideline TG487: MNvitThe final step before acceptance by the OECD consisted of an inter-

laboratory exercise to evaluate different measures of cytotoxicity/cy-tostasis that can be applied when MNvit is performed in the absence ofcytochalasin-B [172]. The use of the MNvit within a battery of tests wasfurther defined by the various regulatory bodies responsible for de-veloping such test strategies.

The OECD Test guideline 487 MNvit was initially adopted in 2010[172]. It gives a thorough description for the MNvit, including sig-nificance, design, technical aspects, statistics and interpretation of re-sults. For additional information, mechanistic interpretation [127] anddetails of protocols were described in separate papers for lymphocytesand other mammalian cells [173,174].

As far as aneuploidy is concerned, the major contribution of ournetwork was to include in the new OECD Test guideline 487 advice ondifferentiating between clastogenic and aneugenic effects: “In additionto using the MNvit test to identify substances that induce micronuclei,the use of immunochemical labeling of kinetochores, or hybridizationwith centromeric/telomeric probes (fluorescence in situ hybridisation(FISH)), also can provide additional information on the mechanisms ofchromosome damage and micronucleus formation. Those labelling andhybridization procedures can be used when there is an increase inmicronucleus formation and the investigator wishes to determine if theincrease was the result of clastogenic and/or aneugenic effects.”.However, kinetochore labeling may not be adequate if interaction of thetest substance with protein synthesis is an issue.

The OECD Test guideline 487 MNvit was revised in 2014-15 in thecontext of an overall review of the OECD Test Guidelines on geno-toxicity and to reflect several years of experience with this test and theinterpretation of the data [172]. The revised version was adopted 29July 2016. The main remarks made in 2014-15 concerning the MNvitwere summarized by Thybaud and colleagues [175]. They essentiallyconfirm the experimental modalities described in the 2010 version and

and accuracy of molecular techniques, the cell-by-cell approach is nowopen to genomics, transcriptomics, proteomics, metabolomics and morerecently epigenomics and methylation pattern analyses, as well as high-content molecular cytogenetic analysis.

Third, as in any toxicity study, the doses matter, and this is espe-cially important for aneuploidy where there may be a very narrowwindow of effective doses both in somatic as well as in germinal cells.Consequences of positive results in any of the tissues should be definedin terms of classification according to international categories, i.e.,possible carcinogens/mutagens/germ cell mutagens. Even without adose-response, aneugens should be regarded as subjects to classifica-tion.

Our studies demonstrated that when the mechanism is well under-stood, a threshold often exists for the induction of aneuploidy. Thequestion remains, however, how to assess and calculate the acceptableexposure level. While the FISH method is adequate to detect non-dis-junction and chromosome loss/gain, improvements allowing thescoring of larger numbers of cells are needed and in progress. Thebenchmark dose was introduced to analyse quantitative dose-effectrelationships and to propose which increases above control levels are"acceptable"; however, current recommendations in genotoxicology arearbitrary (e.g., 10% increase over mean vehicle control) or based onlimited, usually 5–6, data points. This deserves further reflection andconsensus agreements.

When reflecting on the translational impact of our findings on an-euploidy induction and detection, it is important to stress protection ofhuman populations from an increase of genetic load in the progeny andfrom cancer. Our work further supported the utility and availability ofthe well-validated micronucleus assays both in vitro and in vivo, whichin combination with FISH technology allow accurate and sensitive as-sessment of aneuploidy induction in somatic cells. These tests wereinternationally validated and described in OECD guidelines. As far as invitro assays are concerned the necessity of using primary cells or, atleast, karyotypically stable cells should be considered. In vivo it mightbe more relevant to develop multistep carcinogenicity models to assessthe role of aneugenic events during early or later steps and in combi-nation with other (co-)carcinogens and epigenetic effects. These ques-tions are of key importance for cancer prevention and treatment, andthey require major research programmes. Considering the long timerequired to implement a scientifically valuable and validated assay inthe international regulatory landscape, it might be relevant to developas soon as possible an OECD document describing the best proceduresto assess specifically aneuploidy/polyploidy in vitro and in vivo in so-matic and germ cells.

The recent IWGT workgroup, held in 2017 in Japan, assessed therisk of aneugens for carcinogenesis and hereditary diseases andachieved a promising step. It applied the concept of Adverse OutcomePathways (AOPs) to link existing knowledge along the pathway ofcausally connected key events (KE) between two points: the MolecularInitiating Events (MIE) leading to aneuploidy and Adverse Outcomes(AOs). The overall objective is to support regulatory decision making,such as hazard identification and risk assessment, by formally identi-fying the key aneugenic events leading to cancer and hereditary dis-eases [177–179]. However, the major difficulty for hazard and riskrelated to exposure to chemical aneugens remains the choice of ade-quate tests and concentrations to assess the thresholds of effects.

Although the current OECD Test Guideline 487 for in vitro MN de-tection provides a protocol detecting both chromosome breakage andchromosome loss events, it can not be considered optimised for de-tecting the induction of chromosome loss. For designing aneuploidytests, one has to consider that exposures at the highest possible doseswill not maximize the effect. Even without a dose-response, aneugensshould be regarded as subject to classification as possible genotoxicants;there may be vast sensitivity differences between experimental animalsand humans so that dose extrapolations become meaningless.

While a wealth of data for genotoxicity is available in somatic cells,

germ-cell testing remains an important need [161]. The internationalconsensus is that somatic cell tests would likely also detect germ cellmutagens, but limitations of the database recommend caution indrawing conclusions. Germ cell mutagenicity testing would still beneeded for quantitative risk assessment.

Our fascination with the complex scientific questions surroundinganeuploidy has never declined. These include such varied aspects as thepeculiarities of mammalian oogenesis and spermatogenesis, sensitivewindows of exposures affecting the chromosomal constitution, thesignificance of genetic and epigenetic mutation in regulation of germcell formation and the health of offspring, and the molecular players inthe regulation of spindle formation, cell cycle regulation, gene expres-sion and chromosome segregation in oocytes. Questions on oocytequality, developmental competence, and susceptibility to disturbancesby aging, cryopreservation, altered gene expression and lifestyle orenvironmental exposures are still among the most relevant issues inhuman genetics, and they have important implications for medicaltreatments (e.g., assisted reproduction), environmental toxicology,human health of current and coming generations, and animal welfare.

As far as the tumorigenic capacity of each aneuploid karyotype isconcerned, one should realize that in contrast to constitutive aneu-ploidy, where all cells have the same karyotype and thus aneuploidytype, induction of aneuploidy in somatic cells is very heterogeneous.Depending on the specific chromosome imbalance, the presence ofproto-oncogenes or cancer suppressor genes, the functionality of an-euploidy-suppressive mechanisms and the level of the stress inducedwill determine the survival, expansion or death of each cell with aspecific karyotype. Some recent data suggest that high levels of aneu-ploidy might be tumour suppressive due to induction of cell death, incontrast to low levels of aneuploidy for which cells may be permissiveand therefore cancer-prone [202]. Although it is known that aneuploidyis associated with cancer development, uncertainties remain aboutwhether this occurs at early stages, at later stages, or both. In thiscontext, we still face a paradox: the majority of tumours show aneu-ploid karyotypes; aneuploidy level helps grading tumours; chemicalscan induce aneuploidy; but the role of aneugenic chemicals in tumor-igenesis is complex and remains poorly understood [7,28,29].

If we may express one regret, it is probably the lack of sufficientdialogue between the (geno-)toxicologists and the oncologists in thepast. The findings we collected on thresholds of effects for aneugensthat are very often used for chemotherapy could have been helpfulmuch earlier in promoting efficient and personalized therapy.

As far as germ cells are concerned, closer interaction of biologicalresearchers and medical practitioners is recommended in two aspects.First, modern methods developed in human genetics laboratories torecognize inherited syndromes can be applied to experimental animals.This facilitates the bridging between animals and humans. Second,experimental data will be brought to the attention of doctors who ad-vise their patients. One recommendation that gained broad interestamong physicians was the fact that in males the most sensitive stages toaneugens are the meiotic cell divisions. Thus, sexual abstinence for 3–6months after the end of chemical exposure (acute or chronic) will re-duce the risk of having children with an aneuploidy syndrome. We hopethat in the future greater inter-disciplinarity will solve these short-comings.

One area of research that has been neglected so far is the study ofeffects of exposure to multiple agents. This is of great importance forchemotherapeutic, nutritional, environmental and occupational che-micals because that represents the real human world. Aneugens canhave different targets affecting cell division fidelity whereby combi-nations of effects may lead to differences from additivity, which doesnot have anything to do with dose-response curves but with the mul-titude of possible targets and their potentially synergistic interactions.

inter-laboratory comparisons and leading to a solid validation ex-ercise.

- The selected laboratories had already acquired a recognized ex-pertise with some of the available assays.

- It was the right question at the right time. Technological develop-ments in molecular genetics made possible the isolation and label-ling with fluorescent dyes of sequence-specific DNA probes (pan-centromeric, pan-telomeric and chromosome-specific repetitive se-quences). Combining these tools with cytogenetics led to a powerfulcell-by-cell approach to chromosome identification and behaviourwith FISH.

- The accuracy of the refined methods allowed the evaluation ofprecise dose-effect relationships for clastogens versus aneugens bothin vitro and in vivo. Emerging from these studies were the concept ofindirect acting mutagens, the possibility of defining threshold dose-effects and the need for an adequate risk calculation if mechanismsof action are identified.

- The strengths of the collected data and the level of validation ofsome of the newly developed methods permitted the implementa-tion of the results at the regulatory level and resulted in the inclu-sion of aneuploidy induction as a compulsory check for the identi-fication of possible mutagens/carcinogens. One might wonderwhether translational efforts to include research findings into in-ternational regulation/guidelines is a responsibility for a scientist. Inour opinion the answer to this question is yes, in that the scientistsare best qualified to evaluate the quality and the interpretation ofthe data.

- Some of the methods developed and validated for the detection ofchemically induced aneuploidy, in particular FISH and PRINS, wereapplied by us and others to assess chromosomal changes in lym-phocytes of workers occupationally exposed to potential clastogens/aneugens (e.g., benzene) or to grade tumors on the basis of theirchromosome specific aneuploidy (e.g., breast, cervical and pan-creatic tumors) [198–200].

- The extra-European collaborations fostered by the EU-projects, eventhough, unfortunately, there were no means to support such non-European networking at the time.

Of course, our projects also had limitations, some of which were

later addressed and overcome, while some others point to still un-resolved gaps of knowledge. The research on mechanisms leading toaneuploidy could not yet rely on the potent molecular approaches thatbecame available later and often was limited to an understanding of themode of action of tested chemicals rather than to the unraveling themolecular pathways involved. Recently, studying these pathways hasbroadened the spectrum of causes and consequences of chromosomebreaks and chromosome mal-segregation, showing that these processes,initially envisioned as well separated from each other, are indeed oftenintertwined [29,201]. Still unresolved remain the questions of aneu-ploidy as a driving force in cancer development and the assessment ofthe impact of genetic predisposition, lifestyle, and occupational ortherapeutic exposures on the incidence of human germ-cell aneuploidy[7,28,29].

From our collaborative work on the cellular and molecular me-chanisms involved in aneuploidy induction, we would like to emphasizesome general thoughts that may be helpful in the future and in otherdisciplines.

First of all, when trying to progress in understanding, in casu geneticeffects, it is essential not to neglect the basic knowledge. For instance,remembering what chromosomes are when assessing effects of aneu-ploidy. When the consequences of an aneuploid karyotype are con-sidered, it is obvious that it corresponds to a complex sum of variousfactors including the levels and types of gene imbalance induced byspecific chromosome losses/gains. In addition, it will be dependent onthe species and the cell type (germ versus somatic cells) not only forchromosome number and gene content, but also for the structure of thespindle (with or without centrioles). Moreover, constitutive aneuploidy,present from the zygote stage in all somatic cells, might differ sub-stantially from chemically induced aneuploidy that impacts only somecells and/or species with different karyotypes.

Second, it is critical, when technically possible, to perform a cell-by-cell approach, complemented with molecular screening. Cytogeneticshad until the last decade the unique capacity to study cellular, mole-cular and genetic changes cell by cell, and to evaluate the viability ofthe cells studied. It was difficult, time consuming, and required spe-cialized skills. Too often multicellular extract studies were preferred,which produced interesting results, but did not take into account thecell as the biological unit. With the fascinating progress in sensitivity

Fig. 6. The authors during the final stage of preparation of this Reflections paper (sitting left to right: E.M. "Liz" Parry, Micheline Kirsch-Volders, Ilse-Dore Adler; backrow, left to right: Antonella Russo, Francesca Pacchierotti, Ursula Eichenlaub-Ritter).

[36] F.J. de Serres, Aneuploidy as a human health problem of significance in en-vironmental mutagenesis, Environ. Health Perspect. 31 (1979) 1–2.

[37] V.L. Dellarco, K.H. Mavournin, M.D. Waters, Aneuploidy data review committee:summary compilation of chemical data base and evaluation of test methodology,Mutat. Res. Genet. Toxicol. Environ. Mutagen. 167 (1986) 149–169, https://doi.org/10.1016/0165-1110(86)90015-1.

[38] V.L. Dellarco, P.E. Voytek, A. Hollaender (Eds.), Aneuploidy, Basic Life Sciences,Plenum Press, New York, NY, 1985.

[39] D. de Nettancourt, Preface, Mutat. Res. Mol. Mech. Mutagen. 61 (1979), https://doi.org/10.1016/0027-5107(79)90002-2 vii.

[40] M.A. Resnick, B.K. Vig (Eds.), Mechanism of Chromosome Distribution andAneuploidy, Alan R. Liss, New York, NY, 1989.

[41] P. Dustin, Microtubules, Springer Berlin Heidelberg, Berlin, Heidelberg, 1984,https://doi.org/10.1007/978-3-642-69652-7.

[42] Y. Moroi, C. Peebles, M.J. Fritzler, J. Steigerwald, E.M. Tan, Autoantibody tocentromere (kinetochore) in scleroderma sera, Proc. Natl. Acad. Sci. U. S. A. 77(1980) 1627–1631.

[43] T.A. Weinert, L.H. Hartwell, The RAD9 gene controls the cell cycle response toDNA damage in Saccharomyces cerevisiae, Science 241 (1988) 317–322.

[44] R. Li, A.W. Murray, Feedback control of mitosis in budding yeast, Cell 66 (1991)519–531.

[45] M.A. Hoyt, L. Totis, B.T. Roberts, S. cerevisiae genes required for cell cycle arrestin response to loss of microtubule function, Cell 66 (1991) 507–517.

[46] J.M. Parry, D. Sharp, E.M. Parry, Detection of mitotic and meiotic aneuploidy inthe yeast Saccharomyces cerevisiae, Environ. Health Perspect. 31 (1979) 97–111.

[47] E.M. Parry, D.C. Sharp, J.M. Parry, The observation of mitotic division aberrationsin mammalian cells exposed to chemical and radiation treatments, Mutat. Res. 150(1985) 369–381.

[48] I.-D. Adler, Mouse spermatogonia and spermatocyte sensitivity to chemical mu-tagens, Cytogenet. Genome Res. 33 (1982) 95–100, https://doi.org/10.1159/000131732.

[49] U. Kliesch, N. Danford, I.D. Adler, Micronucleus test and bone-marrow chromo-some analysis: a comparison of 2 methods in vivo for evaluating chemically in-duced chromosomal alterations, Mutat. Res. 80 (1981) 321–332.

[50] B.M. Miller, I.D. Adler, Suspect spindle poisons: analysis of c-mitotic effects inmouse bone marrow cells, Mutagenesis 4 (1989) 208–215.

[51] B.M. Miller, I.D. Adler, Application of antikinetochore antibody staining (CRESTstaining) to micronuclei in erythrocytes induced in vivo, Mutagenesis 5 (1990)411–415.

[52] B.M. Miller, I.D. Adler, Aneuploidy induction in mouse spermatocytes,Mutagenesis 7 (1992) 69–76.

[53] I.D. Adler, U. Kliesch, P. van Hummelen, M. Kirsch-Volders, Mouse micronucleustests with known and suspect spindle poisons: results from two laboratories,Mutagenesis 6 (1991) 47–53.

[54] I.D. Adler, Synopsis of the in vivo results obtained with the 10 known or suspectedaneugens tested in the CEC collaborative study, Mutat. Res. 287 (1993) 131–137.

[55] U. Eichenlaub-Ritter, J.B. Tucker, Microtubules with more than 13 protofilamentsin the dividing nuclei of ciliates, Nature 307 (1984) 60–62.

[56] U. Eichenlaub-Ritter, A.C. Chandley, R.G. Gosden, Alterations to the microtubularcytoskeleton and increased disorder of chromosome alignment in spontaneouslyovulated mouse oocytes aged in vivo: an immunofluorescence study, Chromosoma94 (1986) 337–345.

[57] U. Eichenlaub-Ritter, A.C. Chandley, R.G. Gosden, The CBA mouse as a model forage-related aneuploidy in man: studies of oocyte maturation, spindle formationand chromosome alignment during meiosis, Chromosoma 96 (1988) 220–226.

[58] U. Eichenlaub-Ritter, A. Stahl, J.M. Luciani, The microtubular cytoskeleton andchromosomes of unfertilized human oocytes aged in vitro, Hum. Genet. 80 (1988)259–264.

[59] U. Eichenlaub-Ritter, I. Boll, Age-related non-disjunction, spindle formation andprogression through maturation of mammalian oocytes, Prog. Clin. Biol. Res. 318(1989) 259–269.

[60] U. Eichenlaub-Ritter, H. Winking, Nondisjunction, disturbances in spindle struc-ture, and characteristics of chromosome alignment in maturing oocytes of miceheterozygous for Robertsonian translocations, Cytogenet. Cell Genet. 54 (1990)47–54, https://doi.org/10.1159/000132953.

[61] U. Eichenlaub-Ritter, Mechanisms of nondisjunction in mammalian meiosis, Curr.Top. Dev. Biol. 29 (1994) 281–324.

[62] U. Eichenlaub-Ritter, Parental age-related aneuploidy in human germ cells andoffspring: a story of past and present, Environ. Mol. Mutagen. 28 (1996) 211–236.

[63] A. Russo, F. Pacchierotti, P. Metalli, Meiotic non-disjunction induced by fissionneutrons relative to X-rays observed in mouse secondary spermatocytes I. Theresponse of different cell stages to a single radiation dose, Mutat. Res. Fundam.Mol. Mech. Mutagen. 108 (1983), https://doi.org/10.1016/0027-5107(83)90132-X.

[64] A. Russo, F. Pacchierotti, P. Metalli, Nondisjunction induced in mouse spermato-genesis by chloral hydrate, a metabolite of trichloroethylene, Environ. Mutagen. 6(1984), https://doi.org/10.1002/em.2860060507.

[65] R. Costa, A. Russo, M. Zordan, F. Pacchierotti, A. Tavella, A.G. Levis,Nitrilotriacetic acid (NTA) induces aneuploidy in drosophila and mouse germ-linecells, Environ. Mutagen. 12 (1988), https://doi.org/10.1002/em.2860120408.

[66] A. Russo, F. Pacchierotti, Meiotic arrest and aneuploidy induced by vinblastine inmouse oocytes, Mutat. Res. 202 (1988) 215–221, https://doi.org/10.1016/0027-5107(88)90185-6.

[67] A. Manca, B. Bassani, A. Russo, F. Pacchierotti, Origin of aneuploidy in relation todisturbances of cell-cycle progression. I. Effects of vinblastine on mouse bonemarrow cells, Mutat. Res. Fundam. Mol. Mech. Mutagen. 229 (1990), https://doi.

org/10.1016/0027-5107(90)90005-O.[68] P. Leopardi, A. Zijno, B. Bassani, F. Pacchierotti, In vivo studies on chemically

induced aneuploidy in mouse somatic and germinal cells, Mutat. Res. 287 (1993)119–130.

[69] A. Russo, A.G. Levis, The contribution of the mouse spermatid micronucleus assayto the detection of germinal mutagens, Prog. Clin. Biol. Res. 372 (1991) 513–520.

[70] A. Russo, A.G. Levis, Detection of aneuploidy in male germ cells of mice by meansof a meiotic micronucleus assay, Mutat. Res. Lett. 281 (1992), https://doi.org/10.1016/0165-7992(92)90007-5.

[71] F. Marchetti, C. Tiveron, B. Bassani, F. Pacchierotti, Griseofulvin-induced aneu-ploidy and meiotic delay in female mouse germ cells. II. Cytogenetic analysis ofone-cell zygotes, Mutat. Res. Fundam. Mol. Mech. Mutagen. 266 (1992) 151–162,https://doi.org/10.1016/0027-5107(92)90182-2.

[72] G. Hamoir, The discovery of meiosis by E. Van Beneden, a breakthrough in themorphological phase of heredity, Int. J. Dev. Biol. 36 (1992) 9–15, https://doi.org/10.1387/IJDB.1627480.

[73] M. De Brabander, G. Geuens, R. Nuydens, R. Willebrords, J. De Mey, Taxol inducesthe assembly of free microtubules in living cells and blocks the organizing capacityof the centrosomes and kinetochores, Proc. Natl. Acad. Sci. U. S. A. 78 (1981)5608–5612.

[74] A. Deleener, P. Castelain, V. Preat, J. de Gerlache, H. Alexandre, M. Kirsch-Volders, Changes in nucleolar transcriptional activity and nuclear DNA contentduring the first steps of rat hepatocarcinogenesis, Carcinogenesis 8 (1987)195–201.

[75] P. Castelain, A. Deleener, M. Kirsch-Volders, H. Barbason, Cell population kineticsand ploidy rate of early focal lesions during hepatocarcinogenesis in the rat, Br. J.Cancer 60 (1989) 827–833.

[76] F. Van Goethem, M.A. Ghahroudi, P. Castelain, M. Kirsch-Volders, Frequency andDNA content of micronuclei in rat parenchymal liver cells during experimentalhepatocarcinogenesis, Carcinogenesis 14 (1993) 2397–2406.

[77] S. Haesen, M. Timmermans, M. Kirsch-Volders, Induction of micronuclei andkaryotype aberrations during in vivo mouse skin carcinogenesis, Carcinogenesis14 (1993) 2319–2327.

[78] M. Kirsch-Volders, Differential staining of chromosomes and spindle cannot beused as an assay to determine the effect of cancer promoters on primary cultures ofhuman fibroblasts, Mutat. Res. 171 (1986) 177–183.

[79] M. Nijs, M. Kirsch-Volders, Induction of spindle inhibition and abnormal mitoticfigures by Cr(II), Cr(III) and Cr(VI) ions, Mutagenesis 1 (1986) 247–252.

[80] W.L. Wissinger, D.N. Estervig, R.J. Wang, A differential staining technique forsimultaneous visualization of mitotic spindle and chromosomes in mammaliancells, Stain Technol. 56 (1981) 221–226.

[81] L. Verschaeve, K. Vanderkerken, M. Kirsch-Volders, C-banding as a simple tool todiscriminate between micronuclei induced by clastogens and aneugens, StainTechnol. 63 (1988) 351–354.

[82] P. Van Hummelen, A. Deleener, P. Vanparys, M. Kirsch-Volders, Discrimination ofaneuploidogens from clastogens by C-banding, DNA and area measurements ofmicronuclei from mouse bone marrow, Mutat. Res. 271 (1992) 13–28.

[83] J.M. Parry, Studies upon the genetic effects of environmental chemicals: the co-ordinated research programme of the European Economic Community,Mutagenesis 3 (1988) 105–136.

[84] J.M. Parry, A. Sors, The detection and assessment of the aneugenic potential ofenvironmental chemicals: the European Community Aneuploidy Project, Mutat.Res. 287 (1993) 3–15.

[85] J.M. Parry, The analysis of the activity of aneugenic chemicals, from cultured cellsto rodent zygotes: an overview of the Protection of the European Population fromAneugenic Chemicals (PEPFAC project), Mutat. Res. 651 (2008) 1–2, https://doi.org/10.1016/j.mrgentox.2007.10.012.

[86] S. Ellard, E.M. Parry, J.M. Parry, Use of multicolour chromosome painting toidentify chromosomal rearrangements in human lymphocytes exposed to bleo-mycin: a comparison with conventional cytogenetic analysis of Giemsa-stainedchromosomes, Environ. Mol. Mutagen. 26 (1995) 44–54.

[87] J.M. Parry, E.M. Parry, R. Bourner, A. Doherty, S. Ellard, J. O’Donovan, B. Hoebee,J.M. de Stoppelaar, G.R. Mohn, A. Onfelt, A. Renglin, N. Schultz, C. Söderpalm-Berndes, K.G. Jensen, M. Kirsch-Volders, A. Elhajouji, P. Van Hummelen,F. Degrassi, A. Antoccia, D. Cimini, M. Izzo, C. Tanzarella, I.D. Adler, U. Kliesch,P. Hess, The detection and evaluation of aneugenic chemicals, Mutat. Res. 353(1996) 11–46.

[88] M. Fenech, A lifetime passion for micronucleus cytome assays—Reflections fromDown Under, Mutat. Res. 681 (2009) 111–117, https://doi.org/10.1016/j.mrrev.2008.11.003.

[89] A. Vanhauwaert, P. Vanparys, M. Kirsch-Volders, The in vivo gut micronucleus testdetects clastogens and aneugens given by gavage, Mutagenesis 16 (2001) 39–50.

[90] M. De Boeck, P. Hoet, N. Lombaert, B. Nemery, M. Kirsch-Volders, D. Lison, In vivogenotoxicity of hard metal dust: induction of micronuclei in rat type II epitheliallung cells, Carcinogenesis 24 (2003) 1793–1800, https://doi.org/10.1093/carcin/bgg146.

[91] J.D. Tucker, Reflections on the development and application of FISH wholechromosome painting, Mutat. Res. Rev. Mutat. Res. 763 (2015) 2–14, https://doi.org/10.1016/j.mrrev.2014.11.006.

[92] A. Russo, PRINS tandem labeling of satellite DNA in the study of chromosomedamage, Am. J. Med. Genet. 107 (2002) 99–104, https://doi.org/10.1002/ajmg.10102.

[93] D.A. Eastmond, J.D. Tucker, Kinetochore localization in micronucleated cytokin-esis-blocked Chinese hamster ovary cells: a new and rapid assay for identifyinganeuploidy-inducing agents, Mutat. Res. 224 (1989) 517–525.

[94] U. Eichenlaub-Ritter, E. Vogt, S. Cukurcam, F. Sun, F. Pacchierotti, J. Parry,

8. Conclusion

The past EU research efforts emerged in several laboratories fromthe fascination with mitotic and meiotic figures under the microscopeand the discovery of the complexity of the mitotic and meiotic divisionmachinery. It became clear that not only chemicals acting directly onDNA, but also chemicals that interfere with the multiple targets of themitotic/meiotic machinery may be mutagenic and pose a risk forhuman health. Their joined efforts under the leadership of Jim Parry,who was a stimulating coordinator and valued friend, allowed theelucidation of major modes of action of aneugens; the development ofscientifically sound assays to assess aneugens in different tissues, in-cluding germ cells and early embryos; and achieved the internationalvalidation of relevant assays up to their integration into OECD guide-lines, for which the final objective is the protection of the populationfrom aneugenic chemicals. The achieved international validation ofrelevant assays should allow the integration of aneuploidy testing intonational and international (e.g., OECD) mutagenicity test guidelines, inorder to enhance the objective of protecting the human population fromaneugenic chemicals.

Acknowledgements

These studies were supported by the European Commission grantingthe academic and financial freedom for us, our post-doctoral researchassociates, and our graduate research students to conduct these in-vestigations (Programme PRE-ENVPROT 3C, Contract EV3V0549;Programme FP1-ENVPROT 4C, Contracts EV4V0038 and EV4V0064;Programme FP2-STEP, Contract STEP0159; Programme FP3-ENV 1C,Contract EV5V0403; Programme FP4-ENV 2C, Contract ENV4970471;Programme FP5-LIFE QUALITY, Contract QLK4-CT-2000-00058). Wegratefully acknowledge the contributions of all our scientific andtechnical collaborators who made this research possible and successful.Most of their names are found in the references, however not all. Wehope they will find here our deep appreciation for their scientific input,constructive discussions, receptiveness to visiting and working in jointlaboratories, experimental expertise and collegial relationships. We alsoacknowledge colleagues from other European and non-European la-boratories for their openness in collaboration. Grateful thanks toGeorge Hoffmann for his interested support and wise editing in theproduction of this article. We also thank the three readers of themanuscript for their valuable and appreciated comments and sugges-tions.

References

[1] T. Boveri, Zur Frage der Entstehung maligner Tumoren, (1914).[2] T. Boveri, Concerning the Origin of Malignant Tumours by Theodor Boveri.

Translated and annotated by Henry Harris, J. Cell. Sci. 121 (2008) 1–84, https://doi.org/10.1242/jcs.025742.

[3] S. Santaguida, A. Amon, Short- and long-term effects of chromosome mis-segre-gation and aneuploidy, Nat. Rev. Mol. Cell Biol. 16 (2015) 473–485, https://doi.org/10.1038/nrm4025.

[4] R.M. Naylor, J.M. van Deursen, Aneuploidy in cancer and aging, Annu. Rev.Genet. 50 (2016) 45–66, https://doi.org/10.1146/annurev-genet-120215-035303.

[5] A. Capalbo, E.R. Hoffmann, D. Cimadomo, F. Maria Ubaldi, L. Rienzi,Human female meiosis revised: new insights into the mechanisms of chromosomesegregation and aneuploidies from advanced genomics and time-lapse imaging,Hum. Reprod. Update 23 (2017) 706–722, https://doi.org/10.1093/humupd/dmx026.

[6] A. Destouni, J.R. Vermeesch, How can zygotes segregate entire parental genomesinto distinct blastomeres? The zygote metaphase revisited, Bioessays 39 (2017),https://doi.org/10.1002/bies.201600226.

[7] L. Sansregret, C. Swanton, The role of aneuploidy in cancer evolution, Cold SpringHarb. Perspect. Med. 7 (2017) a028373, , https://doi.org/10.1101/cshperspect.a028373.

[8] D. Duelli, Y. Lazebnik, Cell-to-cell fusion as a link between viruses and cancer, Nat.Rev. Cancer 7 (2007) 968–976, https://doi.org/10.1038/nrc2272.

[9] T. Davoli, T. de Lange, The causes and consequences of polyploidy in normaldevelopment and cancer, Annu. Rev. Cell Dev. Biol. 27 (2011) 585–610, https://

doi.org/10.1146/annurev-cellbio-092910-154234.[10] T.L. Orr-Weaver, When bigger is better: the role of polyploidy in organogenesis,

Trends Genet. 31 (2015) 307–315, https://doi.org/10.1016/j.tig.2015.03.011.[11] Q. He, B. Au, M. Kulkarni, Y. Shen, K.J. Lim, J. Maimaiti, C.K. Wong,

M.N.H. Luijten, H.C. Chong, E.H. Lim, G. Rancati, I. Sinha, Z. Fu, X. Wang,J.E. Connolly, K.C. Crasta, Chromosomal instability-induced senescence potenti-ates cell non-autonomous tumourigenic effects, Oncogenesis 7 (2018) 62, https://doi.org/10.1038/s41389-018-0072-4.

[12] G.A. Andriani, V.P. Almeida, F. Faggioli, M. Mauro, W.L. Tsai, L. Santambrogio,A. Maslov, M. Gadina, J. Campisi, J. Vijg, C. Montagna, Whole chromosome in-stability induces senescence and promotes SASP, Sci. Rep. 6 (2016) 35218.

[13] Q. Shi, R.W. King, Chromosome nondisjunction yields tetraploid rather than an-euploid cells in human cell lines, Nature 437 (2005) 1038–1042, https://doi.org/10.1038/nature03958.

[14] M. Kirsch-Volders, A. Vanhauwaert, M. De Boeck, I. Decordier, Importance ofdetecting numerical versus structural chromosome aberrations, Mutat. Res. Mol.Mech. Mutagen. 504 (2002) 137–148, https://doi.org/10.1016/S0027-5107(02)00087-8.

[15] S. Chong, N. Vickaryous, A. Ashe, N. Zamudio, N. Youngson, S. Hemley, T. Stopka,A. Skoultchi, J. Matthews, H.S. Scott, D. de Kretser, M. O’Bryan, M. Blewitt,E. Whitelaw, Modifiers of epigenetic reprogramming show paternal effects in themouse, Nat. Genet. 39 (2007) 614–622, https://doi.org/10.1038/ng2031.

[16] A.H. Yona, Y.S. Manor, R.H. Herbst, G.H. Romano, A. Mitchell, M. Kupiec,Y. Pilpel, O. Dahan, Chromosomal duplication is a transient evolutionary solutionto stress, Proc. Natl. Acad. Sci. 109 (2012) 21010–21015, https://doi.org/10.1073/pnas.1211150109.

[17] A.C. Gerstein, J. Berman, Shift and adapt: the costs and benefits of karyotypevariations, Curr. Opin. Microbiol. 26 (2015) 130–136, https://doi.org/10.1016/j.mib.2015.06.010.

[18] A. Soler, C. Morales, I. Mademont-Soler, E. Margarit, A. Borrell, V. Borobio,M. Muñoz, A. Sánchez, Overview of chromosome abnormalities in first trimestermiscarriages: a series of 1,011 consecutive chorionic villi sample karyotypes,Cytogenet. Genome Res. 152 (2017) 81–89, https://doi.org/10.1159/000477707.

[19] I.A. Uchida, V.C. Freeman, Triploidy and chromosomes, Am. J. Obstet. Gynecol.151 (1985) 65–69.

[20] A. Baumer, D. Balmer, F. Binkert, A. Schinzel, Parental origin and mechanisms offormation of triploidy: a study of 25 cases, Eur. J. Hum. Genet. 8 (2000) 911–917,https://doi.org/10.1038/sj.ejhg.5200572.

[21] M.V. Zaragoza, U. Surti, R.W. Redline, E. Millie, A. Chakravarti, T.J. Hassold,Parental origin and phenotype of triploidy in spontaneous abortions: pre-dominance of diandry and association with the partial hydatidiform mole, Am. J.Hum. Genet. 66 (2000) 1807–1820, https://doi.org/10.1086/302951.

[22] S.I. Nagaoka, T.J. Hassold, Pa. Hunt, Human aneuploidy: mechanisms and newinsights into an age-old problem, Nat. Rev. Genet. 13 (2012) 493–504, https://doi.org/10.1038/nrg3245.

[23] A. Webster, M. Schuh, Mechanisms of aneuploidy in human eggs, Trends Cell Biol.27 (2017) 55–68, https://doi.org/10.1016/j.tcb.2016.09.002.

[24] J. Greaney, Z. Wei, H. Homer, Regulation of chromosome segregation in oocytesand the cellular basis for female meiotic errors, Hum. Reprod. Update 24 (2018)135–161, https://doi.org/10.1093/humupd/dmx035.

[25] D.K. Griffin, C. Ogur, Chromosomal analysis in IVF: just how useful is it?Reproduction 156 (2018) F29–F50, https://doi.org/10.1530/REP-17-0683.

[26] A. Capalbo, E.R. Hoffmann, D. Cimadomo, F. Maria Ubaldi, L. Rienzi, Humanfemale meiosis revised: new insights into the mechanisms of chromosome segre-gation and aneuploidies from advanced genomics and time-lapse imaging, Hum.Reprod. Update 23 (2017) 706–722, https://doi.org/10.1093/humupd/dmx026.

[27] L.M. Zasadil, E.M.C. Britigan, Ba. Weaver, 2n or not 2n: aneuploidy, polyploidyand chromosomal instability in primary and tumor cells, Semin. Cell Dev. Biol. 24(2013) 370–379, https://doi.org/10.1016/j.semcdb.2013.02.001.

[28] L. Sansregret, B. Vanhaesebroeck, C. Swanton, Determinants and clinical im-plications of chromosomal instability in cancer, Nat. Rev. Clin. Oncol. 15 (2018)139–150, https://doi.org/10.1038/nrclinonc.2017.198.

[29] M.S. Levine, A.J. Holland, The impact of mitotic errors on cell proliferation andtumorigenesis, Genes Dev. 32 (2018) 620–638, https://doi.org/10.1101/gad.314351.118.

[30] S.E. McClelland, Role of chromosomal instability in cancer progression, Endocr.Relat. Cancer 24 (2017) T23–T31, https://doi.org/10.1530/ERC-17-0187.

[31] P. Ly, D.W. Cleveland, Rebuilding chromosomes after catastrophe: emerging me-chanisms of chromothripsis, Trends Cell Biol. 27 (2017) 917–930, https://doi.org/10.1016/j.tcb.2017.08.005.

[32] A. Russo, F. Degrassi, Molecular cytogenetics of the micronucleus: still surprising,Mutat. Res. Toxicol. Environ. Mutagen. 836 (2018) 36–40, https://doi.org/10.1016/j.mrgentox.2018.05.011.

[33] H.E. Danielsen, T.S. Hveem, E. Domingo, M. Pradhan, A. Kleppe, R.A. Syvertsen,I. Kostolomov, J.A. Nesheim, H.A. Askautrud, A. Nesbakken, R.A. Lothe,A. Svindland, N. Shepherd, M. Novelli, E. Johnstone, I. Tomlinson, R. Kerr,D.J. Kerr, Prognostic markers for colorectal cancer: estimating ploidy and stroma,Ann. Oncol. 29 (3) (2018) 616–623, https://doi.org/10.1093/annonc/mdx794.

[34] T. Potapova, G. Gorbsky, The consequences of chromosome segregation errors inmitosis and meiosis, Biology (Basel) 6 (2017) 12, https://doi.org/10.3390/biology6010012.

[35] M.R. Cosenza, A. Cazzola, A. Rossberg, N.L. Schieber, G. Konotop, E. Bausch,A. Slynko, T. Holland-Letz, M.S. Raab, T. Dubash, H. Glimm, S. Poppelreuther,C. Herold-Mende, Y. Schwab, A. Krämer, Asymmetric centriole numbers at spindlepoles cause chromosome missegregation in cancer, Cell Rep. 20 (2017)1906–1920, https://doi.org/10.1016/j.celrep.2017.08.005.

Mutagens and Carcinogens. Genotoxicity under extreme culture conditions. Areport from ICPEMC task group 9, Mutat. Res. 257 (1991) 147–205.

[143] R. Langenbach, P.B. Smith, C. Crespi, Recombinant DNA approaches for the de-velopment of metabolic systems used in in vitro toxicology, Mutat. Res. 277(1992) 251–275.

[144] S. Ellard, Y. Mohammed, S. Dogra, C. Wölfel, J. Doehmer, J.M. Parry, The use ofgenetically engineered V79 Chinese hamster cultures expressing rat liver CYP1A1,1A2 and 2B1 cDNAs in micronucleus assays, Mutagenesis 6 (1991) 461–470.

[145] C. Crofton-Sleigh, A. Doherty, S. Ellard, E.M. Parry, S. Venitt, Micronucleus assaysusing cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressingfive human cytochromes P-450 and microsomal epoxide hydrolase, Mutagenesis 8(1993) 363–372.

[146] A.T. Doherty, S. Ellard, E.M. Parry, J.M. Parry, An investigation into the activationand deactivation of chlorinated hydrocarbons to genotoxins in metabolicallycompetent human cells, Mutagenesis 11 (1996) 247–274.

[147] C.L. Crespi, F.J. Gonzalez, D.T. Steimel, T.R. Turner, H.V. Gelboin, B.W. Penman,R. Langenbach, A metabolically competent human cell line expressing five cDNAsencoding procarcinogen-activating enzymes: application to mutagenicity testing,Chem. Res. Toxicol. 4 (1991) 566–572.

[148] A.T. Doherty, S. Ellard, E.M. Parry, J.M. Parry, A study of the aneugenic activity oftrichlorfon detected by centromere-specific probes in human lymphoblastoid celllines, Mutat. Res. 372 (1996) 221–231.

[149] M. Kirsch-Volders, M. Aardema, A. Elhajouji, Concepts of threshold in mutagenesisand carcinogenesis, Mutat. Res. 464 (2000) 3–11.

[150] B.M. Miller, H.F. Zitzelsberger, H.U. Weier, I.D. Adler, Classification of micro-nuclei in murine erythrocytes: immunofluorescent staining using CREST anti-bodies compared to in situ hybridization with biotinylated gamma satellite DNA,Mutagenesis 6 (1991) 297–302.

[151] L. Renzi, F. Pacchierotti, A. Russo, The centromere as a target for the induction ofchromosome damage in resting and proliferating mammalian cells: assessment ofmitomycin C-induced genetic damage at kinetochores and centromeres by a mi-cronucleus test in mouse splenocytes, Mutagenesis 11 (1996) 133–138, https://doi.org/10.1093/mutage/11.2.133.

[152] G. Schriever-Schwemmer, I.D. Adler, Differentiation of micronuclei in mouse bonemarrow cells: a comparison between CREST staining and fluorescent in situ hy-bridization with centromeric and telomeric DNA probes, Mutagenesis 9 (1994)333–340.

[153] A. Elhajouji, M. Lukamowicz, Z. Cammerer, M. Kirsch-Volders, Potential thresh-olds for genotoxic effects by micronucleus scoring, Mutagenesis 26 (2011)199–204, https://doi.org/10.1093/mutage/geq089.

[154] G.E. Johnson, S.H. Doak, S.M. Griffiths, E.L. Quick, D.O.F. Skibinski, Z.M. Zaïr,G.J. Jenkins, Non-linear dose-response of DNA-reactive genotoxins: re-commendations for data analysis, Mutat. Res. 678 (2009) 95–100, https://doi.org/10.1016/j.mrgentox.2009.05.009.

[155] Z. Cammerer, M.M. Schumacher, M. Kirsch-Volders, W. Suter, A. Elhajouji, Flowcytometry peripheral blood micronucleus test in vivo: determination of potentialthresholds for aneuploidy induced by spindle poisons, Environ. Mol. Mutagen. 51(2010) 278–284, https://doi.org/10.1002/em.20542.

[156] I. Decordier, E. Cundari, M. Kirsch-Volders, Survival of aneuploid, micronucleatedand/or polyploid cells: crosstalk between ploidy control and apoptosis, Mutat. Res.651 (2008) 30–39, https://doi.org/10.1016/j.mrgentox.2007.10.016.

[157] I. Decordier, E. Cundari, M. Kirsch-Volders, Mitotic checkpoints and the main-tenance of the chromosome karyotype, Mutat. Res. 651 (2008) 3–13, https://doi.org/10.1016/j.mrgentox.2007.10.020.

[158] S. Frias, P. Van Hummelen, M.L. Meistrich, X.R. Lowe, F.B. Hagemeister,M.D. Shelby, J.B. Bishop, A.J. Wyrobek, NOVP chemotherapy for Hodgkin’s dis-ease transiently induces sperm aneuploidies associated with the major clinicalaneuploidy syndromes involving chromosomes X, Y, 18, and 21, Cancer Res. 63(2003) 44–51.

[159] H.G. Tempest, E. Ko, P. Chan, B. Robaire, A. Rademaker, R.H. Martin, Spermaneuploidy frequencies analysed before and after chemotherapy in testicularcancer and Hodgkin’s lymphoma patients, Hum. Reprod. 23 (2008) 251–258,https://doi.org/10.1093/humrep/dem389.

[160] W. van Dorp, R. Haupt, R.A. Anderson, R.L. Mulder, M.M. van den Heuvel-Eibrink,E. van Dulmen-den Broeder, H.I. Su, J. Falck Winther, M.M. Hudson, J.M. Levine,W.H. Wallace, Reproductive function and outcomes in female survivors of child-hood, adolescent, and young adult cancer: a review, J. Clin. Oncol. 36 (2018)2169–2180, https://doi.org/10.1200/JCO.2017.76.3441.

[161] C.L. Yauk, M.J. Aardema, J. Van Benthem, J.B. Bishop, K.L. Dearfield,D.M. DeMarini, Y.E. Dubrova, M. Honma, J.R. Lupski, F. Marchetti,M.L. Meistrich, F. Pacchierotti, J. Stewart, M.D. Waters, G.R. Douglas, Approachesfor identifying germ cell mutagens: report of the 2013 IWGT workshop on germcell assays, Mutat. Res. Toxicol. Environ. Mutagen. 783 (2015) 36–54, https://doi.org/10.1016/j.mrgentox.2015.01.008.

[162] F.J. de Serres, J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens.Report of the International Collaborative Program. Progress in Mutation Research,vol. 1, Elsevier Science, Amsterdam, 1981.

[163] J. Ashby, J.M. Parry, C.F. Arlett, M.M. Coombs, The UKEMS collaborative geno-toxicity trial organisation, selection criteria and preparation of the chemicals,Mutat. Res. 100 (1982) 1–6.

[164] D.J. Kirkland, The role of the UKEMS in the development of testing guidelines,Mutagenesis 17 (2002) 451–455.

[165] DHSS, Guidelines for the Testing of Chemicals for Mutagenicity, Report on Healthand Social Subjects no. 24, HM Stationery Offic, London, UK, 1981.

[166] DOH Department of Health, Guidance for the Testing of Chemicals forMutagenicity, Committee on Mutagenicity of Chemicals in Food, Consumer

Products and the Environment., HM Stationery Office, London, UK, 2000.[167] M. Kirsch-Volders, T. Sofuni, M. Aardema, S. Albertini, D. Eastmond, M. Fenech,

M. Ishidate, E. Lorge, H. Norppa, J. Surrallés, W. von der Hude, A. Wakata, Reportfrom the in vitro micronucleus assay working group, Environ. Mol. Mutagen. 35(2000) 167–172.

[168] M. Kirsch-Volders, T. Sofuni, M. Aardema, S. Albertini, D. Eastmond, M. Fenech,M. Ishidate, S. Kirchner, E. Lorge, T. Morita, H. Norppa, J. Surrallés,A. Vanhauwaert, A. Wakata, Report from the in vitro micronucleus assay workinggroup, Mutat. Res. Toxicol. Environ. Mutagen. 540 (2003) 153–163, https://doi.org/10.1016/j.mrgentox.2003.07.005.

[169] W. von der Hude, S. Kalweit, G. Engelhardt, S. McKiernan, P. Kasper, R. Slacik-Erben, H.G. Miltenburger, N. Honarvar, R. Fahrig, B. Görlitz, S. Albertini,S. Kirchner, D. Utesch, F. Pötter-Locher, H. Stopper, S. Madle, In vitro micro-nucleus assay with Chinese hamster V79 cells - results of a collaborative studywith in situ exposure to 26 chemical substances, Mutat. Res. 468 (2000) 137–163.

[170] E. Lorge, V. Thybaud, M.J. Aardema, J. Oliver, A. Wakata, G. Lorenzon, D. Marzin,SFTG international collaborative study on in vitro micronucleus test I. Generalconditions and overall conclusions of the study, Mutat. Res. 607 (2006) 13–36,https://doi.org/10.1016/j.mrgentox.2006.04.006.

[171] R. Corvi, S. Albertini, T. Hartung, S. Hoffmann, D. Maurici, S. Pfuhler, J. vanBenthem, P. Vanparys, ECVAM retrospective validation of in vitro micronucleustest (MNT), Mutagenesis 23 (2008) 271–283, https://doi.org/10.1093/mutage/gen010.

[172] OECD, Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECDPublishing, Paris, 2014, https://doi.org/10.1787/9789264224438-en.

[173] M. Fenech, M. Kirsch-Volders, A.T. Natarajan, J. Surralles, J.W. Crott, J. Parry,H. Norppa, D.A. Eastmond, J.D. Tucker, P. Thomas, Molecular mechanisms ofmicronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian andhuman cells, Mutagenesis 26 (2011) 125–132, https://doi.org/10.1093/mutage/geq052.

[174] M. Fenech, N. Holland, E. Zeiger, W.P. Chang, S. Burgaz, P. Thomas, C. Bolognesi,S. Knasmueller, M. Kirsch-Volders, S. Bonassi, The HUMN and HUMNxL interna-tional collaboration projects on human micronucleus assays in lymphocytes andbuccal cells–past, present and future, Mutagenesis 26 (2011) 239–245, https://doi.org/10.1093/mutage/geq051.

[175] V. Thybaud, E. Lorge, D.D. Levy, J. van Benthem, G.R. Douglas, F. Marchetti,M.M. Moore, R. Schoeny, Main issues addressed in the 2014-2015 revisions to theOECD Genetic Toxicology Test Guidelines, Environ. Mol. Mutagen. 58 (2017)284–295, https://doi.org/10.1002/em.22079.

[176] Y. Sakuratani, M. Horie, E. Leinala, Integrated approaches to testing and assess-ment: OECD activities on the development and use of adverse outcome pathwaysand case studies, Basic Clin. Pharmacol. Toxicol. (2018), https://doi.org/10.1111/bcpt.12955.

[177] A. Lynch, et al., Targets and Mechanisms of Chemically Induced Aneuploidy. Part Iof the Report of the 2017 IWGT Workgroup on Assessing the Risk of Aneugens forCarcinogenesis and Hereditary Diseases, (2018) Submitted.

[178] F. Pacchierotti, et al., Aneuploidy in Germ Cells. Part II of the Report of the 2017IWGT Workgroup on Assessing the Risk of Aneugens for Carcinogenesis andHereditary Diseases, (2018) Submitted.

[179] D. Tweats, et al., Role of Aneuploidy in the Carcinogenic Process. Part III of theReport of the 2017 IWGT Workgroup on Assessing the Risk of Aneugens forCarcinogenesis and Hereditary Diseases, (2018) Submitted.

[180] D.R. Larson, T. Misteli, The genome—seeing it clearly now, Science 357 (2017)354–355, https://doi.org/10.1126/science.aao1893.

[181] H.D. Ou, S. Phan, T.J. Deerinck, A. Thor, M.H. Ellisman, C.C. O’Shea, ChromEMT:visualizing 3D chromatin structure and compaction in interphase and mitotic cells,Science 357 (2017), https://doi.org/10.1126/science.aag0025 eaag0025.

[182] G.J. Gorbsky, The spindle checkpoint and chromosome segregation in meiosis,FEBS J. 282 (2015) 2471–2487, https://doi.org/10.1111/febs.13166.

[183] A.J. Holland, D.W. Cleveland, Losing balance: the origin and impact of aneuploidyin cancer, EMBO Rep. 13 (2012) 501–514, https://doi.org/10.1038/embor.2012.55.

[184] I. Vitale, L. Galluzzi, M. Castedo, G. Kroemer, Mitotic catastrophe: a mechanismfor avoiding genomic instability, Nat. Rev. Mol. Cell Biol. 12 (2011) 385–392,https://doi.org/10.1038/nrm3115.

[185] B.G. Lambrus, A.J. Holland, A new mode of mitotic surveillance, Trends Cell Biol.27 (2017) 314–321, https://doi.org/10.1016/j.tcb.2017.01.004.

[186] K. Crasta, N.J. Ganem, R. Dagher, A.B. Lantermann, E.V. Ivanova, Y. Pan, L. Nezi,A. Protopopov, D. Chowdhury, D. Pellman, DNA breaks and chromosome pul-verization from errors in mitosis, Nature 482 (2012) 53–58, https://doi.org/10.1038/nature10802.

[187] K.J. MacKenzie, P. Carroll, C.A. Martin, O. Murina, A. Fluteau, D.J. Simpson,N. Olova, H. Sutcliffe, J.K. Rainger, A. Leitch, R.T. Osborn, A.P. Wheeler,M. Nowotny, N. Gilbert, T. Chandra, M.A.M. Reijns, A.P. Jackson, CGAS surveil-lance of micronuclei links genome instability to innate immunity, Nature 548(2017) 461–465, https://doi.org/10.1038/nature23449.

[188] M. Terradas, M. Martín, A. Genescà, Impaired nuclear functions in micronucleiresults in genome instability and chromothripsis, Arch. Toxicol. 90 (2016)2657–2667, https://doi.org/10.1007/s00204-016-1818-4.

[189] F. Pacchierotti, U. Eichenlaub-Ritter, Environmental hazard in the aetiology ofsomatic and germ cell aneuploidy, Cytogenet. Genome Res. 133 (2011) 254–268,https://doi.org/10.1159/000323284.

[190] J. Reichmann, B. Nijmeijer, M.J. Hossain, M. Eguren, I. Schneider, A.Z. Politi,M.J. Roberti, L. Hufnagel, T. Hiiragi, J. Ellenberg, Dual-spindle formation in zy-gotes keeps parental genomes apart in early mammalian embryos, Science 361(2018) 189–193, https://doi.org/10.1126/science.aar7462.

Exposure of mouse oocytes to bisphenol A causes meiotic arrest but not aneu-ploidy, Mutat. Res. 651 (2008) 82–92, https://doi.org/10.1016/j.mrgentox.2007.10.014.

[95] G.E. Johnson, E.M. Parry, Mechanistic investigations of low dose exposures to thegenotoxic compounds bisphenol-A and rotenone, Mutat. Res. Toxicol. Environ.Mutagen. 651 (2008) 56–63, https://doi.org/10.1016/j.mrgentox.2007.10.019.

[96] A. Elhajouji, P. Van Hummelen, M. Kirsch-Volders, Indications for a threshold ofchemically-induced aneuploidy in vitro in human lymphocytes, Environ. Mol.Mutagen. 26 (1995) 292–304.

[97] A. Zijno, F. Marcon, P. Leopardi, R. Crebelli, Analysis of chromosome segregationin cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent da-mage resulting from low dose exposure to spindle poisons, Mutagenesis 11 (1996)335–340.

[98] E.M. Parry, J.M. Parry, C. Corso, A. Doherty, F. Haddad, T.F. Hermine, G. Johnson,M. Kayani, E. Quick, T. Warr, J. Williamson, Detection and characterization ofmechanisms of action of aneugenic chemicals, Mutagenesis 17 (2002) 509–521.

[99] A. Elhajouji, F. Tibaldi, M. Kirsch-Volders, Indication for thresholds of chromo-some non-disjunction versus chromosome lagging induced by spindle inhibitors invitro in human lymphocytes, Mutagenesis 12 (1997) 133–140.

[100] R.R. Marshall, M. Murphy, D.J. Kirkland, K.S. Bentley, Fluorescence in situ hy-bridisation with chromosome-specific centromeric probes: a sensitive method todetect aneuploidy, Mutat. Res. 372 (1996) 233–245.

[101] A.D. Tates, A.J.J. Dietrich, N. de Vogel, I. Neuteboom, A. Bos, A micronucleusmethod for detection of meiotic micronuclei in male germ cells of mammals,Mutat. Res. Lett. 121 (1983) 131–138, https://doi.org/10.1016/0165-7992(83)90111-2.

[102] J. Lähdetie, M. Parvinen, Mieotic micronuclei induced by X-rays in early sper-matids of the rat, Mutat. Res. Mol. Mech. Mutagen. 81 (1981) 103–115, https://doi.org/10.1016/0027-5107(81)90091-9.

[103] B.W. Collins, D.R. Howard, J.W. Allen, Kinetochore-staining of spermatid micro-nuclei: studies of mice treated with X-radiation or acrylamide, Mutat. Res. Lett.281 (1992) 287–294, https://doi.org/10.1016/0165-7992(92)90023-B.

[104] M. Kallio, T. Sjoblom, J. Lahdetie, Effects of vinblastine and colchicine on male ratmeiosis in vivo: disturbances in spindle dynamics causing micronuclei and meta-phase arrest, Environ. Mol. Mutagen. 25 (1995) 106–117.

[105] M. Kallio, J. Lahdetie, Analysis of micronuclei induced in mouse early spermatidsby mitomycin C, vinblastine sulfate or etoposide using fluorescence in situ hy-bridization, Mutagenesis 8 (1993) 561–567.

[106] A.M.M. Tommasi, S. De Conti, M.M.M. Dobrzyńska, A. Russo, Evaluation andcharacterization of micronuclei in early spermatids of mice exposed to 1,3-buta-diene, Mutat. Res. Fundam. Mol. Mech. Mutagen. 397 (1998) 45–54, https://doi.org/10.1016/S0027-5107(97)00194-2.

[107] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, I.-D. Adler, D.H. Blakey,S.D. Dertinger, G. Krishna, T. Morita, A. Russo, S. Sutou, In vivo rodent ery-throcyte micronucleus assay. II. Some aspects of protocol design including re-peated treatments, integration with toxicity testing, and automated scoring,Environ. Mol. Mutagen. 35 (2000), https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<234::AID-EM10>3.0.CO;2-L.

[108] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, D.H. Blakey, S.D. Dertinger,L. Abramsson-Zetterberg, G. Krishna, T. Morita, A. Russo, N. Asano, H. Suzuki,W. Ohyama, D. Gibson, In vivo erythrocyte micronucleus assay. III. Validation andregulatory acceptance of automated scoring and the use of rat peripheral bloodreticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 627 (2007), https://doi.org/10.1016/j.mrgentox.2006.08.010.

[109] I.-D. Adler, D. Anderson, R. Benigni, U.-H.H. Ehling, J. Laehdetie, F. Pacchierotti,A. Russo, A.D.D. Tates, Synthesis report of the step project detection of germ cellmutagens, Mutat. Res. Fundam. Mol. Mech. Mutagen. 353 (1996) 65–84, https://doi.org/10.1016/0027-5107(95)00240-5.

[110] F. Pacchierotti, I.-D. Adler, D. Anderson, M. Brinkworth, N.A. Demopoulos,J. Lähdetie, S. Osterman-Golkar, K. Peltonen, A. Russo, A. Tates, R. Waters,Genetic effects of 1,3-butadiene and associated risk for heritable damage, Mutat.Res. Fundam. Mol. Mech. Mutagen. 397 (1998), https://doi.org/10.1016/S0027-5107(97)00199-1.

[111] P. Van Hummelen, X.R. Lowe, A.J. Wyrobek, Simultaneous detection of structuraland numerical chromosome abnormalities in sperm of healthy men by multicolorfluorescence in situ hybridization, Hum. Genet. 98 (1996) 608–615.

[112] R.H. Martin, E. Ko, K. Chan, Defection of aneuploidy in human interphase sper-matozoa by fluorescence in situ hybridization (FISH), Cytogenet. Genome Res. 64(1993) 23–26, https://doi.org/10.1159/000133552.

[113] J.M. Holmes, R.H. Martin, Aneuploidy detection in human sperm nuclei usingfluorescence in situ hybridization, Hum. Genet. 91 (1993) 20–24.

[114] A.J. Wyrobek, I.D. Adler, Detection of aneuploidy in human and rodent spermusing FISH and applications of sperm assays of genetic damage in heritable riskevaluation, Mutat. Res. 352 (1996) 173–179, https://doi.org/10.1016/0027-5107(95)00249-9.

[115] A. Baumgartner, T.E. Schmid, C.G. Schuetz, I.D. Adler, Detection of aneuploidy inrodent and human sperm by multicolor FISH after chronic exposure to diazepam,Mutat. Res. 490 (2001) 11–19.

[116] I.D. Adler, T.E. Schmid, A. Baumgartner, Induction of aneuploidy in male mousegerm cells detected by the sperm-FISH assay: a review of the present data base,Mutat. Res. Fundam. Mol. Mech. Mutagen. 504 (2002) 173–182, https://doi.org/10.1016/S0027-5107(02)00090-8.

[117] A.J. Wyrobek, T.E. Schmid, F. Marchetti, Cross-species sperm-FISH assays forchemical testing and assessing paternal risk for chromosomally abnormal preg-nancies, Environ. Mol. Mutagen. 45 (2005) 271–283, https://doi.org/10.1002/

em.20121.[118] H. Yin, E. Baart, I. Betzendahl, U. Eichenlaub-Ritter, Diazepam induces meiotic

delay, aneuploidy and predivision of homologues and chromatids in mammalianoocytes, Mutagenesis 13 (1998) 567–580.

[119] Y. Shen, I. Betzendahl, F. Sun, H.R. Tinnenberg, U. Eichenlaub-Ritter, Non-in-vasive method to assess genotoxicity of nocodazole interfering with spindle for-mation in mammalian oocytes, Reprod. Toxicol. 19 (2005) 459–471, https://doi.org/10.1016/j.reprotox.2004.09.007.

[120] R. Ranaldi, G. Gambuti, U. Eichenlaub-Ritter, F. Pacchierotti, Trichlorfon effectson mouse oocytes following in vivo exposure, Mutat. Res. 651 (2008) 125–130,https://doi.org/10.1016/j.mrgentox.2007.10.010.

[121] F. Sun, I. Betzendahl, K. Van Wemmel, R. Cortvrindt, J. Smitz, F. Pacchierotti,U. Eichenlaub-Ritter, Trichlorfon-induced polyploidy and nondisjunction in mouseoocytes from preantral follicle culture, Mutat. Res. 651 (2008) 114–124, https://doi.org/10.1016/j.mrgentox.2007.10.008.

[122] F. Sun, I. Betzendahl, F. Pacchierotti, R. Ranaldi, J. Smitz, R. Cortvrindt,U. Eichenlaub-Ritter, Aneuploidy in mouse metaphase II oocytes exposed in vivoand in vitro in preantral follicle culture to nocodazole, Mutagenesis 20 (2005)65–75, https://doi.org/10.1093/mutage/gei010.

[123] F. Pacchierotti, R. Ranaldi, U. Eichenlaub-Ritter, S. Attia, I.-D. Adler, Evaluation ofaneugenic effects of bisphenol A in somatic and germ cells of the mouse, Mutat.Res. 651 (2008) 64–70, https://doi.org/10.1016/j.mrgentox.2007.10.009.

[124] M. Kirsch-Volders, A. Vanhauwaert, U. Eichenlaub-Ritter, I. Decordier, Indirectmechanisms of genotoxicity, Toxicol. Lett. 140–141 (2003) 63–74.

[125] F. Pacchierotti, I.-D. Adler, U. Eichenlaub-Ritter, J.B. Mailhes, Gender effects onthe incidence of aneuploidy in mammalian germ cells, Environ. Res. 104 (2007)46–69, https://doi.org/10.1016/j.envres.2006.12.001.

[126] F. Sun, H. Yin, U. Eichenlaub-Ritter, Differential chromosome behaviour inmammalian oocytes exposed to the tranquilizer diazepam in vitro, Mutagenesis 16(2001) 407–417.

[127] M. Kirsch-Volders, G. Plas, A. Elhajouji, M. Lukamowicz, L. Gonzalez, K. VandeLoock, I. Decordier, The in vitro MN assay in 2011: origin and fate, biologicalsignificance, protocols, high throughput methodologies and toxicological re-levance, Arch. Toxicol. 85 (2011) 873–899, https://doi.org/10.1007/s00204-011-0691-4.

[128] B. Verdoodt, I. Decordier, K. Geleyns, M. Cunha, E. Cundari, M. Kirsch-Volders,Induction of polyploidy and apoptosis after exposure to high concentrations of thespindle poison nocodazole, Mutagenesis 14 (1999) 513–520.

[129] M. Casenghi, R. Mangiacasale, M. Tuynder, P. Caillet-Fauquet, A. Elhajouji,P. Lavia, S. Mousset, M. Kirsch-Volders, E. Cundari, p53-independent apoptosisand p53-dependent block of DNA rereplication following mitotic spindle inhibi-tion in human cells, Exp. Cell Res. 250 (1999) 339–350, https://doi.org/10.1006/excr.1999.4554.

[130] I. Decordier, L. Dillen, E. Cundari, M. Kirsch-Volders, Elimination of micro-nucleated cells by apoptosis after treatment with inhibitors of microtubules,Mutagenesis 17 (2002) 337–344.

[131] F. Marchetti, J.B. Mailhes, L. Bairnsfather, I. Nandy, S.N. London, Dose-responsestudy and threshold estimation of griseofulvin-induced aneuploidy during femalemouse meiosis I and II, Mutagenesis 11 (1996) 195–200, https://doi.org/10.1093/mutage/11.2.195.

[132] E. Fragouli, D. Wells, A.E. Iager, U.A. Kayisli, P. Patrizio, Alteration of gene ex-pression in human cumulus cells as a potential indicator of oocyte aneuploidy,Hum. Reprod. 27 (2012) 2559–2568, https://doi.org/10.1093/humrep/des170.

[133] A.E. Czeizel, C. Elek, S. Gundy, J. Métneki, E. Nemes, A. Reis, K. Sperling,L. Tímár, G. Tusnády, Z. Virágh, Environmental trichlorfon and cluster of con-genital abnormalities, Lancet (Lond. Engl.) 341 (1993) 539–542.

[134] S. Cukurcam, F. Sun, I. Betzendahl, I.-D. Adler, U. Eichenlaub-Ritter, Trichlorfonpredisposes to aneuploidy and interferes with spindle formation in in vitro ma-turing mouse oocytes, Mutat. Res. 564 (2004) 165–178, https://doi.org/10.1016/j.mrgentox.2004.08.008.

[135] T.C. Hsu, J.C. Liang, L.R. Shirley, Aneuploidy induction by mitotic arrestants.Effects of diazepam on diploid Chinese hamster cells, Mutat. Res. 122 (1983)201–209.

[136] M. Izzo, A. Antoccia, F. Degrassi, C. Tanzarella, Immunofluorescence analysis ofdiazepam-induced mitotic apparatus anomalies and chromosome loss in Chinesehamster cells, Mutagenesis 13 (1998) 445–451.

[137] P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas,B.F. Thomas, T.J. Hassold, Bisphenol a exposure causes meiotic aneuploidy in thefemale mouse, Curr. Biol. 13 (2003) 546–553.

[138] A. Muhlhauser, M. Susiarjo, C. Rubio, J. Griswold, G. Gorence, T. Hassold,P.A. Hunt, Bisphenol a effects on the growing mouse oocyte are influenced bydiet1, Biol. Reprod. 80 (2009) 1066–1071, https://doi.org/10.1095/biolreprod.108.074815.

[139] S. Lenie, R. Cortvrindt, U. Eichenlaub-Ritter, J. Smitz, Continuous exposure tobisphenol A during in vitro follicular development induces meiotic abnormalities,Mutat. Res. 651 (2008) 71–81, https://doi.org/10.1016/j.mrgentox.2007.10.017.

[140] F. Marchetti, A. Massarotti, C.L.C.L. Yauk, F. Pacchierotti, A. Russo, The adverseoutcome pathway (AOP) for chemical binding to tubulin in oocytes leading toaneuploid offspring, Environ. Mol. Mutagen. 57 (2016) 87–113, https://doi.org/10.1002/em.21986.

[141] U. Eichenlaub-Ritter, U. Winterscheidt, E. Vogt, Y. Shen, H.-R. Tinneberg,R. Sorensen, 2-methoxyestradiol induces spindle aberrations, chromosome con-gression failure, and nondisjunction in mouse oocytes, Biol. Reprod. 76 (2007)784–793, https://doi.org/10.1095/biolreprod.106.055111.

[142] D. Scott, S.M. Galloway, R.R. Marshall, M. Ishidate, D. Brusick, J. Ashby,B.C. Myhr, International Commission for Protection Against Environmental

[191] C.L. Yauk, J. Lucas Argueso, S.S. Auerbach, P. Awadalla, S.R. Davis,D.M. DeMarini, G.R. Douglas, Y.E. Dubrova, R.K. Elespuru, T.W. Glover,B.F. Hales, M.E. Hurles, C.B. Klein, J.R. Lupski, D.K. Manchester, F. Marchetti,A. Montpetit, J.J. Mulvihill, B. Robaire, W.A. Robbins, G.A. Rouleau,D.T. Shaughnessy, C.M. Somers, J.G. Taylor, J. Trasler, M.D. Waters, T.E. Wilson,K.L. Witt, J.B. Bishop, Harnessing genomics to identify environmental determi-nants of heritable disease, Mutat. Res. Rev. Mutat. Res. 752 (2013) 6–9, https://doi.org/10.1016/j.mrrev.2012.08.002.

[192] D. Babariya, E. Fragouli, S. Alfarawati, K. Spath, D. Wells, The incidence andorigin of segmental aneuploidy in human oocytes and preimplantation embryos,Hum. Reprod. 32 (2017) 1–12, https://doi.org/10.1093/humrep/dex324.

[193] S. Lu, C. Zong, W. Fan, M. Yang, J. Li, A.R. Chapman, P. Zhu, X. Hu, L. Xu, L. Yan,F. Bai, J. Qiao, F. Tang, R. Li, X.S. Xie, Probing meiotic recombination and an-euploidy of single sperm cells by whole-genome sequencing, Science 338 (2012)1627–1630, https://doi.org/10.1126/science.1229112.

[194] C. Templado, F. Vidal, A. Estop, Aneuploidy in human spermatozoa, Cytogenet.Genome Res. 133 (2011) 91–99, https://doi.org/10.1159/000323795.

[195] C. Dominguez-Brauer, K.L. Thu, J.M. Mason, H. Blaser, M.R. Bray, T.W. Mak,Targeting mitosis in cancer: emerging strategies, Mol. Cell 60 (2015) 524–536,https://doi.org/10.1016/j.molcel.2015.11.006.

[196] K.A. Knouse, T. Davoli, S.J. Elledge, A. Amon, Aneuploidy in cancer: seq-ing an-swers to old questions, Annu. Rev. Cancer Biol. 1 (2017) 335–354, https://doi.org/10.1146/annurev-cancerbio-042616-072231.

[197] S.F. Bakhoum, B. Ngo, A.M. Laughney, J.A. Cavallo, C.J. Murphy, P. Ly, P. Shah,R.K. Sriram, T.B.K. Watkins, N.K. Taunk, M. Duran, C. Pauli, C. Shaw,K. Chadalavada, V.K. Rajasekhar, G. Genovese, S. Venkatesan, N.J. Birkbak,N. McGranahan, M. Lundquist, Q. LaPlant, J.H. Healey, O. Elemento, C.H. Chung,N.Y. Lee, M. Imielenski, G. Nanjangud, D. Pe’er, D.W. Cleveland, S.N. Powell,J. Lammerding, C. Swanton, L.C. Cantley, Chromosomal instability drives

metastasis through a cytosolic DNA response, Nature 553 (2018) 467–472,https://doi.org/10.1038/nature25432.

[198] B. Verdoodt, I. Charlette, B. Maillet, M. Kirsch-Volders, Numerical aberrations ofchromosomes 1 and 17 in tumor cell lines of the exocrine pancreas as determinedby fluorescence in situ hybridization, Cancer Genet. Cytogenet. 94 (1997)125–130.

[199] B. Verdoodt, P. Castelain, C. Bourgain, M. Kirsch-Volders, Improved detection ofDNA aneuploidy in primary breast cancer using quantitative DNA image analysisin combination with fluorescent in situ hybridization technique, Histochem. J. 27(1995) 79–88.

[200] P. Segers, S. Haesen, P. Castelain, J.J. Amy, P. De Sutter, P. Van Dam, M. Kirsch-Volders, Study of numerical aberrations of chromosome 1 by fluorescent in situhybridization and DNA content by densitometric analysis on (pre)-malignantcervical lesions, Histochem. J. 27 (1995) 24–34.

[201] A. Russo, F. Pacchierotti, D. Cimini, N.J. Ganem, A. Genescà, A.T. Natarajan,S. Pavanello, G. Valle, F. Degrassi, Genomic instability: crossing pathways at theorigin of structural and numerical chromosome changes, Environ. Mol. Mutagen.56 (2015) 563–580, https://doi.org/10.1002/em.21945.

[202] A.D. Silk, L.M. Zasadil, A.J. Holland, B. Vitre, D.W. Cleveland, B.A. Weaver,Chromosome missegregation rate predicts whether aneuploidy will promote orsuppress tumors, Proc. Natl. Acad. Sci. 110 (2013) E4134–E4141, https://doi.org/10.1073/pnas.1317042110.

[203] A. Russo, In vivo cytogenetics: mammalian germ cells, Mutat. Res. Mol. Mech.Mutagen. 455 (2000) 167–189, https://doi.org/10.1016/S0027-5107(00)00115-9.

[204] T.E. Schmid, W. Xu, I.D. Adler, Detection of aneuploidy by multicolor FISH inmouse sperm after in vivo treatment with acrylamide, colchicine, diazepam orthiabendazole, Mutagenesis 14 (1999) 173–179, https://doi.org/10.1093/Mutage/14.2.173.

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