COMMENTARY

The emperor wears no clothes in the field of carcinogen risk assessment:ignored concepts in cancer risk assessment

James E.Trosko� and Brad L.Upham

National Food Safety Toxicology Center, Department of Pediatrics andHuman Development, Michigan State University, East Lansing,MI 48824, USA

The following is a position paper challenging the paradigmthat ‘carcinogen 5 mutagen’, and that the current rodentbioassay to predict risks to human cancers is relevant anduseful. Specifically, we review current observations con-cerning carcinogenesis that might lead to another approachfor assessing the identification of human carcinogenichazards and the risk assessment that chemicals mightpose. We give a brief review of the multistage and multi-mechanism process of cancer in a tissue that involves notonly genotoxic but also epigenetic events, and the import-ance of stem and progenitor cells in the development ofcancer. We focus on the often ignored ‘epigenetic’ effectsof carcinogens and the role of cell communication systemsin epigenetically altering gene expression that leads to animbalance of cell proliferation, differentiation and apop-tosis in a tissue that can contribute to the cancer process. Todraw attention to the fact that the current paradigm andpolicy to test toxic chemicals is often misleading and incor-rect, we discuss how oxidative stress, in spite of the DNAdamaging data, most probably contributes to cancer at theepigenetic level. Additionally, we briefly review how thismutagenic concept has greatly diverted attention awayfrom doing research on the lower molecular weight, non-genotoxic, polycyclic aromatic hydrocarbons (PAHs), andhow these low molecular weight PAHs are etiologicallymore relevant to the disease potential of environmentalmixtures such as cigarette smoke.

Introduction: ‘Cancer is not a disease of a singlecell’—limitations of a reductionalist’s view

‘ . . .The cancer problem is not merely a cell problem, it is aproblem of cell interaction, not only within tissues, but alsowith distal cells in other tissues. But in stressing the wholeorganism, we must also remember that the integration of nor-mal cells with the welfare of the whole organisms is broughtabout entirely by molecular messages acting on molecularreceptors’ (1).

The goal of this rather iconoclastic challenge is todraw attention to the current paradigm and policy to testtoxic chemicals that are correlated with cancer. Specifically,most still believe that cancer-causing chemicals are linked toDNA damage and mutations and that the current rodent bioas-say to predict hazards and assess risks to human cancersis relevant and useful. While it could be argued that this

‘carcinogen 5 mutagen’ paradigm has long been challenged,one needs only to examine the huge number of papers that arestill published where this paradigm still drives the use of in vitro‘genotoxicity’ assays that are misinterpreted, and where bothgovernment regulatory agencies and pharmaceutical/industriallabs still require the use of these questionable assays. Specif-ically, we wish to review current observations concerningcarcinogenesis that might lead to another approachfor assessing the human carcinogenic risk that chemicalsmight pose.

Along with Potter’s insight and the old adage, ‘The whole isalways greater than the sum of its parts’, any best approach forassessing the human carcinogenic risk after chemical exposurewill be confronted by no easy solutions. To begin with, theparadigm, ‘carcinogens as mutagens’ (2), illustrates the pointwe wish to make, namely, that carcinogenesis is ‘more’ thanmutagenesis! What would that ‘more’ be? Clearly, when a cell(an adult stem cell; a progenitor cell or a terminally differen-tiated cell) is exposed to any chemical, there is a possibility ofDNA damage and mutations in either or both nuclear andmitochondrial DNA. In addition, cell death by either necrosisor apoptosis could be the result. Finally, epigenetic alterationscould ensue which, then, could alter the cell’s commitment tocell division, cell differentiation, apoptosis, adaptive responsesof differentiated cells or senescence.

In the recent past, the term, ‘epigenetic mechanism’, was avague, non-universally accepted term. However, moleculardemonstrations have now shown that genes in cancer cellscan be abnormally expressed or that certain toxic/carcinogenicchemicals, without DNA-damaging capacity or mutagenicpotential, can alter the patterns of gene expression by modi-fications of methylation and acetylation of DNA and histones(3). Almost without reference to the introduction of DNAmicro-array technology, which measures altered gene expres-sion, much attention has been unconsciously focused on the roleof epigenetic changes occurring during the carcinogenic pro-cess. It is as though a kind of risk assessment ‘schizophrenia’occurs in that the combination of using so-called in vitro geno-toxicity assays, rodent bioassays, detection of DNA lesions andmutations in cells of tumors and the monitoring of altered geneexpression, using powerful DNA micro-array technologies, co-exist without critical examinations of what is being measured.What does it all mean and what does it do to the prevailingparadigm shaping the risk assessment of potentially cancer-causing chemicals?

At the same time that all these cancer-associated chemicalspresumably inducing lesions in DNA of various cell types intissues of exposed animals, they induce intracellular signalingand alter gap junctional intercellular communication (GJIC)(4). The broadest definition of an ‘epigenetic’ change inducedin a cell is that which alters the expression of the informa-tion of the genome at the transcriptional, translational or

�To whom correspondence should be addressed. Tel: 11 517 432 3100, Ext. 188; Fax: 11 517 432 6340; Email: james.trosko@ht.msu.edu

# The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

All rights reserved. For permissions, please email: journals.permissions@oupjournals.org 81

Mutagenesis vol. 20 no. 2 pp. 81--92, 2005 doi:10.1093/mutage/gei017Advance Access publication 22 March 2005

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post-translational levels. It could occur as a heritable trans-criptional change in proliferating cells or as a change in theexpression of a stem cell to terminally differentiate or apop-tose. However, altered gene expression is usually precededby changes at the cell signaling level that governs transcrip-tional changes, as well as alterations in cell--cell signalingwithin tissues. While epigenetic alterations are sometimesdefined as ‘inherited’ non-mutagenic alterations found in theexpression of genes, it must be recalled that in vivo, chemicalsaffect the few stem cells, the progenitor and terminally differ-entiated cells. Stem cells induced to differentiate or apoptoseby toxic chemicals, rather than proliferate, do so by epigeneticmechanisms when the cells do not proliferate. Moreover,when a terminally differentiated cell responds to a potentiallytoxic chemical, it might express ‘stress’ genes. In addition,proliferating cells in Go phase can be induced to alter its geneexpression transiently, to express genes for initiating cell cycleentrance but which return to the original transcription stateafter proliferation. Therefore, we chose to include the broaderdefinition of epigenetic changes, which can occur in bothproliferating and non-proliferating cells. The fact is thatmany chemicals can contribute to the carcinogenic processwithout inducing mutations or cell necrotic death. DDT, phe-nobarbital, saccharin, peroxisome proliferators, etc. are allexperimentally known to contribute to the carcinogenic processin rodents.

Therefore, the real challenge is: ‘How can one assess theepigenetic potential of a chemical’s contribution to humancancer when it must be measured either in vitro or in experi-mental animals?’ No 2D in vitro assay, using either normal,primary rodent or human cells, or any immortalized normal orcancer cell line, can mimic the in vivo, human in situ conditionof complex interactions between stem cells, proliferating pro-genitor cells and terminally differentiated cells within a tissueand between tissues (i.e. stromal--epithelial interactions) (5).Moreover, historic evidence has demonstrated the limitationsof using experimental animals (6) and epidemiological studiesfor a number of obvious reasons.

The fundamental issues raised by this challenge seemto include: (i) carcinogenesis in animals and human beings isa multistage, multimechanism process (i.e. the initiation/promotion/progress model) (7); (ii) while experimental animalsand human beings are fundamentally and biologically alike, inprinciple, they differ in many ways that could affect any one orall those stages; (iii) in both animals and human beings, indi-vidual genetic-, developmental stage-, gender-, dietary-, lifestyle- and environmental-factors can, and do, influence each ofthese stages; (iv) since the experimental animal and humanbeing consist of a hierarchical/cybernetic organization of mul-ticell types, each interacting with cells within and betweentissues (8), use of cell-free, molecular targets, and 2D cellcultures, using either ‘pure’ animal or human primary orimmortalized/tumor cells, will never mimic the in vivo situation(9). This is not to suggest that the use of molecular/biochemicalor 2D cell cultures of primary or immortalized rodent or humancells will not generate a much needed mechanistic understand-ing of the carcinogenic process, as has experimental rodentcarcinogenesis studies, particularly using the initiation/promo-tion/progress models, as well as transgenic and specific geneknock-outs/knock-ins; (v) delineation of mechanisms of eachstage of carcinogenesis will be needed, particularly the poten-tial of thresholds for epigenetic agents; (vi) the study of thein vitro mechanisms of carcinogenesis that might be identical

to the carcinogenesis process in human beings; and (vii) therole of mixtures of carcinogenic factors (both endogenousand exogenous) that could influence any of the three steps ofcarcinogenesis to be additive, synergistic or antagonistic. Aswonderful as these approaches have been and will continueto be, more will be needed.

The irony of this analysis is that, while we argue that currentin vitro assays to measure mutagenesis, cytotoxicity or even‘epigenetic’ potential of chemicals are not adequate for accur-ate extrapolation to humans, the use of current animal bio-assays will also fall far short of adequacy. Since space willnot permit an attempt for rationalizing the use of 3D, humanadult stem cells (9) to attempt to breach this impossible task,the bottom line is that the use of human stem cells is where webelieve that this field must aim for.

Limitations of animal bioassays

While not wedded to using animal bioassays for the pre-testingof new chemicals for their potential risk in contributing tohuman carcinogenesis, one has to remember that today’s bioas-say protocol assumes that the induction of cancer in rodents,after an exposure to sublethal concentrations for the lifetime ofthe animal, generates a meaningful risk extrapolation informa-tion to human beings. However, such assumptions are overlysimplistic. Since carcinogenesis is a multistage, multimechan-ism process, one would have to either assume that (i) the highdose-exposure of the single chemical being tested can induceall the mechanisms by itself; or (ii) the high dose of the chem-ical upsets the normal homeostatic control of the physiologicalstate of the animal, which, in turn, affects one aspect of themultistage process.

Given that the cells of a tumor appear to be monoclonallyderived from a single cell (10), and in spite of their heterogen-eity and genomic instability, and given that ‘initiators’ seem toinduce a stable, irreversible event, both mutagenesis (a irre-versible change in the qualitative or quantitative nature of thegenome) and a stable ‘epigenetic’ mechanism (an alteredexpression of a gene) can contribute to the initiation phase ofcarcinogenesis. Short of directly testing changes in DNAsequences after exposure to an agent that is or could be aninitiator, the problem is that the use of short-term in vitroassays to detect phenotypic changes, which are used as surro-gates for mutations, will always generate too many false posi-tives (11,12). Even measuring mutations in the cells of tumorscreated in rodents after exposure to a carcinogenic agent couldbe interpreted incorrectly if the agent acted to select aspontaneously generated mutation in an oncogene or tumorsuppressor gene (13).

Agents that can promote tumors appear to act ‘epigenetic-ally’, i.e. the process of promotion can be interrupted; appear tohave threshold levels of action; and can be ameliorated byantipromoters (14). In addition to being species-, gender- andorgan-specific, including developmental stage-specificity, pro-moting agents can act to select, clonally, the initiated cell bymitogenesis and/or blockage of apoptosis. Irritation or celldeath or cell removal by surgery, can also act as a promotingcondition of the initiated cell (14).

Those agents, that, after the terminal stages of carcinogen-esis, impart invasive, metastatic and angiogenic properties(progression phase of carcinogenesis), seem to do so in a stablemanner. Both irreversible mutagenic and stable epigeneticevents could contribute to this phase.

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Blinded by the ‘carcinogen as mutagen’ paradigm,epigenetic mechanisms have been ignored!

As these reviewers look back at this complex problem, we seewhere Hanahan and Weinberg’s comment is extremely relev-ant to the challenge: ‘those researching the cancer problem willbe practicing a dramatically different type of science than wehave experienced over the past 25 years. Surely much of thischange will be apparent at the technical level. But ultimately,the more fundamental change will be conceptual’ (15). Wethink the major paradigm that has paralyzed any reasonablesolution to understand the tens of thousands of published can-cer studies is the paradigm ‘carcinogens as mutagens’ (2). It isthe result of the classic ‘lamp post effect’. We look for the lostkeys under the lamp post because that is where there is light.Based on the fact that (i) mutations (gene and chromosomal)are found in cancers in animals exposed to chemicals (ii) thesechemicals are sometimes tested positive in the accepted andeasy to run, but highly misleading, in vitro assays to detect‘mutations’ (using phenotypic markers as surrogates forchanges in DNA sequences) or (iii) predispositions to cancersdo occur in humans that inherit germ-line mutations, it wasassumed that mutagenesis was the ‘end all, be all’ for carcino-genesis. It goes without much examination that, by not usinganimal bioassays in an initiation/promotion/progression proto-col, and by not testing chemicals at the extreme limits of theirtoxic tolerance, which is unrealistic for real-life human expos-ures, no realistic mechanistic understanding can be derived foruse in any risk assessment model. (Here we assume the bestcancer risk assessment model must include mechanistic data.)The high-dose protocol used to test potential chemical carcino-gens, and misclassification of the presumed mechanism ofcarcinogen action of the chemical used, based on in vitrogenotoxicity assays, which, themselves, are chock-full of lim-itations and artifacts, cannot lead to any concise interpretationof the underlying mechanism of action. Viewing the use ofinsensitive in vitro assays used to understand the equivalentinsensitive rodent bioassays can be likened to the blind leadingthe blind.

Promoters as inducers of oxidative stress do not makethem ‘genotoxic’

Even when the promotion process appeared to involve a mito-genic or inhibited apoptotic process to expand the initiated cell(assumed to be induced by a chemical mutagenic/carcinogen,to ‘save’ the paradigm), investigators claimed that mutationsneeded to be fixed during the promotion process. While thishypothesis is, in part, true, it does not explain the mechanismsof agents that stimulate mitogenesis or inhibit apoptosis, bothbiological processes involved in the promotion phase (16). AsWeinstein has pointed out, ‘ . . . extensive proliferation occursduring normal fetal and embryonic development, as well as inthe continuous renewal in the adult of the entire skin,gastrointestinal epithelium and bone marrow . . . Thus, excess-ive cell proliferation per se is probably not the exclusive caus-ative factor in human breast cancer’ (17). Mutagens usuallyinduce necrosis and stop cell proliferation. After his studies onmutations in human tissues, Thilly (13) concluded that ‘directmeasurements of kinds and numbers of point mutations havefound no clear relationship with exposure to environmentalagents, save for sunlight in the skin’. He goes on to offer analternative explanation of those environmental chemicals, clas-sified as ‘mutagens’ from short-term tests for genotoxicity,

wherein they select pre-existing cells with mutations ratherthan induce oncomutations. This supports the hypothesis pre-viously suggesting that most chemical ‘carcinogens’, tested inanimal bioassays and in in vitro genotoxicity assays, are epi-genetic agents which are tumor promoters, not ‘epigenetic’carcinogens (14). Spontaneously initiated cells, induced byeither errors in repair of endogenous or exogenous agents orby errors in replication of normal DNA templates, are probablythose that are promoted by the so-called ‘carcinogenicmutagens’.

In order that it might not be missed, we suggest that chem-icals, associated with carcinogenesis, might indeed induceDNA damage at high concentrations in certain kinds of cells,which probably die or will never divide (‘dead cells do not giverise to cancers’), and which might induce DNA lesions inmitochondrial DNA, are probably ‘epigenetic’ in nature, notmutagenic or genotoxic. If these concentrations are neededin vitro to induce DNA lesions and the recovery of phenotypicchanges called mutations, the same concentrations in vivowould probably induce acute tissue damage, if not the deathof the organism. To demonstrate that electrophiles of metab-olized chemicals can interact with naked DNA or findingDNA lesions in extracted DNA from animal organs containingcells, most of which are probably not the target cells for cancer,do not constitute rigorous proof of the chemical’s mechanismof action as an animal carcinogen. In other words, it would behighly unlikely that these so-called genotoxic chemical car-cinogens do contribute to carcinogenesis as the mutagens of themutations found in the oncogenes or tumor suppressor genes ofthe tumor found in the chemically treated animal. The latterstatement should justify the claim that this review should berather ‘iconoclastic’ and a ‘paradigm-buster’.

Cha et al. (18) also reported that N-nitroso-N-methylurea-induced rat mammary tumors arise from cells with pre-existingoncogenic Hras1 gene mutations. While this and other similarstudies have been swept under the regulatory rug, it alsodemonstrates that a chemical, interpreted as a mutagen throughin vitro assays and shown to be a carcinogen in a bioassay, wasassumed to induce mutations found in oncogenes in the tumorsof the chemically exposed animal. The chemical was mostlikely an epigenetic agent that promoted a pre-existing spon-taneously initiated cell. Even several in vitro transformationassay studies, which again seem to be ignored, have provideddata that challenge the prevailing paradigm that a chemical,which induces rodent transformation in vitro, must be a muta-gen. Brookes et al. (19) and Mass and Austin (20) showed that7,12-dimethylbenz[a]anthracene (DMBA), the ‘quintessential’chemical mutagenic/carcinogen, did not mutate the Ki-ras andHa-ras oncogene of the DMBA-transformed cells.

Apparently, many chronic diseases affiliated with oxidativestress, such as cancer, are not always a consequence of tissuenecrosis, DNA mutations, or protein damage but rather, owingto an altered gene expression through epigenetic mechanisms(16,21). Another example that oxidative reactions do not con-tribute primarily to the genotoxic, initiating phase of cancer,are the results of rodents treated with organic and hydrogenperoxides in the two--stage cancer model systems, in whichthese compounds exhibited tumor promoting and not initiat-ing activity (22--25), indicating that these oxidants are notmutagens but rather epigenetic toxicants. In the past 15 years,considerable research in oxidative stress has shifted fromunderstanding how oxidations lead to macromolecular dam-age, to comprehending how reactive oxygen species (ROS)

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reversibly control the expression of genes at non-cytotoxicdoses (26). In this respect, at least 127 genes and signal trans-ducing proteins have been reported to be sensitive to reductiveand oxidative (redox) states in the cell (26).

Although many intracellular signaling pathways are knownto be redox-sensitive, the most studied signal transductionfactors are mitogen-activated protein kinases (MAPK) andnuclear factor-kB (NF-kB) (26--28). These two pathwayseither directly or indirectly transduce most redox responses(26). MAPK is not only activated by ROS (29) but actuallyrequires the presence of endogenously produced H2O2 (30).This is one of several studies demonstrating that endogenousgrowth factors (extracellular ligands) generate ROS, whichare then required downstream in intracellular signaling tosuccessfully transmit their signals to the nucleus (31). Asmentioned above, the successful transmission of an extracellu-lar signal from the membrane to the nucleus through intracel-lular signaling pathways in solid tissue cell types is alsodependent upon intercellular signals through gap junctions(16,21). Not surprisingly, ROS have also been demonstratedto reversibly inhibit GJIC at non-cytotoxic levels (32). If gapjunctions were not closed, then the H2O2 generated by extra-cellular ligands could escape through gap junctions into neigh-boring cells, thereby potentially diluting to a subthreshold levelthat would be insufficient for MAPK-dependent activation oftranscription factors. These examples demonstrate how extra-,intra- and inter-cellular signaling pathways might interactto coordinate the epigenetic expression of genes in responseto ROS.

Antioxidants have also been demonstrated to serve as sub-cellular messengers for normal cell function (26). For example,a major H2O2-scavenging pathway is the two-electron reduc-tion of H2O2 catalyzed by glutathione peroxidase, whichclearly serves as a protective role against peroxide-dependentoxidative injury. However, depletion of intracellular pools ofglutathione (GSH), by inhibiting the rate-limiting step of itsbiosynthesis, paradoxically reverses the biological effect ofH2O2 in several systems. For example, inhibition of GJIC(32), induction of c-jun (33) and activation of NF-kB (34)by H2O2 was completely reversed when the cellular systemswere depleted of GSH, which indicates that these signalingpathways not only required H2O2 but also GSH. Inhibition ofGJIC and the induction of early-response genes are hallmarksof tumor promotion and in the results just described, a reduc-tion in the natural antioxidant GSH could also potentiallyprotect a cell from proliferative responses to extracellular lig-ands. Apparently, assessment of the true risk that oxidativestress poses to human health will need to move beyondthe genotoxic, DNA-damaging paradigm, and incorporate anunderstanding of how oxidative reactions contribute to theepigenetic expression of genes.

Polycyclic aromatic hydrocarbons: the mutagenicconcept falls short of a comprehensive understandingof carcinogenic potential

In many cases, it has been argued that tumor-promoting chem-icals can induce oxidative stress. Ergo, by definition by some,they are ‘mutagens’, yet when tested in in vitro assays, theyprove not to be DNA damaging agents or mutagens (TCDD,TPA, DDT, phenobarbital, peroxisome proliferators, etc.). Anexcellent example is pentachlorophenol, which has been shownto induce oxidative stress (35), to be a tumor promoter and not

an initiator in mouse liver (36), and can inhibit GJIC at non-mutagenic, non-cytotoxic conditions, both in vitro and in vivo(37). Another example is chemicals from cigarette smoke, longbeen thought to be primarily a carcinogenic mutagen, becauseof the identification of high molecular weight (five- to seven-ringed), mutagenic polycyclic aromatic hydrocarbons (PAHs)such as benzo[a]pyrene. Yet, the most abundant PAHs incigarette smoke are the low molecular weight fractions, par-ticularly the three- and four-ringed PAHs in which the methyl-ated anthracenes and phenanthrenes are 62 times higher thanbenzo[a]pyrene and benzo[e]pyrene (Table I) (38).

Considering that the fraction of cigarette smoke containingthe three- and four-ringed PAHs is highly co-carcinogenicwhen applied to the skin of mice treated with benzo[a]pyrene(Figure 1) (39), and that cigarette smoke is a strong promoterand a weak complete carcinogen (40--43), suggest that thisfraction could significantly contribute to cancer. Ten to fifteenyears after giving up smoking, the ex-smoker faces the samelow risk of developing cancer of the upper digestive tract, thelung, the pancreas and the urinary tract as the non-smoker(Figure 2) (44). This fact strongly suggests that cigarettesmoke contributes to the non-genotoxic and reversible phasesof cancer. Contribution of cigarette smoke to the non-genotoxicphase of cancer is particularly important. Early work on thecarcinogenicity of cigarette smoke condensates strongly indi-cated that the neutral fractions, which contained primarilyPAHs, were the most carcinogenic fractions, but that theconcentrations of the most prominent complete carcinogens,

Table I. Concentration of selected PAHs in cigarette smoke as reportedby Severson et al. (38)

PAH ng/cigarette

Methylphenanthrene and anthracene 1494Methylpyrene 203Phenanthrene 362Anthracene 173Pyrene 162Fluoranthene 123Benz[a]anthracene, chrysene, triphenylene 76Benzo[a]pyrene and benzo[e]pyrene 24

623

Fig. 1. Co-carcinogenic activity of the tar fractions of cigarette smokecontaining 2-, 3-, 4- and 41 ringed PAHs as reported from Hoffmannet al. (39). BaP, benzo[a]pyrene.

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i.e. benzo[a]pyrene, was far too low to account, by themselves,for the carcinogenic activity of the condensates (40,45). Fur-thermore, the much less studied area of co-carcinogenesis is acloser fit to the extended exposure of human smoking than thecomplete carcinogenic nature of selected PAHs from cigarettesmoke condensates (40). Therefore, understanding the bio-logical effects of these three- and four-ringed PAHs, whichare prevalent in cigarette smoke and possess co-carcinogenicactivity, on cell signaling pathways relevant to the epigenetic,non-genotoxic phase of cancer is important. These three- andfour-ringed PAHs are not only the most prevalent PAHs incigarette smoke but also in many environmental systems andfood products.

To date, we have successfully demonstrated that many PAHsinhibit GJIC in pluripotent mammalian epithelial cells (46--49).The most significant results indicated that three- and four-ringPAHs containing bay regions or bay-like regions are far moreactive in inhibiting GJIC than the isomers that do not havethese structural motifs. We would like to note that the term ‘bayregion’ refers to the pocket formed by the stereically hinderedregion created by an angular benzo ring and, similarly, the term‘bay-like’ region is used to describe the angular pocket formedat the top of the benzene ring by a methyl- or chloro-group(Figure 3). These bay regions are not in reference to thoseformed by the well-known DNA reactive diolepoxides butrather refer to the unmetabolized parent structure. Our focuswas on the epigenetic effects of PAHs, which does not requirethe metabolic activation of a PAH to a chemically more react-ive electrophilic compound. The epigenetic effects of PAHs, inparticular, are poorly understood and the toxic risks of unmeta-bolized PAHs need to be reassessed at this non-genotoxic level.

More specifically, we demonstrated that the 1- and 9-methyl-or 1- and 9-chloroanthracene or 1,9-dimethylanthracene(1-meA, 9-meA, or 1-Cl-A, 9-Cl-A or DMA), which formbay-like regions inhibit GJIC as well as phenanthrene thatcontains a bay region, whereas the 2-methyl or 2-chloro-anthracene (2-meA, 2-Cl-A), which have linear configurations,do not inhibit GJIC (50). The same exact pattern of GJICinhibition with respect to dose and time that was observedwith the chlorinated-anthracene isomers compared with thenon-chlorinated PAH isomers suggests that the structure is

more important than the chemical reactivity of these com-pounds, considering that the chlorine substitution convertsthese PAHs from a neutral to a more nucleophilic compound.The methyl versus chloroanthracenes is chemically very dif-ferent but structurally quite similar, suggesting a common typeof receptor. Moreover, multiple bay-like regions do not signi-ficantly increase the potency of the compound to inhibitGJIC when compared with a compound that contains a singlebay-like region. For example, 9-methylanthracene and 9,10-dimethylanthracene possesses multiple bay-like regions, while1-methylanthracene possesses only one bay-like region; yet,these compounds all had similar dose--response curves. Inhibi-tion of GJIC by these PAHs was also a reversible process,which is consistent with the reversible nature of tumor promo-tion in vivo. Inhibition occurred in a short time period for all thePAHs, indicating that the gap junctions are being modified atthe post-translational level.

Although inhibition of GJIC may contribute to the mitogenicevents of a promoter by removing an initiated cell from growthsuppression, other epigenetic events such as the actual activa-tion of a mitogenic-signaling pathway are also required. Wehave published results showing that GJIC-active (inhibitory)PAHs activate MAPK signaling pathways, while the GJIC-inactive PAHs do not induce MAPK (50). The kinetic resultsalso indicate that GJIC activity was affected before MAPKinduction (a difference of 5 versus 15 min). These results arealso consistent with the hypothesis that a quiescent cell mustfirst be removed from growth suppression through inhibition ofGJIC and at the same time or before the onset of mitogenicevents.

The significances of these results are: (i) the three- and four-ringed PAHs, which have been determined to be non-mutagenic, are biologically active by altering cell signalingthat favors proliferation; (ii) ignoring these molecular signalingevents greatly underestimates the potential risk of these com-pounds to human health, particularly cancer; (iii) they are moreconsistent with the in vivo results of rodent systems exposed totobacco smoke, indicating that combusted tobacco mixtures arestrong tumor promoters but very weak initiators and complete

Fig. 3. Structure--activity relationship of three-ringed PAHs.

Fig. 2. The decline of lung cancer in ex-smokers as reported fromWynder et al. (97).

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carcinogens; (iv) they are more consistent with the reversiblenature of tobacco smoke as indicated by the drastic decrease incancer risk after cessation of smoking; (v) they offer novelmolecular targets for the design of new chemopreventativeand therapeutic strategies. Traditionally, the risk of theselower molecular weight PAHs to human health have beenignored precisely because of their non-genotoxic properties,and is a classic case of how the ‘carcinogens 5 mutagens’paradigm has greatly contributed to the underestimation of thetrue risk that environmental and food borne compounds pose tohuman health.

The need for a mechanistic, biological cancer-riskassessment model

One major implication of knowing the mechanism of action ofa potential chemical (Is it a mutagen, cytotoxicant or epigenetictoxicant?) is for epidemiological studies and risk analyses.Ignoring for the moment the contentious debate on ‘chemicalmutagenic--carcinogenic initiators (e.g. Do they exist?; Arethere threshold levels of exposure?; Do they cause the muta-tions in oncogenes and tumor suppressor genes in tumorsof animals exposed to the chemical’, etc.?), cytotoxicants canlead to cancers by their ability to induce compensatory hyper-plasia, an indirect tumor promoting stimuli for surviving initi-ated cells. More importantly, it is the measured opinion of thesecommentators that most chemicals associated with tumors inexposed animals or human populations are epigenetic in char-acter, by selectively cloning out of the target tissue pre-existinginitiated cells. As tumor promoters, these chemicals do work atthreshold levels (51) and must be given in a regular, sustainedand chronic fashion in the absence of antitumor promoters.Irregular exposures, exposures at subthreshold levels, orexposures that are interrupted (as stopping cigarette smoking)or are simultaneously present with antitumor promoters, willmodify the predicted outcomes. Even knowing the mechanismof action, but without understanding the characteristics of theepigenetic agents, might lead to erroneous predictions of risk.E. Franco et al. (98) have implied that, in spite of its successfultrack record, epidemiology as a discipline has become thefocus of considerable controversy concerning its usefulnessand limitations to identify cancer-causing exposures. This isbecause few cancer epidemiological studies incorporate themultistage, multimechanism concept of carcinogenesis in thedesign, execution and interpretation of their studies. The stud-ies that have done so (52,53), are yet to incorporate the newer‘ignored hallmarks’ of cancer, such as the stem cell as the‘target cells’ of cancer and the complex set of interactions towhich Potter refers (54). Even the term, ‘molecular epidemi-ology’ seems to miss the point that Potter makes in his quote.The DNA sequence alone will never predict with certainty therisk of a cancer in an individual in a population exposed to achemical.

‘Good news’--‘bad news’ as the conundrum ofchemical carcinogenesis

To these commentators, the ‘emperor wears no clothes in thefield of chemical carcinogenesis’ when it comes to the selectiveview of using chemical carcinogen data to support one’sassessment of the dangers for a particular chemical’s ability to‘cause’ cancer (55). It has been noted for some time that manychemicals, while being associated with the induction of cancersin animal bioassays, can, at the same time, in the same animal,

protect against other cancers or demonstrate the limitations ofthe animal bioassays as adequate predictors of potential humancarcinogenesis (56). Even more striking is the observation thatthe same chemical, in this case, phenobarbital, a valuablehuman anti-epileptic drug, can promote liver cancers in rodentsif given after the initiation of a post-weaned rat, but it sup-presses liver tumors if given to an initiated pre-weaned rat(57). In addition, the tumors of these differentially treated ratswere, themselves, different histologically. This suggests thatthe target cells for the initiation stage of the pre- and post-treated rats were, themselves, different.

Phenobarbital appears to act as a promoter when it inhibitsgap junction intercellular communication (58). The livertumors of the pre-weaned, initiated rat were ‘embryonic-like’(basophilic), suggesting that they might have originated fromliver stem cells, which do not seem to have expressed connexingenes or functional gap junctions, similar to other adult stemcells (54). Therefore, these pre-weaned rat tumors would not bepromoted by phenobarbital, since they have no gap junctions.Agents that cause mitogenesis of stem cells probably stimu-lated the promotion of these basophilic tumors. It seems to be afact that tumors can be characterized by a lack of functionalheterologous or homologous GJIC (54). In fact, the classicHeLa and MCF-7 cancer cell lines are cells that do not expresstheir connexin genes (59,60), whereas many other cancer cellsdo have expressed connexins that are rendered non-functionalbecause of some expressed oncogene.

Since phenobarbital was used as the example, it is interestingto note that, had regulations been in effect during the drugdevelopment and safety evaluation of this drug, it mightnever have been given human use approval because it is oneof the classic promoters for rodent liver tumors. Phenobarbitalcan ‘induce’ liver tumors in non-‘initiated’ animals at highdoses and with chronic treatment. After decades of humanuse, there has been no epidemiological evidence of liver orhead/oral cancers in human beings.

Stem cells and cell communication as ignoredhallmarks of cancer

Guided by the insight of V.R.Potter in the aforementionedquote, it should be clear that carcinogenesis occurs in acomplex in vivo environment where the single ‘target’ cellexists in a tissue where there are cell--matrix interactions,stromal--epithelial interactions (5) through soluble extracellularsignals between a few adult stem cells, their finite-life, pro-genitor daughter cells, and their lineage-N-stage, terminallydifferentiated cells and any invasive, inflammatory-relatedcells. Consequently, any cell-free, in vitro reductionalisticapproach to study mechanisms of carcinogenesis must care-fully re-integrate components before any extrapolation to thein vivo human situation. First, and foremost, resolution must bereached on the classic problem, ‘Is the ‘‘target’’ cell for initi-ation of the carcinogenic process a stem cell (61) or anydifferentiated cell that can ‘‘de-differentiate’’?’ (62). Second,the understanding of the complex integration of extra-, intra-and gap junctional intercellular communication in the targetorgan (Figure 4) must be accomplished before we can truly finda biologically based risk assessment from non-human systemsto individual human beings.

While the problem is yet to be resolved, the list of cancer‘hallmarks’ (11) includes the phenotype of ‘immortalization’as an early characteristic of all cancer cells. This implies a

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resistance to apoptosis and blockage of terminal differenti-ation, which are, also, ‘hallmarks’ of cancer cells. Since stemcells are usually defined as cells that are naturally ‘immortal’until they are induced to terminally differentiate or to ‘mortal-ize’, it is observed that one explanation to support the stem celltheory of cancer is by ‘initiating’ an adult, immortal stemcell wherein the initiated stem cell stays immortal, rather thanbeing induced to become ‘immortal’, a prediction of the ‘de-differentiation’ hypothesis. Recent observations have shownthat immortal, normal human breast epithelial stem cells canbe prevented from terminally differentiating by SV40 large-Ttransfection as well as preventing neoplastic transformation(61). The discovery of ‘cancer stem’ cells (63), while notrigorously excluding the ‘de-differentiation’ hypothesis, ismore easily explained using Ockham’s razor, by the stem cellhypothesis.

Recently, the well-studied human carcinogen, benzene,which most, including the authors of this study, have thoughtwas a mutagen (directly or via its metabolites) has been shownto differentially induce the cell death of human bone marrowCD34 hematopoietic progenitor cells (64). Yet, Reddy et al.(99) had already shown that benzene-treated mice did notexhibit any DNA adduct formation in the bone marrow. Infact, in the previous study, apoptosis was observed, suggestinga benzene-induced signal transduction-altered induction of theapoptosis signaling-gene system.

The best approach to be used

Assuming the aforementioned concepts that have been used tocriticize our past and current approach to evaluate the risk tocancer after chemical exposure suggests the ‘standard animal’bioassay, and along the current short-term tests for ‘mutageni-city’, are, in large part, misinterpreted, we would recommendthat their use in the future be discontinued. A recent series ofinvited papers has focused on assessing the current protocols

used to determine the carcinogenic risk of chemicals (65). Inthe interim period before the development of a more human-relevant assay, any chemical that might need testing should betested as having ‘epigenetic’ potential in vitro or in vivo. As a‘carcinogen’, it should be tested as a tumor promoter. Thisapproach might be done using either (i) the classic initiation/promotion models in rodent systems (possibly using only ion-izing radiation as initiators of internal organs or UV light forskin initiation, followed by the test chemical) or (ii) theconnexin32-knockout mouse (66,67). In the latter, the KOCx32 is already a ‘constitutive promoter’ of the mouse liver.Therefore, by testing new chemicals as a potential ‘initiator’,one does not need to follow the initiation with a known pro-moter (68). Testing the new chemical as a promoter in thismouse after it has been initiated with ionizing radiation shouldnot enhance the tumor frequency over that of the initiated-onlyanimals. However, it should be noted that even this system isnot free from potential misinterpretation, in that these animalsare highly susceptible to chemical carcinogenesis. If chemicalsare not mutagenic, then how can these chemicals increase theinitiation process? To be consistent with the hypothesis beingput forward here, the chemical initiator that increases livertumors in the Cx32 KO mouse might affect, epigenetically,pre-existing mutated cells (i.e. ‘oval’ cells) in such a manner asto allow them to escape tumor suppression in a liver tissueenvironment without Cx32, a tumor suppressor gene. More-over, if most of the carcinogens act as promoters, testing themas liver promoters in this mouse should be ‘negative’, whereasin the wild-type mouse, it would be positive after the liver wasexposed to some ‘initiator’.

Ultimately, we recommend that a new emphasis be placedon the development and validation of several normal humanstem cell 3D in vitro assays (human lung, liver, breast, prostate,kidney, brain, hematopoeitic, etc.) to test for cytotoxic andepigenetic endpoints (altered cell proliferation, differentiation,apoptosis, methylation changes, cell--cell communication) atnon-cytotoxic levels (3,69). These should be used to identify ifany threshold levels of change are seen for these endpoints atnon-cytotoxic levels.

Three dimension ‘organoid’ and differentialsensitivity of 2D and 3D cultures

Cells in vivo exist in a dynamic, interactive 3D milieu.Jacks and Weinberg have stated: ‘The notion that cellulartransformation and tumor progression involve the cooperativeeffects of proliferative signaling pathways and antiapoptoticpathways has been well studied in standard monolayerculture and in some in vivo models. However, the 3D culturesystem has the distinct advantage that it takes into accountphysiologically relevant interactions while being amenable tofacile manipulation and biochemical analyses’ (9). Use of 2Din vitro assays to measure mutagenesis, cytotoxicity (necrosisand apoptosis) and epigenetic alteration of gene expression are,clearly, not perfect surrogates to replicate the in vivo situation.Moreover, the 2D in vitro assay uses proliferating cells usually(either primary or immortalized cells). In addition, theseculture systems are usually of one cell type, albeit, evenhere, the cells are not necessarily genotypically or phenotypic-ally homogenous. To make matters even more complex whentrying to extrapolate results from in vitro to in vivo assays,many of these toxicity assays are performed on cells in twodimensions when in log phase, a situation usually not seen

Fig. 4. Gap junctions in cellular homeostasis. Extracellular signals, such asgrowth factors, toxicants and cell adhesion molecules that vary for each celltype, interact with membrane receptors, which then activate intracellularsignal transduction pathways that induce the transcription of genes (A,B,C)through activated transcription factors. These intracellular pathways operateunder cascade and scaffolding systems that cross communicate with eachother in controlling the expression of genes that direct the proliferation,differentiation and apoptosis of cells within a tissue. These multiplecheckpoints are further modulated by intercellular signals traversing gapjunctions, thereby maintaining the homeostatic state of a tissue. Abnormalinterruption of these integrated signaling pathways by food-related andenvironmental toxicants results in diseased states, such as cancer.

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in vivo except during massive tissue growth or hyperplasiaduring wound healing.

In the last decade, more innovative techniques (70--75) arebeing used to reconstruct some of the 3D dynamics seen in vivo,such as organotypic cultures (76), co-culture of several celltypes (77), formation of 3D ‘organoids’ (61), insert wells todetect soluble factors that might identify stromal--epithelialcommunication (78), and embryonic and adult stem cells fortesting toxicants (69,79). In all tissues, there exist three basiccell types, the few adult stem cells, the majority committedprogenitor or transit cells with a finite life span and the termin-ally differentiated cells. The ‘niche’ in which the adult stemcell resides controls its behavior, in addition to feedback sig-nals from the terminally differentiated daughter cells (80), inaddition to environmental factors, such as the oxygen tensionand calcium levels (81), and other nutrients. Most in vitroassays used to detect the toxicity of chemicals are done at logphase and high oxygen levels, in media with high calciumlevels and complex growth factors, such as serum. All thesefactors create conditions in the tested cells that do not mimicthe physiological state of the potential target cells in vivo.Many studies have shown that the same cells tested in 2D logphase conditions, confluent 2D conditions or 3D systems do notyield the same toxic result (82). No one has performed such anin vitro study with a proper mixture of human stem cells, theirdifferentiated progenitor daughter cells and terminally differ-entiated progeny. This ought to be the ultimate goal for futurerisk assessment after chemical (or radiation) exposure from thein vitro to in vivo human situation. Such a study can be theclosest experimental risk assessment that can be performed inhumans. Even in this case, the limitations of individual geneticbackground, and other complex interacting factors will neverbe mimicked for extrapolation to a particular individual. Itmight be the best for which one can strive.

Stromal--epithelial interactions as one example ofextracellular communication linked to GJIC: hormones,growth factors, cytokines, extracellular matrixmodulation of gap junctions

With the use of co-culture conditions and the development ofnew growth conditions for epithelial cell cultures, clear evi-dence of stromal--epithelial interactions have been observed,which influences the biological control of cell behavior (83). Inaddition, in the case of stem cells that have the potential toproliferate either symmetrically or asymmetrically, identifyingthose factors that could control which manner of cell division astem cell will take is crucial. Recently, endothelial cells, co-cultured with neural stem cells were shown to stimulate theself-renewal of cells and expand neurogenesis (78). Even thelong-term effects of radiation to induce a persistent increase inthe plasma levels of pro-inflammatory cytokines owing to animbalance of CD4 T cells (84) again illustrates the interactionof different cell types on each other in the complex carcino-genic process.

Modulating cell--cell communication through GJIC plays anessential role in the development of most epithelial and fibro-blasts progenitor cells in vivo (85). Endogenous factors that canmodulate (increase or decrease GJIC) include growth factors(86), various cytokines and hormones (87), as well as extracel-lular matrices (88). Modulation of GJIC between coupled cellscan influence cell proliferation, cell differentiation and apop-tosis, as well as synchronize electronic and metabolic functions

(16,89). The complex coordination of extracellular matrix,growth factors and nutrients in the medium can influence theexpression and function of the gap junction proteins, as well asthe state of differentiation of cells.

Redox disturbances of homeostatic control of cellproliferation/differentiation/apoptosis

Inhibition of GJIC and apoptosis, altered differentiation, andstimulation of cell proliferation of these cells in the 3D culturesof normal human epithelial cells should be the closest we canget to mimicking the in vitro condition of the in vivo humansituation. Any chemical testing positive at a given non-cytotoxic dose could be tested in a rodent system as a tumorpromoter of initiated breast, liver, skin systems during a val-idation and species comparison phase. If a chemical is testedrigorously as being negative for any of the cytotoxic and epi-genetic endpoints, even at the highest tolerated level, non-cytotoxic levels will not prove the chemical is ‘safe’. However,it might be the best we would ever do.

When a chemical enters the human body, its ultimate fate onits potential biological and health effects will depend on anumber of factors (genetic, gender, developmental state, targetorgan, cell type, cell cycle status, destination, endogenousmetabolic fate, interaction with other endogenous/exogenousfactors, concentration at target site, acute or chronic exposurepatterns). The human being is a complex hierarchy of ahomeostatic--cybernetic system of interacting negative andpositive signals from stem, progenitor and terminally differen-tiated cells in and between various organ systems. When chem-icals in our food, medications, environment and life-stylechoices interfere with this delicate orchestration of homeostaticcontrol of cell proliferation, differentiation, apoptosis andadaptive responses of our terminally differentiated cells, thereis the potential of either a biological and a health effect or both.Disruption of this complex cellular communication systemduring embryonic and fetal development could lead to lethalityor birth defects, impair neonatal and adolescent development,and could lead to maturation arrest and reproductive/neurological/behavioral dysfunctions, as well as diabetes, andafter initiation of single cells could lead to cancer and athero-genesis (90--92). Although it must be stressed that while allhealth effects caused by exposures to chemicals have an un-derlying biological basis, not all biological responses as a resultof these chemical exposures lead to health effects.

One lesson from these mechanistic studies that might applyto understanding epidemiological interpretations of humanintervention studies, particularly with potential chemo-preventive agents correlated with reduced diseases in animalsor humans consuming certain nutrients or diets, is that addingsupplements to individuals who are ‘deficient’ might be bene-ficial. However, adding these supplements to individuals whoare ‘sufficient’ for these supplements might not show anyimprovements. In fact, if the supplements are added at dosesthat are ‘pharmacological’ rather than ‘physiological’, theremight even be detrimental health effects. The real tasks hereare to determine the amount of the ‘sufficient’ levels that confernormal health in each individual (developmental stage, gender,genetic polymorphism, etc.).

The recent perception in various disease studies has detecteda potential role of the inflammatory process with variouschronic diseases, such as cancer, atherosclerosis, diabetes,etc. (93--95). Inflammation is a quintessential example of an

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extracellular communication process, involving secreted fac-tors from one cell type to another cell type, evolutionarilydesigned for adaptive purposes, but which, if sustained in achronic fashion, can be very maladaptive. Triggering this in-flammatory process or chemically mimicking it in a sustainedmanner could lead to various health consequences. It should benoted that the classic tumor promoter, TPA, was shown to be aninflammatory inducer, induce oxidative stress in cells (96), yetnot shown to be a mutagen. In addition, many of the antitumorpromoters have antioxidant activity.

This suggests that there might be a shared underlying com-ponent to many chronic diseases associated with sustainedchronic inflammation that probably influences the tumor pro-motion phase of carcinogenesis. This, again, suggests that themost efficacious intervention strategy for chemoprevention isthe use of antioxidants during the promotion phase of carcino-genesis to prevent or delay the initiated cells from accruing thenecessary ‘hallmarks’ of cancer.

Summary as providing mechanistic insights for abiological approach to cancer risk assessmentof chemicals

A story is told of Albert Einstein’s lecture to a lay audienceabout his recent ‘Theory of Relativity’. After his lecture, areporter in the lecture hall came up to him and said, ‘ProfessorEinstein, now that you physicists understand the workings ofthe universe, don’t you think it is complicated?’ Einsteinthought for a moment and replied, ‘Young man, when youknow nothing of the universe, it is, indeed, complicated. How-ever, when you begin to understand, it is MERELY COM-PLEX!’ The same could be said today of our understandingof the carcinogenic process. In the face of ignorance, it is,indeed, complicated. However, now that we are beginning tounderstand the process, it is merely complex. With that as thebackdrop of our challenge, and with the statement by the lateRobert Good, the cancer immunologist, ‘It does not matterwhether a hypothesis is right or wrong, but rather does itstimulate good experiments?’, we feel it is time to criticallyre-examine the hypothesized role of chemical carcinogens asDNA damaging agents/mutagens and the carcinogen-inducedeffects on the epigenetic control of stem cell development(Figure 5).

When an animal or a human being is exposed to a chemical,it is distributed to various tissues, in which are three differentkinds of cells, the few adult stem cells, the finite-limited pro-genitor cells and the terminally differentiated cells. These cellsinteract with each other through both extra- and gap junctionalintercellular communication mechanisms. While some chem-icals are metabolized to electrophiles and these chemicals candamage DNA, as well as other cellular molecules, they do notnecessarily damage the three types of cells in an equivalentfashion. Evaluating tissues that have but a few adult stem cellsin the tissue and finding DNA lesions, one cannot conclude thatthese lesions were in the cells that led to the cancers, let aloneto the mutations found in any tumor in that tissue. Even the useof sensitive DNA micro-array technology to detect gene ex-pression changes found in affected tissues, actually detectsmostly primary changes in the non-stem or speculated targetcell for cancer or the secondary or tertiary changes found in thedifferent cell types owing to an upset in homeostatic regulationof the communication mechanisms among the different celltypes.

One assumption is that the few adult stem cells are thecancer-target cells. If these cells do not have metabolizingenzymes, or if they do or do not repair their DNA the sameway as the other type of cells, any conclusions, concerning theidentification of that DNA lesion, or the mutation found in anyoncogene or tumor suppression gene of the tumor from thattissue, is a real intellectual stretch.

It cannot be argued that chemicals do not influence cancersformed in animals exposed to chemicals or cancers found inindividuals of a population exposed to a chemical. Clearly, thecancer-‘causing’ chemical did contribute in some way to theformation of that tumor. If the mechanism of action is notthrough mutagenesis, and only in those cases exposed to highconcentrations of a chemical or microbial or parasitic toxins,which could kill cells in a sustained fashion leading to chronicinflammation and cytotoxicity (alcoholism or hepatitis, asexamples), then some epigenetic mechanism must be attributedto its ‘carcinogenicity’. Given that the tumor promotion phase

Fig. 5. Summary statement: in response to environmental and food bornetoxicants and toxins that may induce oxidative stress, the stem and progenitorcells can be affected at the (1) genotoxic level, leading to somatic mutations.However, the significance of genotoxicity has been questioned (13) andgenotoxic level also leads to cytotoxicity. (2) Cytotoxic (necrotic) levels,acute death of cells in a tissue by toxic compounds often results in thecompensatory release of cytokines that affect distal cells. (3) Epigeneticlevel, toxic agents can directly affect the expression of genes at thetranscriptional, translational and post-translational levels by altering theintegrated cell signaling systems controlling gene expression resulting in (4),the interruption of the homeostatic balance of a tissue by disturbing theequilibrium between apoptosis, proliferation and differentiation. Suchimbalances lead to (5), chronic disease such as cancer. Assessment ofepigenetic events and the use of stem cells have numerous applications inhuman health.

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in human beings is the result of a sustained exposure to a non-genotoxic chemical at a threshold level and the absence ofantitumor promoters, this phase of carcinogenesis must beconsidered as the most efficacious place of chemoprevent-ive intervention. If, at least, the inflammatory process plays arole in some tumor promoting chemicals, anti-inflammatoryagents could play a role in the antipromotion phase of carcino-genesis. However, even if a little bit of these antioxidant/anti-inflammatory agents can be beneficial, more might not bebetter because its potential of assisting individuals deficientin anti-oxidants might not assist those that are ‘sufficient’. Infact, the antioxidant properties of these agents might becomepro-oxidants under different conditions found in the individual.

These chemicals, which prove to be carcinogenic throughsome epigenetic mechanism, must be viewed as having prop-erties of all tumor promoters, having the ‘hallmarks’ of species,gender, tissue and cell-type specificity; must work at thresholdlevels or above these levels; must be used in a sustained,chronic fashion; and must be found in individuals absence ordeficient in antioxidants. Moreover, these chemicals do notaffect cells in a vacuum. They interact with other exogenousand endogenous chemicals that could be additive, synergisticor antagonistic to the chemical of interests. This explains, inlarge part, why two genetically identical individuals, exposedto the same amount and duration of a specific chemical, will notexhibit the same risk to cancer.

Finally, any epidemiological or risk assessment analysis of achemical’s potential to induce cancer must take into accountthe epigenetic mechanisms of action of chemicals. While it isbeyond the scope of this analysis to translate the implicationsof chemical carcinogens acting as epigenetic toxicants in riskassessment models, the future of cancer risk assessment mod-eling must integrate the characteristics of epigenetic toxicantson initiated stem cells in tissues. Some of these factors include:(i) threshold concentrations by which these chemicals work aspromoters; (ii) sustained and chronic exposures; (iii) absenceof antipromoters; and (iv) species, gender and developmentalstage-specificity. Recognition of potential differentialresponses of the different cell types to the chemical of interestmust be taken into account. Moreover, this is a complex or‘systems’ view of how these chemicals not only alter complexsignaling within cells, but also, the complex signaling betweendifferent cell types within an organ and the interaction ofsignals between organs within an organism. Finally, the con-cepts of the role of adult stem cells and of secreted- and gapjunctional intercellular communication must be considered inviewing the pathogenesis of cancer (100).

Acknowledgement

Supported by an NIEHS Superfund Basic Science Program grant to JET(PA42 ES04911).

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Received on January 4, 2005; revised on February 15, 2005;accepted on February 16, 2005

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