Pinto 2002 Int J Radiat Biol

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8 int. j. radiat. biol 2002, vol. 78, no. 5, 375 ± 388

Quanti� cation of radiation induced DNA double-strand breaks inhuman � broblasts by PFGE: testing the applicability of randombreakage models

M. PINTO, K. M. PRISE* and B. D. MICHAEL

(Received 22 June 2001; accepted 1 November 2001)

Abstract. DNA after radiation exposure (Ward 1995, 1998) .Purpose: To assess the applicability of methods of quanti� cation Because of the nature of energy deposition along theof double-strand breaks (DSB) based on the random breakage tracks of electrically charged particles, densely ioniz-paradigm, measuring yield and distribution of DSB induced by ing radiation is expected to induce complex lesionsvarying radiation quality.

(Burkart et al. 1999) . Among these are DSB, whichMaterial and methods: 240 kVp X-rays and 238Pu a-particles wereused to induce DSB in AG01522B primary human � broblasts. are expected to be more diYcult to repair (WardDNA molecular weight distributions were resolved by means of 1985, 1998, Goodhead 1994) . Indeed, recent studiesthree pulsed-� eld gel-electrophoresis (PFGE) protocols, which, where repair of radiation-induced DSB is measuredwhen combined together, allowed separation and quanti� cation

have shown slower kinetics with increased linearof double-stranded fragments between 5.7 Mbp and 12 kbp.energy transfer (LET), or a higher proportion ofSeveral analytical methods quanti� ed the DSB yields.

Results : Data showed signi� cant diVerences in the fragmentation DSB rejoining slowly (Stenerlow et al. 2000) . Therepatterns according to radiation quality. For both X-rays and a- is evidence for higher frequencies of misrejoined DSBparticles, it was observed that the shape of the fragmentation with increased LET (Kuhne et al. 2000) and/or morepro� les deviates from the prediction of a random breakage

breaks left unrejoined after long incubation timesmechanism. This is in contrast to other studies where sparselyionizing radiations appeared to distribute breaks uniformly post-irradiation (Ritter et al. 1977, Belli et al. 2000,throughout the genome. Deviations from random breakage were Stenerlow et al. 2000) . These studies have demon-more evident after high linear energy transfer (LET) radiation, strated that not all DSB appear to be treated in thewhich showed an excess of breaks <1 Mbp and a de� cit in

same way by cells. For several years, quanti� cationthe production of fragments >1 Mbp, a value that could beof DSB induction frequencies has focused on totaldose-dependent.

Conclusions : Current methods of DNA fragmentation analysis yields of breaks. Before PFGE techniques wereafter induction of DSB may lead to contradictory conclusions employed, it was generally found that the total yieldon both DSB yields and distributions. This study showed that of DSB showed little variation with radiation qualitythe application of diVerent DSB quanti� cation methods, derived

(Belli et al. 1994) . With the advent of PFGE, diVer-from random breakage or supported by its concepts, resulted indiVerent radiation biological eVectivenesses (RBE) for the induc- ences in DSB yields with LET began to be measured,tion of DSB, depending on how these methods were employed. but they showed RBE signi� cantly smaller than thoseTo compare experimental results from diVerent laboratories, for higher-order biological eVects (Lobrich et al. 1993,care should be taken to provide as many details as possible about

Prise et al. 1998) . This diVerence could partly bethe application of methods of quanti� cation of DNA damage.related to the method used for the quanti� cation ofFor all the methods used, total DSB yields resulted in RBE less

than those for mutation induction or reproductive cell death, DSB (Prise et al. 1998, Stenerlow et al. 1999, Hoglundsuggesting that total DSB yields only gave a limited indication and Stenerlow 2001) . It is also likely that the damageof the severity of the in� icted damage. Production of correlated

measured with the available experimental techniquesbreaks on the chromatin loop structures by single particle-trackis not the ultimate determinant of cellular end-pointstraversals may explain the deviations observed between experi-

mental data and the predictions of the random breakage (Olive 1998) . Characterization of the most relevantparadigm. initial lesions is thus extremely important. At the

DNA level, these DSB are thought to be clusters ofstrand breaks and base damages, described by Ward1. Introduction(1981) as locally multiply damaged sites (LMDS). At

It is currently accepted that DNA double-strand the level of the higher-order chromatin structures,breaks (DSB) are important lesions induced in cellular Rydberg (1996) proposed that regionally multiply

damaged sites (RMDS) may also be relevant.*Author for correspondence; e-mail: [email protected] of DSB may result in an additional chal-Gray Cancer Institute, PO Box 100, Mount Vernon Hospital,

Northwood HA6 2JR, UK. lenge, independent of their individual complexity

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online © 2002 Taylor & Francis Ltdhttp://www.tandf.co.uk/journals

DOI: 10.1080/0955300011011094 1

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8 376 M. Pinto et al.

( Johnston and Bryant 1994) . PFGE and constant the medium was replaced with fresh a-MEM for1 day incubation before irradiation. Typically, an� eld gel-electrophoresis (CFGE) were not designed

to detect and characterize the various orders of average of 0.02 dpm was incorporated per cell.complexity of damage referred to as LMDS, thougha fraction of these may be detected as additional 2.2. Irradiationbreaks upon treatment with speci� c enzymes, suchas Nth and Fpg. Nevertheless, PFGE can be used to Before irradiation, aMEM was replaced with fresh

20 mm HEPES-buVered aMEM with cells kept onmeasure multiple DSB that are clustered arounddistances that are within the region of detection of ice. For 238Pu a-particle irradiation (incident energy

3.5 MeV) the volume-averaged LET was calculatedthe technique; for example, track-correlated DSBintroduced in higher-order chromatin looped struc- as 110 keV mm Õ 1 (Folkard et al. 1989). X-irradiation

was performed on a Pantak IV system (240 kVp)tures from a few tens of kbp to several Mbp. Accurateknowledge of the fragment size distributions can also equipped with a 0.25 mm Cu and a 0.5 mm Al

� lters with cells attached on Petri dishes kept on ice.be a valuable tool for testing diVerent models ofchromatin structure and its interaction with charged- Dose rates were 0.88 Gy s Õ 1 for a-particles and

0.027 Gy s Õ 1 for X-rays.particle tracks. The complex scenario of chargedparticle tracks intersecting several orders of chro-matin organization predicts that methods for the 2.3. Pulsed-�eld gel electrophoresisquanti� cation of DSB based on random breakagemay be an oversimpli� cation, and should be applied Immediately after irradiation, cell monolayers were

washed repeatedly with PBS kept at 4 ß C, beforewith care. The main purpose of this work was toassess the applicability of random breakage-based incubation with 0.25% (w/v) trypsin/EDTA at 4 ß C

for 10 min. Trypsin/EDTA was then neutralized bymethods for the quanti� cation of DSB. In the analysisof our data, it was shown that when diVerent methods adding fresh cold a-MEM. Typically, two dishes were

used per dose for X-rays, while six to eight dishesbased on the same paradigm or making use of itsconcepts are used to quantify DSB, diVerent yields, were employed per dose when irradiating with a-

particles to accumulate a suYcient dpm signal in thedistribution of DSB and RBE can be found.gels. Cell suspensions were then pelleted by centrifu-gation (700g, 4 ß C) and then re-suspended in approxi-mately 30 ml cold PBS. Plugs preparation and2. Materials and methodselectrophoresis, for protocols I and II, were per-2.1. Cell culture formed according to Newman et al. (1997) , with thefollowing modi� cations. Samples were gently mixedAG01522B primary human � broblasts were rou-

tinely grown in a-modi� ed minimum essential with Gilson pipettes (which had had their ends cutto avoid excess shear), an operation that took nomedium (Sigma, Poole, UK), in an atmosphere

of 95% air and 5% CO2 at 37 ß C. The medium longer than 1 min in the waterbath at 37 ß C tominimize DSB repair during samples preparation.was supplemented with 20% (w/v) foetal calf

serum (Sigma), 100 mg ml Õ 1 benzyl penicillin After lysis, plugs were stored in 1 3 TE buVer(10 mmol dm Õ 3 Tris, 1 mmol dm Õ 3 EDTA, pH 7.5)and 100 mg ml Õ 1 streptomycin (Gibco, Hemel

Hempstead, UK), 200 mm l-glutamine (Gibco), at 4 ß C for a maximum of 2 days before electrophor-esis. For PFGE protocol III, plugs were loaded in1 3 non-essential amino-acids (Sigma) and a

mixture of deoxynucleotides (2-deoxyadenosine, 1.0% (w/v) rapid agarose gels, prepared using1.0 3 TBE buVer, and electrophoresed in a 0.5 3 TBE2-deoxycytidine, 2-deoxyguanosine, thymidine, aden-

osine, cytidine, guanosine, uridine). For experiments, running buVer at 6 V cm Õ 1 , 120 ß pulses ramped from50 to 60 s. With a total run time of 18 h, PFGEcells were seeded in 60 mm plastic Petri dishes for

X-irradiation or, for a-irradiation, in custom-made protocol III was mainly used to improve markerseparation in the region between 1 Mbp and 48 kbp.27 mm glass-walled dishes consisting of a base of

3 mm thick mylar � lm. After a few hours, to allow All the PFGE runs were carried out in a BioRadCHEF DRIII unit with the buVer temperature main-for cell attachment, cells were radiolabelled with

0.05 mCi ml Õ 1 [2-14C]thymidine (speci� c activity tained at 13.5ß C by circulation through a chiller unit.After electrophoresis, gels were stained in52 mCi mmol Õ 1 , Amersham International,

Amersham, UK). Cells were grown in radiolabelled 0.5 mg ml Õ 1 ethidium bromide overnight followed bydestaining in fresh buVer (TAE or TBE) for up tomedia from 7 to 10 days to reach cell con� uence,

with 90–95% of the cells synchronized in G0 -phase 2 h. Gel images were captured on a UV transillumin-ator with a black-and-white digital camera (Cohu).(� ow cytometry measurements, not shown) and then

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8 377Quanti�cation of DNA DSB by PFGE

The gel lanes were then cut across the direction of can derive the frequency ni of the number-averagefragment size in section i. This quantity is usefulDNA migration, according to the position of known

molecular weight markers, for improved repro- when performing regression analysis with parametricmethods (see Section 2.4.2). One can also calcu-ducibility of the measurements. The markers em-

ployed were: Schizosaccaromyces pombe (3.5–5.7 Mbp), late the number of fragments in section i, N(Mi ) 5 n(Mi ) 3 DMi :Saccaromyces cerevisiae (225 kbp–2.2 Mbp), 8–48 kbp

standard (all BioRad, Hemel Hempstead, UK),48 kbp l ladder (48 kbp–1.2 Mbp, Pharmacia, ni ; n(Mi ) .

H Fi

Mi DMi(3)

Amersham, UK) and 1 kbp standard (500 bp–12 kbp,GIBCO, Hemel Hempstead, UK). The gel sectionswere then melted and prepared for scintillation N i ; N (Mi ) .

H Fi

Mi(4)

counting as described in Newman et al. (1997) . Lanesin which radioactive samples had not been loaded The number of fragments in a section directly quanti�eswere employed to monitor background activities. the DSB present in that gel segment (see Section 2.4.3Agarose gel sections of size comparable with those for a description of the ‘direct quanti� cation’ methodcut on the radioactive lanes were treated normally and details of how to extrapolate its estimates to totalas described above. The activities (dpm) averaged yields of DSB per cell).over several non-radioactive samples, per experi- Several groups have used regression analysis withment, were then used as the dpm background, to be analytical functions to quantify the DSB induced byautomatically subtracted by the scintillation counter radiation. The non-linear regressions with all thefrom the dpm of the radioactive samples. functions described below were performed using the

Levenberg–Marquardt algorithm on a statistics2.4. Quanti�cation of DSB computer package (e.g. Microcal Origin).

For quanti� cation of DSB described in the follow-2.4.1. Fraction of the activity released (FAR) approach.ing sections, the dpm signal measured in each gelWith the exception of the ‘direct quanti� cation’segment was transformed in fraction Fi of the totalmethod, all the DSB quanti� cation approaches usedactivity:in this work were derived from the random breakageparadigm. The most common implementation ofFi 5

dpm(sectioni )

Sj dpm(sectionj )(1)

these concepts is referred to as FAR described byBlocher (1990) , where the amount of DNA smallerwhere Sj dpm(sectionj ) is the total number of dpm than a � xed threshold size (normally the gel exclusionin a gel lane, including the well. Assuming that [2- size determined by the electrophoresis protocol used)14C]-thymidine is uniformly incorporated throughout is measured as a fraction of the total DNA contentthe DNA, Fi also represents the fraction of DNA per cell. The analytical function provided for datamass present in the section i. Mass-fractions were� tting is, in the original notation:then converted into frequency or number of breaks by

means of the following relationship:F

< k 5 1 Õ expA Õrk

n B ·C1 1rk

n A1 Õk

nBD (5)Fi .

n (Mi ) 3 Mi 3 DMi

H(2)

Based on random breakage, equation (5) predicts themass F

< k of DNA below a threshold size k, due toEquation (2) relates the fraction of mass Fi to thefragmentation of a polymer (in this case a chromo-frequency n (which is normalized for the molecularsome) of initial size n after the induction of r breaks.weight range of the gel section, DMi ) of the number-A variation of this method, employing the so calledaverage fragment size M in section i, the molecular‘Q’ function, was proposed by Cedervall and Kallmanweight range DMi in the section and the total mass(1994, and references therein) and reviewed sub-of DNA present per cell, set to the genome size H.sequently (Cedervall et al. 1995, Cedervall andAssuming, for simplicity, that the distribution ofRadivoyevitch 1996, Cedervall and Lange 1998) .fragment sizes in each section i is governed byBoth methods integrate the signal available overrandom breakage, the goodness of the approximationlarge ‘windows’ of fragment sizes. Since largerreported in equation (2) depends on the validity offragments weigh proportionally more in the inte-the inequality DMi /Mi % 1 (calculation not shown),gration, the calculation of the yields of DSB is notwhich translates into the need of cutting as manysensitive to the contribution of breaks that appearthin sections as possible, considering the limitation

in the sensitivity of the assay. From equation (2) one close to each other (approximately this translates to

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< 200 kbp). Nevertheless, one can choose the molecu- the background damage is required. This is oftencarried out by subtracting the experimental pro� leslar weight threshold size below which to integrate,(mass-intensities or frequencies) which seems to beto include or exclude the signal from the largeran over-simpli� cation (see Pinto et al. 2000) . Startingfragments as shown in � gure 2 and in Hoglund et al.from the ‘broken stick’ concepts, an alternative(2000). Thus, with the FAR analysis, a molecularmethod was previously derived called background-weight region is selected for the regression to estimatedependent random breakage (BDRB) (Pinto et al.what the total DSB yield per cell ‘would’ be to2000) , which does not require subtraction of experi-reproduce the experimental � ndings in the restrictedmental pro� les. Testing the BDRB model as derivedregion. Estimation of the total yield of DSB fromin Pinto et al. (2000) on an extensive dataset did notmeasurements in a restricted molecular weightalways prove successful. The reason for this wasregion is quite general of the analytical approachesfound to be due to an extrapolation needed in thepresented in this and the following section.model for its derivation. In the original derivation ofRecently, more sophisticated approaches havethe BDRB model, the background frequency distribu-been used that have been referred to as DSB frag-tion is � rst described empirically by means of thementation analysis, focusing on the actual shapes offunctionthe fragmentation pro� les. The main advantage over

integral approaches (as FAR or Q-function) is that y (S ) 5 A · SB (7)the problem of weighing a cumulative signal for the

where S is the molecular weight of the frag-larger fragments sizes is eliminated. Experimentalments found in the unirradiated control samples, andstudies employing fragmentation analysis have shownthe parameters A and B are estimated using thethe importance of measuring the amount of everexperimental data.smaller fragments produced during irradiation

Once A and B from equation (7) are estimated,(Lobrich et al. 1996, Rydberg 1996, Prise et al. 1998,their values are incorporated in a second � tting2001, Rydberg et al. 1998, Newman et al. 1997, 2000,function, to be used on the pro� les observed afterHoglund et al. 2000, Hoglund and Stenerlow 2001).irradiation, hence the name ‘background dependent’.It is generally found that, after fragmentation ana-Fragmentation data obtained from PFGE are onlylysis, more DSB were detected than with integral available in a restricted molecular weight region,approaches. Several analytical parametric methods are because of the limitation of the technique itself. Thecurrently available for fragmentation analysis, either derivation of the BDRB model needed a full descrip-derived from the random breakage paradigm or tion of the background pro� les, including the mole-making use of its assumptions. Some of these are cular weight region that remains in the gel wells. Tobrie� y reviewed below. estimate the amount of DNA present outside theregion where size-markers are available, and particu-

2.4.2. ‘Broken stick’ and ‘background-dependent random larly above the largest molecular weight marker size,breakage’ methods. The ‘broken stick’ method an extrapolation of the function y (S ) in the region of(Contopoulou et al. 1987, Cook and Mortimer 1991) the gel well was required. For some experiments,provides the function reported in equation (6). This background distributions varied such that extrapola-describes the frequency n of fragments of size x when tion led to an over or underestimation of the amountm breaks are introduced randomly in a chromosome of DNA in the well, based on the genome size H.of initial size S. Instead, in this study it was assumed that the function

y (S ) written above had to be representative of thebackground pro� le only up to the gel wells, that is,n (x, m, S ) 5

m

SC2 1 mA1 Õx

SBDexpAÕmx

S B (6)below the exclusion size e. Above e it was assumedthat 46 ‘virtual’ chromosomes were present, having

We have previously reviewed the use of formula (6) all the same (for mathematical convenience) size C ¾to analyse fragmentation patterns measured experi- diminished from the original ‘intact’ average sizementally (Pinto et al. 2000). In particular, it was (H/46). The size-reducing factor (FARes in equationobserved that the ‘broken stick’ method, as do most 9) was chosen to depend on the extent of theof the fragmentation analysis methods available, manipulation-induced fragmentation, as measuredfocuses on radiation-induced DSB. As such, it does from the unirradiated control samples using thenot consider the breaks present in the unirradiated values of A and B . From equation (7), the backgroundcontrol samples, due to the manipulation procedures frequency distribution becomes:(background breaks) but independently of the radi-ation-induced breaks. To estimate radiation-induced y (S ) 5 G ASB for S ×[1, e]

46 d (S Õ C ¾ ) for S >e(8)

DSB yields, a correction of the experimental data for

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where small minority. It was in fact estimated that for themeasured patterns to be due to the fragmentation ofonly a fraction of the cells present, these should haveC ¾ 5

H

46· (1 Õ FARes ) (9)

experienced so many DSB, hence their DNA wouldbe so highly fragmented, that it would be impossibleandto detect any fragment larger than a few hundredsof kbp in an ordinary PFGE experiment. The experi-FARes 5

A

H·eB+2 Õ 1

B 1 2(10) mental data from this and other investigations (e.g.

Hoglund et al. 2000) show instead a signi� cantThis change for the function y (S) introduces a dis- amount of Mbp-sized fragments in the gel lanes ofcontinuity in e, which does not aVect the analytic the unirradiated controls.behaviour of the BDRB � tting function in the experi- Considering the very large number of DSB that ismental data region. With this modi� cation for the measured in the unirradiated controls (in the orderdescription of the background pro� le, the � tting of thousands per cell), it seems unlikely that thefunction provided by the BDRB method was fragmentation pro� les measured with PFGE in thesere-derived in the same way as described in Pinto samples re� ect the status of a cell at the time ofet al. (2000) , to become: irradiation. Instead, it seems more likely that DSB

are introduced during the PFGE procedures, possiblyn (x, m, H, A, B, e) 5 A ·

m

H Cm

H

e2 +B Õ x2 +B

2 1 Bafter the DSB induced by radiation. This suggeststhat the two actions occur in the opposite order asin the derivation of the BDRB model, but modelling

1e1 +B Õ x1 +B

1 1 B A2 Õmx

HBD expAÕmx

HB this ‘reversed’ condition is complex. In fact, althoughthere have been a few attempts to reproduce themeasured background fragments size distribution

1 46·m

HC2 1 Am

HB · (C ¾ Õ x)D expAÕmx

HB with ad-hoc probability functions for the localizationof the background-induced DSB in the interphasechromatin (Khvostunov and Andreev 2001) , the

1 AxB expAÕmx

HB (11) actual mechanism underlying the observed patternsremains unknown. The problem is solved if the twomechanisms are considered as acting independently,Equation (11) is already normalized to the number

of DSB present due to its semi-empirical nature, as in which case the result does not depend on theorder of the two actions. The scenario can then bediscussed in Pinto et al. (2000). For the special case

when B is either Õ 1 or Õ 2, the formula changes as successfully described as was done in the derivationof the BDRB method.detailed in the former derivation. In equation (11), e

is the exclusion size, C ¾ was de� ned in equation (9)and H , the diploid genome size, is estimated to be 2.4.3. Direct quanti�cation of DSB. Regression analysis

of experimental data from a limited molecular weight6.4 Gbp for human cells (International HumanGenome Sequencing Consortium 2001). With the region, using functions derived from the random

breakage paradigm, allows estimation of the totalmodi� ed BDRB function as in equation (11), resultsof regression analysis were much more reproducible yields of breaks per cell, as shown with the FAR and

BDRB methods. DSB can also be quanti� ed bythan with the original BDRB formula, and no longerbiased by the description of y (S) in each experiment, means of direct conversion of mass fraction F i into

numbers of breaks N i , using equation (4). Addingas � gure 5 shows.The basis of the BDRB method lies in the assump- up the N i for all the sections available in the gel

allows counting of the number of fragments presenttion that radiation induces breaks on a distributionof fragments of varying size, counting many double- in the region covered by the markers in the PFGE

run. Since fragmentation of the human chromosomesstranded fragments, rather than a few intact chromo-somes, as other methods assume. In the derivation may lead to DNA fragments of size greater than the

largest molecular weight markers resolved in the gel,of the BDRB method, this distribution of fragmentsis present before irradiation and can be observed in this direct conversion per se underestimates the total

number of DSB per cell. To extrapolate to total DSBthe unirradiated control samples. It is assumed in themodel that the fragmentation patterns observed in yields, some assumptions are needed. For example,

it is convenient to assume that the number of breaksthe unirradiated controls are representative of thebackground damage present in every cell of the popu- present above the PFGE protocol-speci� c exclusion

size e, in the gel wells, is negligible compared to thatlation that is under investigation, rather than in a

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measured in the gel lanes. In experiments measuringthe initial induction of DSB, this is referred to as ahigh-dose approximation (Hoglund et al. 2000,Stenerlow et al. 2000), because at high doses very fewlarge fragments ( large enough to remain trapped inthe wells) are left. For DSB repair experiments, afterlong incubation times, double stranded fragmentswill have been joined to each other to the extent thata signi� cant proportion of them may be above the10 Mbp limit of detection, and the high-doseapproximation would be inappropriate.

Because of the limited sensitivity of PFGE, it isnot possible to detect any signal from fragmentssmaller than approximately 10 kbp, even at very highdoses such as 100 Gy. The limited sensitivity below10 kbp may be in part due to the loss of smallerfragments by diVusion out of the plugs. Failure todetect signal in these regions may not necessarilyimply that the number of DSB is negligible. Forextrapolating from the estimates of the ‘direct conver-

Figure 1. When DSB are measured directly in a limited molecu-sion’ method to the total number of breaks per cell,lar-weight region (see Section 2.4.3), the ‘broken stick’one can use the ‘broken stick’ model, assuming method can be used to estimate the total yield of DSB

random breakage, and equation (6) to make an per cell. In the solid curve relative to [10 kbp–5.7 Mbp],estimate of what fraction of the total DSB is being if 2000 DSB are measured, 2300 are predicted to be

distributed in the entire genome. The under-estimationmeasured in a window [M1 , M2 ]. Integration ofDm is then approximately 15%. When 2000 DSB areformula (6) between M1 and M2 yields:measured in a more restricted window [1 kbp–1 Mbp]

DSB(m, H, M 1 , M 2 ) 5 (m 1 1) (dotted curve), the error made because of under-estimation is >100%.

·CexpA ÕmM 1

H BÕ expA ÕmM 2

H BDvitro. With the aid of three PFGE protocols, it waspossible to measure the production of DNA fragments

1m

H·CM 2 expAÕ

mM 2

H B by radiation-induced DSB between 12 kbp and5.7 Mbp. The lower limit is mainly set by the sensitiv-ity of detection of 14C disintegrations, whereas the

Õ M 1 expAÕmM 1

H BD (12) upper limit is due to PFGE techniques’ intrinsicdiYculties in extracting very large DNA fragments.

For most PFGE conditions (M 1 and M 2 % H , the The following sections report results and data analysisDNA content of the diploid genome), the term in with three of the four methods presented previously.the � rst part of equation (12) is dominant, thus thesecond part may be ignored. The function in equa-

3.1. FAR analysistion (12) is plotted in � gure 1 for reference, for twochoices of [M 1 , M 2 ]. Once the number of DSB ( y- For data analysis with the FAR approach, proposedaxis in � gure 1) has been estimated from the experi- by Blocher, equation (5), FAR were calculated belowmental data by ‘direct conversion’, the curve shown 12 diVerent molecular weight thresholds, eight ofmay be used to solve, graphically, for the total which are shown in � gure 2 in separate panels (a–h).extrapolated yield of breaks m (x-axis). This may be They were then corrected by subtracting the corres-done, for example, with the aid of data plotting

ponding FAR measured in the unirradiated controls.software.This assumes a linear behaviour of the FAR depend-Solving for m may also be done numerically, usingence with the breaks induced which appears to beequation (12). The under-estimation of m is alsoan over-simpli� cation (Blocher 1990) . Subtractedshown as Dm in � gure 1.FAR, averaged from several experiments, are shownin � gure 2. When large fragments are included in

3. Results the FAR integration, the total DSB yields were higherfor X-rays than for a-particles (panels a–c). How-This study measured DNA fragmentation patterns

after induction of DSB in human cellular DNA in ever, as the threshold size was reduced to below

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Figure 2. (a–h) FAR (with controls subtracted) for a-particles ( ) and X-rays ( ) with each panel representing a diVerent exclusionsize (k in equation 5). (i) Means Ô SE of the least-square regression analysis using equation (5) for a range of threshold sizesincluding those depicted in (a–h). In some cases for the X-ray data, � ts to full dose–response curves were not performed ( ] )but are those obtained for FAR 115 Gy only. For these points only, the errors shown are propagated from the FAR SEM (n 5 6).

approximately 1 Mbp (panel d) the yields of DSB lines that are non-horizontal. With the exclusion ofthe data shown at molecular weight thresholds 48.5,induced by a-particles increased above those of X-rays

(e–h). At the largest threshold size studied, the FAR 97 and 147.5 kbp, where the experimental uncertain-ties appear to be dominant, apparent (i.e. on thesignal is weighted to the larger DNA fragments, and

therefore for more sparsely located breaks. Widely semilogarithmic scales as shown in panel i) linearregressions in the range 225 kbp–5.7 Mbp resulted inseparated breaks are expected to be produced more

eVectively after sparsely ionizing radiation. By slopes diVerent from zero at 68% level of con� dence(B 5 Õ 9 Ô 1 for X-rays, 22 Ô 2 for a-particles). Itlowering the threshold size, sparse breaks are no

longer contributing to the integrated signal, and the should be stressed that this was observed not onlyfor a-particles, but also for X-rays, indicating thatFAR becomes more sensitive to the closely associated

breaks, which are expected to be produced more X-rays can induce DSB that are non-randomly dis-tributed. Results obtained with the FAR analysiseVectively by densely ionizing radiation.

Figure 2 also summarizes the estimates for the method, shown in panel (i), are also summarizedin table 1.extrapolated total DSB yield, based on regression

analysis, with varying threshold size, as shown inpanel (i). If random breakage was occurring, the 3.2. Fragmentation analysis (BDRB and directestimate for the total DSB would be independent of quanti�cation)the threshold size used for the FAR integration, witha constant yield that would be seen as a horizontal The use of fragmentation analysis may help to

understand the mechanisms of induction of DSB byline in panel (i) (Hoglund et al. 2000) . Moving from5.7 Mbp to lower molecular weights in panel (i), the interaction of charged-particle tracks and chro-

matin structures (Hoglund and Stenerlow 2001).down to 200 kbp, the data points appear as lying on

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Table 1. Results obtained by the FAR method.

DSB·Gy Õ 1 Gbp Õ 1

Radiation quality <5.7 Mbp <4.6 Mbp <3.5 Mbp <1.6 Mbp <1.02 Mbp <825 kbp

110 keV mm Õ 1 3.4 Ô 0.2 3.9 Ô 0.2 4.6 Ô 0.2 5.9 Ô 0.1 6.9 Ô 0.1 6.8 Ô 0.2a-particles

240 kVp X-rays 4.5 Ô 0.3 4.9 Ô 0.1 5.32 Ô 0.06 5.4 Ô 0.2 6.0 Ô 0.2 5.6 Ô 0.2a-particle RBE 0.76 Ô 0.07 0.80 Ô 0.04 0.86 Ô 0.04 1.09 Ô 0.04 1.15 Ô 0.04 1.21 Ô 0.06

<685 kbp <450 kbp <225 kbp <147.5 kbp <97 kbp <48.5 kbp

110 keV mm Õ 1 7.2 Ô 0.2 7.8 Ô 0.1 8.1 Ô 0.2 8.5 Ô 0.4 9.0 Ô 0.8 13 Ô 1a-particles

240 kVp X-rays 5.8 Ô 0.2 (D) 6.4 Ô 0.3 (D) 6.6 Ô 0.3 6.7 Ô 2 (D) 8 Ô 3 (D) 10 Ô 1a-particle RBE 1.24 Ô 0.05 1.22 Ô 0.06 1.23 Ô 0.06 1.27 Ô 0.04 1.1Ô 0.4 1.3Ô 0.2

Yields of DSB Gy Õ 1 Gbp Õ 1 for X-rays and a-particles calculated using the FAR method for 12 diVerent molecular weight thresholdsusing equation (5) with means Ô SEM shown. In some cases, ( ] ), corresponding to particular molecular weight thresholds, as shownwith the same symbol in panel 2(i), the data were obtained by only considering the FAR at 115 Gy (n 5 6). Also shown are thecalculated RBE with SE.

With fragmentation analysis, the DNA mass signal is and 100 Gy, respectively. With three PFGE protocolsavailable, three molecular weight regions for thenot integrated between a given molecular weight

threshold and the edge of the gel, as with FAR. application of the BDRB method were selected,reported in table 2. ‘A’ and ‘B’ vary with the experi-Several ‘windows’ are identi� ed by molecular weight

markers, and the signal in each window is integrated, ment, thus the analysis has to be performed on eachexperiment separately. The results of regression ana-to be monitored individually. It is ideal to have

several, narrow ‘windows’ to detect any � ne structure lysis from several experiments can be then comparedand averaged. Some experiments provided measure-in the molecular weight distribution, as well as to

avoid making rough approximations during data ments of DNA fragmentation pro� les at several doses.In this case, the BDRB method was applied to dataanalysis (see, for example, equation (2) for the conver-

sion from mass of DNA to number of fragments). for each dose value available. The results of theseBDRB regressions, for the same experiment, wereNevertheless, the sensitivity of the ordinary PFGE

assays sets a lower limit to the width of the windows, plotted against dose, and a linear regression wascarried out (assuming linearity of DSB inductionbelow which it would be impossible to detect any

radiation-induced DSB. with dose, forcing the lines to pass through the originof the axes on the plots) to calculate the total DSByield per cell, per gray. This is shown in � gure 4 for3.2.1. BDRB method. With the aid of equations (2–4),

one can transform the measured signal, which is a typical experiment.Five diVerent doses per experiment were used forproportional to the DNA mass, to other quantities.

The frequency of DSB per base pair is shown, for this multidose BDRB analysis (10, 50, 100, 150,200 Gy for a-particles, 11.5, 57.5, 115, 173, 230 Gytwo particular experiments, in � gure 3. On the x-axis,

the mean molecular weight of the ‘windows’ chosen for X-rays). Figure 5(a) shows the results of the BDRBregressions on each of the experiments carried outin the gels is plotted.

To use the BDRB method on the datasets shown in this study, expressed as total extrapolated DSByields per Gy and per cell (or per Gbp). A diVerentin � gure 3, one � rst needs to estimate the intercept

‘A’ and the slope ‘B’ from the unirradiated controls. symbol is used in � gure 5(a) for each radiation qualityand each of the three molecular weight regions inThe best estimates for these parameters are then

inserted into equation (11), the background-depend- which the method was applied. Further, a diVerentsymbol is used to distinguish between experimentsent � tting function provided by the BDRB method.

A non-linear regression performed using the that were analysed with the BDRB method at asingle dose versus multidose, given the same radiationLevenberg–Marquardt algorithm is then performed

to estimate the total yield of breaks, assuming random quality and the same molecular weight region ofanalysis.breakage. Figure 3 shows the results of this non-

linear regression with the BDRB method on two The results for the DSB yields obtained with theBDRB method, plotted in � gure 5 were then aver-experimental datasets, one relative to X-rays (a) and

the other relative to a-particles (b), at the doses 115 aged, grouping experimental data for X-rays and

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Figure 3. Application of the BDRB method to data from two independent experiments where 115 Gy doses of X-rays (a) or 100 Gydoses of a-particles (b) were used to induce DSB. For both panels, are single determinations for the frequency of DNAfragments, per unit base pair per cell, measured in the irradiated samples and not corrected for the background damage. arethe corresponding values measured in the unirradiated controls. Dotted lines correspond to linear regressions on the unirradiatedcontrol pro� les to estimate the parameters ‘A’ and ‘B’ of (7), needed for the non-linear regression with equation (11). Solid linesare the result of the non-linear regressions restricted to data-points >100 kbp.

a-particles, for each molecular weight region. For theaveraging procedure, results from experiments ana-lysed at a single dose were grouped together withexperiments that were analysed at multiple dosepoints (for the same radiation quality and samemolecular weight region analysed) since there wasno evidence for better accuracy on the results fromone group of experiments over the other. Theseaveraged estimates for the total extrapolated DSByields obtained with the BDRB method aresummarized in table 2, expressed as mean Ô SEM.

3.2.2. Direct quanti�cation. Independently of the FARand BDRB analysis, total yields of DSB per cell havealso been quanti� ed by means of the ‘direct quanti-Figure 4. Example of linear regression of BDRB results evalu-� cation’ method described in Section 2.4.3. For eachated on data at a � xed dose, versus dose, for the same

experiment. Each with error bars shown (visible where experiment, measurements of DNA mass-intensitylarger than the symbols) is the estimate, with its SE, were converted by means of equation (4) to theobtained applying the BDRB method on a dataset at a number of fragments in all the molecular weight�xed dose, as shown in � gure 3. After data from the same regions available between the smallest and largestexperiment have been analysed at the various dose points

molecular-weight markers available for that experi-available, the BDRB estimates are plotted versus thecorresponding dose, and a linear regression is made to ment [M1 , M2 ]. As with the analysis of data usingestimate the breakage yield per unit dose. For the experi- the BDRB method described in Section 3.2.1, experi-ment shown, where X-rays were used to induce DSB, mental data were analysed in three diVerent molecu-the breakage yield was estimated as (35.3 Ô 1.2) DSB lar weight intervals. From the number of breaksGy Õ 1 cell Õ 1 . This, together with the results from other

measured between M1 and M2 , i.e. added togetherexperiments analysed in the same way, is plotted in� gure 5. from all the sections available and corrected by

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Table 2. Results obtained by the BDRB method applied on data from several independent experiments.


Radiation quality 225 kbp–5.7 Mbp 225 –825 kbp 225–285 kbp

110 keV mm Õ 1 a-particles 7.0 Ô 0.3 ( , ) 7.6Ô 0.9 ( , ) 9 Ô 2 ( )240 kVp X-rays 5.5 Ô 0.4 ( , ) 4.9Ô 0.2 ( ] ) 6.2 Ô 1.4 ( )a-particles RBE 1.3 Ô 0.1 1.6Ô 0.2 1.5 Ô 0.5

Each experiment provided data that were analysed in one of the three molecular weight regions shown. Results of the BDRBanalysis on ‘homologous’ experiments were then averaged, and the mean Ô SEM are shown in separate columns. Results from individualexperiments, in the same experimental regions reported, were plotted in � gure 5(a). The symbols shown in parentheses indicate thatthe averages were obtained from experiments that are marked with the same symbols in � gure 5(a).

subtraction of the corresponding background breaks, the yields of breaks. More recently, the apparent lowlevels of background damage, measured by FARthe total yield of breaks per cell was extrapolated

using equation (12) and � gure 1. With the aid of methods, has been found to conceal a very high levelof DSB (Hoglund et al. 2000, Pinto et al. 2000) whendata-plotting software, the solution for the total yield

of DSB was derived graphically. Experimental data fragmentation analysis is performed. The origin ofthese DSB is still unclear. Rydberg (2000) showedwere analysed exclusively at 115 Gy for X-rays and

100 Gy for a-particle irradiation. Results of analysis that radiation-induced heat-labile sites may be con-verted to DSB during PFGE lysis carried out atwith the ‘direct quanti� cation’ method, extrapolated

to total yields of DSB, are shown in � gure 5(b) for 50 ß C. Whether endogenous heat-labile sites areconverted to DSB in the same way is unknown.each separate experiment. Results for the total extra-

polated yield from each independent experiment There has been increasing experimental evidencethrowing doubt on the validity of FAR approaches.were then averaged, grouping together experiments

that were analysed in the same molecular weight Above all, the lack of correlation between RBEmeasured for the total initial yields of DSB andinterval. Average estimates for DSB yields per cell

evaluated using the ‘direct quanti� cation’ method reproductive cell death or mutation (Heilmann et al.1995, Prise et al. 1998, 2001) for diVerent radiationare shown in table 3, together with a-particles RBE

for DSB induction. qualities. In the attempt to overcome this problem,studies employing PFGE techniques have movedTable 4 summarizes the estimated DSB yields for

both a-particles and X-rays with all the three methods toward a new type of analysis: observation of thefragmentation patterns after induction and repair ofused in this study. For each method of analysis and

each radiation quality, results are shown for two DSB (Lobrich et al. 1996, Newman et al. 1997, 2000,Stenerlow et al. 1999, Hoglund et al. 2000, Hoglundmolecular weight regions analysed, each with errors

indicating a 68% con� dence limit. The � rst estimate and Stenerlow 2001) to include DSB that FAR withMbp-sized thresholds fails to detect. The main aimof the two, shown on the left column for each method

in table 4, is generally the lower and was obtained is to � nd correlations between radiation quality andproduction of subclasses of initial damage, such aswhen the larger DNA fragments were included in

the analysis. The second estimate was instead DSB in proximity, e.g. within the same chromatinloop structures. Indeed, these studies have found thatobtained when large DNA fragments were excluded

from the data analysis, giving more weight in the the production of fragments of critical sizes, thoughtto correspond to the interaction of charged particleevaluation of the DSB yields to the smaller DNA

fragments. tracks with the chromatin looped structures, shows amore pronounced dependence on LET, whereas totalyields of DSB show little variation with LET.4. Discussion In this work, several mathematical methods wereemployed to quantify the initial induction of DSB byPFGE initially used the FAR approach which gave

a larger observable molecular weight spectrum, com- ionizing radiation. The yields of DSB presented heredepend on the method used (FAR, BDRB and directpared with CFGE, very low levels of background

damage ( < 5% of the signal available) and relatively quanti� cation). Also, for each method, the estimatefor the extrapolated total yield of DSB depended ongood sensitivity ( < 5 Gy). It is known, however, that

FAR is not linearly related to the number of breaks which molecular weight region of the experimentaldata was used in the non-linear regression, asinduced, i.e. the dose (Blocher 1990) , and that an

accurate mathematical model is needed to calculate reported in table 4. This was shown with the FAR

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radiation. This has only been observed in somestudies (Hoglund and Stenerlow 2001) , as in mostcases DSB induced by X-rays or c-rays could beadequately described by random inductionmechanisms (Newman et al. 1997, 2000).

To test the reproducibility of the random breakagepredictions, implemented in the FAR approach, analternative and independent method of quanti� cationof DSB was employed. The BDRB method is basedon random breakage and was used to analyse frag-mentation patterns at � xed dose points. In this case,the random breakage model is used to predict theshape of the experimental distributions over thewidest possible molecular weight range. Figure 3showed two examples of BDRB regressions, wherethe � tting region was deliberately limited to fragmentssizes >100 kbp. When smaller fragments wereincluded in the BDRB regressions, the estimates forthe extrapolated total DSB yields were always largerthan in the case shown in the picture. When includingfragments <100 kbp in the analysis, the BDRB

Figure 5. (a) Results of the BDRB regression analysis performed curves are forced to predict fragmentation in excessseparately on each of the experimental datasets available to what the upper molecular weight regions wouldin the study. Symbols, with error bars, are best value Ô SE suggest, thus yielding larger estimates for m, the totalfrom regression analysis with equation (11). Full symbols

DSB yield. Just as the yields estimated with the FARshow extrapolated total yields of DSB from 100 Gy a-particle irradiation data, open symbols relate to 115 Gy, method were larger the smaller the threshold size,X-rays data. Experimental interval 225 kbp–5.7 Mbp: the yields obtained from the BDRB method weresingle BDRB regression of X-ray data ( ), or BDRB larger when fragments <100 kbp were included inX-ray dose–response ( ), single regression of a-particles the analysis (data not shown). Overall, this con� rmsdata ( ) or a-particles BDRB dose–response ( ).

that it is not possible to describe uniquely an observedExperimental interval 225–825 kbp: single BDRB regres-sion of X-ray data ( ] ), a-particles BDRB single regression fragmentation pattern with a method based on( ), a-particles BDRB dose–response ( ). Experimental random breakage. As � gure 3 shows, with a-particles,interval 225–285 kbp: X-ray BDRB dose–response ( ), below 500 kbp there is an increased signal relative toa-particles BDRB dose–response ( ). Results obtained controls than that observed from X-rays. The higherfrom the BDRB analysis shown in this plot are averaged

eVectiveness in the production of fragments of theseand summarized in table 2. (b) Results obtained with the‘direct quanti� cation’ method applied to each separate sizes has often been interpreted as due to the produc-experiment. Open symbols: X-ray single dose 115 Gy, tion of correlated breaks at chromatin loop structuresbold symbols: a-particles single dose 100 Gy. Error by particle track traversals (Newman et al. 1997,bars are SE from a single experiment. Interval Hoglund et al. 2000) . With the BDRB model, it was48 kbp–5.7 Mbp: X-rays ( ), a-particles ( ), experi-

also found that the � tting curves often fell above themental interval 12–825 kbp: X-rays ( ] ), a-particles ( ).Interval 12–225 kbp: X-rays ( ), a-particles ( ). Values experimental data for a-particle-induced DSB, in theshown are reported, averaged for each group, in table 2 region >1 Mbp. This supports the idea that denselyand are also compared with those obtained with other ionizing radiation is less likely to produce Mbp-sizedmethods in table 4. fragments, originating from sparsely located DSB.

This was also observed with the FAR approach whenthe largest fragment sizes were included in the ana-method in table 1 and � gure 2 where, by lowering

the molecular-weight threshold sizes, the extrapol- lysis. The yields obtained with the BDRB approachare reported in table 2 for three molecular weightated total DSB yields increased as more distant

breaks were excluded. Data analysis with the FAR regions analysed, and in table 4 for comparison withthose calculated by FAR analysis and ‘direct quanti-approach showed that the total DSB yields for a-

particles vary between 3.5 and 13 DSB per Gy and � cation’. a-particles RBE obtained with the FARmethod with Mbp-sized molecular weight thresholdsGbp (table 1) depending on whether the signal is

integrated below 5.7 Mbp or below 48 kbp. Results are signi� cantly diVerent from those obtained withthe BDRB method.from FAR analysis in this study also show deviations

from a random induction of DSB for sparsely ionizing The third method used was ‘direct quanti� cation’.

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Table 3. Results of the application of the ‘direct quanti� cation’ method with the supplementary use of random breakage to extrapolateto total DSB yield per cell.


Radiation quality 48 kbp–5.7 Mbp 12–825 kbp 12–225 kbp

110 keV mm Õ 1 a-particles 5.6 Ô 0.2 ( ) 8.2 Ô 0.4 ( ) 10 Ô 2 ( )240 kVp X-rays 4.7 Ô 0.2 ( ) 5.5 Ô 0.5 ( ] ) 6 Ô 2 ( )a-particle RBE 1.19 Ô 0.07 1.5 Ô 0.1 1.6 Ô 0.5

Analysis is restricted to the 115 Gy dataset for X-ray experiments and the 100 Gy dataset for a-particle irradiation experiments.Values are the mean Ô SEM from the results of the application of the ‘direct quanti� cation’ method on each separate experiment,shown in � gure 5(b). Means were calculated separately per three molecular weight regions. Results from individual experiments areplotted in � gure 5(b), with a diVerent symbol for each molecular weight region reported. The symbols shown in parentheses indicatethat the averages were obtained from experiments that are marked with the same symbols in � gure 5(b).

Table 4. Summary of total extrapolated DSB yields.

DSB·Gy Õ 1 GbpÕ 1

FAR BDRB Direct quanti� cation

Radiation quality <5.7 Mbp <48 kbp 225 kbp–5.7 Mbp 225–285 kbp 48 kbp–5.7 Mbp 12–225 kbp

110 keV mm Õ 1 a-particles (3.4 Ô 0.2) (13 Ô 1) (7.0 Ô 0.3) (9 Ô 2) (5.6 Ô 0.2) (10 Ô 2)240 kVp X-rays (4.5 Ô 0.3) (10 Ô 1) (5.5 Ô 0.4) (6.2Ô 1.4) (4.7 Ô 0.2) (6 Ô 2)a-particle RBE (0.76 Ô 0.07) (1.3 Ô 0.2) (1.3 Ô 0.1) (1.5Ô 0.5) (1.19 Ô 0.07) (1.6 Ô 0.5)

Total yield of DSB per cell, for X-rays and a-particles, calculated from data analysis with the FAR approach, the BDRB methodand the direct quanti� cation of DSB, adapted from tables 1–3. For each method, two estimates for the DSB yields are shown, with itsSE for the FAR method and with SEM for the ‘direct quanti� cation’ and the BDRB method. The smaller DNA fragments werecontributing less than the larger fragments to the � rst estimate, shown on the left, for each method. For the second estimate, theanalysis was more focused on the contribution of the smaller DNA fragments.

Although this method is not derived by random resulting RBE for DSB yields can dramaticallychange from <1 to values >1, from the same experi-breakage concepts, it is also supported by random

breakage when extrapolating to total DSB yields per mental data sets, depending on analysis method. The‘direct quanti� cation’ method, under conditions satis-cell, to avoid risk of signi� cant underestimation of

the total DSB yields. With this method, experiments fying the ‘high dose’ approximation, could be pre-ferred over the other methods used in this study,that allowed counting breaks leading to the smallest

fragment sizes resulted in the highest estimates for since it does not make assumptions regarding theDSB induction mechanism, which otherwise maythe total DSB yield, as shown in table 3. This provides

additional evidence for non-random breakage. have to be considered to be random.Random breakage concepts are not only largelyTable 4 shows that RBE obtained by ‘direct quanti-

� cation’ do not diVer signi� cantly from those used for assessing the extent of initial DNA damage.FAR dose–response curves are often employed asobtained with the BDRB method. As found in

Stenerlow et al. (1999, 2000) , both the methods of calibration curves for the number of DSB presentduring rejoining (e.g. Tabocchini et al. 2000, Friedlfragmentation analysis in this study lead to DSB

yields per cell larger than those obtained by conven- and Kellerer 2001) based on several assumptions.When using a fragmentation model based on randomtional FAR with Mbp thresholds.

The main conclusion from this work is that breakage to describe the changes in the molecularweight distributions during rejoining, it is implicitlymethods of DSB analysis based on random breakage

should be used carefully, specifying exact details of assumed that DSB are ‘removed’ (1) independentlyof the size of the fragment they relate to, (2) independ-the analysis performed. Given a DSB quanti� cation

method, its application to an experimental dataset ently of their vicinity to other DSB and (3) in anycase they will be removed, even it they have beencan lead to signi� cantly diVerent results depending

on the molecular weight region observed. By using involved in complex misrejoining events to restitutethe original distribution of intact chromosomesseveral DSB quanti� cation methods, each supported

by random breakage concepts to estimate total DSB (Pinto et al., in preparation). Experimental evidencefor DSB misrejoining events and formation ofyields per cell, this study suggests that random break-

age concepts are not universally applicable. The chromosome aberrations together with micronuclei

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P., 1994, DNA double-strand breaks induced by low energyformation, after exposure to ionizing radiation, indi-protons in V79 cells. International Journal of Radiation Biology,cates that these assumptions may not hold. RBE for65, 529–536.the induction of DSB by diVerent radiation ions are Belli, M., Cherubini, R., Dalla Vecchia, M., Dini, V.,

generally <2, even if sophisticated implementations Moschini, G., Signoretti, C., Simone, G., Tabocchini,M. A. and Tiveron, P., 2000, DNA DSB induction andof random breakage are used, as suggested in thisrejoining in V79 cells irradiated with light ions: a constantstudy and reviewed by Prise et al. (2001) . For the� eld gel electrophoresis study. International Journal of Radiationinterpretation of DNA repair experiments and, moreBiology, 76, 1095–1104.generally, for a comprehensive description of the BloÈcher , D., 1990, In CHEF electrophoresis a linear induction of

eVects of ionizing radiations, it is of paramount dsb corresponds to a nonlinear fraction of extracted DNAwith dose. International Journal of Radiation Biology, 57, 7–12.importance to characterize the relevant initial lesions

Burkart, W., Jung, T. and Frasch, G., 1999, Damage pattern asand to � nd a reliable and user-friendly method toa function of radiation quality and other factors. Comptesquantify them.rendus de l’Academie des sciences—Serie III, 322, 89–101.Computer models have become available to inter- Cedervall, B. and Kallman, P., 1994, Randomly distributed

pret the interaction of particle tracks with higher- DNA double-strand breaks as measured by pulsed � eld gelelectrophoresis: a series of explanatory calculations. Radiationorder chromatin structures (Friedland et al. 1998, 1999,and Environmental Biophysics, 33, 9–21.Sachs et al. 1998, 1999, Ponomarev et al. 2000) or with

Cedervall, B. and Lange, C. S., 1998, Analysis of DNA massthe 30 nm chromatin � bre (Holley and Chatterjeedistributions in pulsed-� eld gels to determine DNA dsb1996) supported by experimental measurements induction and rejoining kinetics. In Abstracts of the 46th Annual

(Rydberg 1996, Rydberg et al. 1998) . Sophisticated Meeting of the Radiation Research Society (Nashville: RadiationResearch Society), P20–379.models of chromatin organisation at larger scales are

Cedervall, B. and Radivoyevitch, T., 1996, Methods for analysisalso being developed and tested experimentally (Sachsof DNA fragment distributions on pulsed � eld gel electro-et al. 1995, Yokota et al. 1995, Ostashevsky 1998, 2000)phoresis gels. Electrophoresis, 17, 1080–1086.and could help with understanding the formation of Cedervall, B., Wong, R., Albright, N., Dynlacht, J., Lambin,

initial clustered damage and chromosome aberrations. P . and Dewey, W. C., 1995, Methods for the quanti� cationof DNA double-strand breaks determined from the distribu-Progress in these � elds is promising but will need thetion of DNA fragment sizes measured by pulsed-� eld gelsupport of experimental techniques to validate theelectrophoresis. Radiation Research, 143, 8–16.models and to detect the most critical types of initial

Contopoulou, C. R., Cook, V. E. and Mortimer, R. K., 1987,damage. Owing to the complexity of the cell nuclear Analysis of DNA double strand breakage and repair usingarchitecture, chromatin structure, charged-particles orthogonal � eld alternation gel electrophoresis. Yeast, 3,

71–76.track deposition patterns and interpretation of theCook, V. and Mortimer , R., 1991, A quantitative model of DNAinitial damage by the cellular repair machinery,

fragments generated by ionizing radiation, and possiblecooperation of modelling and experimental studiesexperimental applications. Radiation Research, 125, 102–106.may be the only approach to follow in order to help Folkard , M., P rise, K. M., Vojnovic, B., Davies, S., Roper,

understand the mechanism of radiation-induced cell M. J. and Michael, B. D., 1989, The irradiation of V79mammalian cells by protons with energies below 2 MeV.injury.Part I: Experimental arrangement and measurements of cellsurvival. International Journal of Radiation Biology, 56, 221–237.

Friedl, A. A. and Kellerer , A. M., 2001, Comments onAcknowledgements‘Underestimation of the small residual damage when meas-

The authors thank Dr Andrea Malcolmson for uring DNA double-strand breaks (DSB): is the repair ofradiation-induced DSB complete?’ International Journal ofperforming � ow-cytometry measurements of AG-Radiation Biology, 77, 249–254.01522B cell-cycle distribution at con� uence and

Friedland, W., Jacob, P ., P aretzke, H. G., Merzagora, M. andMr Stuart Gilchrist for 238Pu a-particle and 240 kVp Ottolenghi, A., 1999, Simulation of DNA fragment distri-X-rays dosimetry. They also thank Dr G. Simone butions after irradiation with photons. Radiation andfor providing details of PFGE protocol III. M. P. is Environmental Biophysics, 38, 39–47.

Friedland, W., Jacob, P ., P aretzke, H. G. and Stork, T., 1998,grateful to his colleagues, Dr H. C. Newman andMonte Carlo simulation of the production of short DNAProfessor M. Durante for introducing him to thefragments by low-linear energy transfer radiation using� eld of Radiation Research. The work was supported higher-order DNA models. Radiation Research, 150, 170–182.

by the Cancer Research Campaign, the Sir Samuel Goodhead, D. T., 1994, Initial events in the cellular eVects ofScott of Yews Trust and the Nuclear Fission Safety ionizing radiations: clustered damage in DNA. International

Journal of Radiation Biology, 65, 7–17.Program of the European Community.Heilmann, J., Taucher-Scholz, G. and Kraft, G., 1995,

Induction of DNA double-strand breaks in CHO-K1 cellsby carbon ions. International Journal of Radiation Biology, 68,References153–162.

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