A Paradigm Shift is Required for the Risk Assessment of Potential Human Health After Exposure to Low Level Chemical Exposures

Abstract

Chemicals are known to be associated with birth defects, cancer, cardiovascular diseases, immunological, reproductive, and neurological disorders. In response to recent reviews of limitations of current concepts and techniques for toxicity testing, this commentary challenges the paradigm that chemicals are directly responsible for DNA damage in the genomic–nuclear DNA in relevant cells of the human body. This challenge is not that mutations do not play roles in human-inherited or somatic diseases but that chemical exposures bring about disease end points by epigenetic mechanisms or by alterations in adult stem cell numbers in utero (ie, the Barker hypothesis) or postnatally, by selecting preexisting mutated cells. Classic concepts, that is, multistage, multimechanism process of carcinogenesis, stem cell theory of cancer, and newer and ignored concepts, such as cancer stem cells and cell−cell communication, will be used to support the view that the toxic effect of chemicals is mediated by nonmutagenic mechanisms at human relevant exposures.

A Call for a “Vision” for Toxicity Testing in the 21st Century

It now is becoming crystal clear that not only in vivo and in vitro toxicity assays have fallen short of human risk assessment after exposures to chemicals, but also for rigorous interpretation of underlying mechanisms. Long before the recent publication of Toxicity Testing in the 21st Century: A Vision and a Strategy, ¹ it was pointed out that there were major problems in the animal bioassays, in vitro toxicity assays, and the broad conceptual strategy to test chemicals for their potential toxicities and the extrapolation from both the in vitro and rodent bioassays to human risk assessment. ² As pointed out in the Toxicity Testing in the 21st Century: A Vision and a Strategy, ¹ it is critical that information from toxicity testing for public health and regulatory decisions be related to the basic mechanism of toxicity of the chemical on relevant human targeted cells and how that mechanism of toxicity be related to the pathogenesis of any particular disease consequence.

The National Research Council (NRC) report, Toxicity Testing in the 21st Century: A Vision and a Strategy, ¹ was compiled for the simple reason that current concepts and techniques to assess the toxicities of chemicals and pharmaceutical drugs have been shown to have serious limitations. This is borne out in a quote from the report: “Using the results of animal tests to predict human health effects involves a number of assumptions and extrapolations that remain controversial.” A number of responses to this report have begun to appear, both in international symposia and in publications. ^{3 –16}

Another quote of the NRC report, which highlights a key recommendation, will be addressed in this “commentary,” namely, “Today, toxicological evaluation of chemicals is poised to take advantage of the on-going revolution in biology and biotechnology … making it increasingly possible to study the effects of chemicals using cells, cellular components, and tissues-preferably of human origin-rather than whole animals.” However, the essence of a subsequent assessment of the same issues in the NRC report and the European Union regulation (Registration, Evaluation, Authorization and Restriction of Chemical [REACH]) concluded, “The testing of substances for adverse effects on humans and the environment needs a radical overhaul if we are to meet the challenges of ensuring health and safety.” ¹²

That this commentary might be viewed as relevant, rather than as another example of chasing windmills, is seen in a series of articles coming out of a symposium entitled Use and Application of Action in Cancer Risk Assessment. ¹⁷ In that series of articles, even though the introductory overview stated, “Due to significant scientific advances on the origin of cancer (both mutagenic and nonmutagenic), there is a great deal of national and international interests in the application of mode of action (MOA) to risk assessment,” little was explicitly discussed about the epigenetic mechanisms associated with chemicals associated with cancers. Although several of these articles addressed nonmutagenic mechanisms of carcinogenicity of chemicals that were associated with cancers via bioassay systems and epidemiological links, none of these articles took on the challenge that the carcinogenicity of these chemicals might have been due to the nongenotoxic or epigeneticmode of action, primarily by promoting preexisting spontaneously or radiation-induced mutations. In other words, possibly, all chemicals associated with the appearance of cancers might not damage DNA to cause mutations, but rather they act by promoting the preexisting mutated cells.

Ultimately, to make the causal connection between exposure to a chemical and a human disease, one, conceptually, must consider (a) the analytical detection of chemicals during in vivo exposure, as well as the chemical being biologically relevant in the target cell or tissue; (b) the possible mechanisms by which a chemical could bring about cellular toxicity; (c) the possible mechanisms of pathogenesis of any human disease in vivo (How do birth defects, cancers, atherosclerosis, cardiovascular diseases, cataracts, immunological reactions, reproductive dysfunctions, and neurological diseases come about?); (d) the manner by which the mechanism of chemical toxicity might interact with the mechanism of the pathogenesis of a given human disease; and (e) how this interaction of a toxicological mechanism might be modified (enhanced or abrogated) by mixture interactions of either or both endogenous and exogenous dietary/environmental chemicals.

Nature of the Paradigm Challenge: Chemical Toxicities Are Not Due to Genotoxicity

To make it explicitly clear, this challenge is not suggesting that DNA damage or an error of DNA replication, leading to the formation of a mutation, does not occur, nor that mutations do not contribute to either heritable or somatic human diseases. What this challenge involves is the idea that chemicals that do contribute to human diseases do so via either epigenetic or cytotoxic mechanisms.

To understand the rationale for this radical idea that a toxic chemical that contributes to a disease end point, such as a cancer that has mutated genes in the cells of the tumor, we are claiming that the mutation was not produced directly by the alleged chemical. Specifically, we are hypothesizing that the toxic chemical selects out of the exposed tissue, a previously mutated cell. From whence do these previously mutated cells come? One might speculate they arose spontaneously by “errors in ordinary DNA replication” or from radiation, such as UV light. A brief review will be presented as to how toxic chemicals could, in principle, react to be a toxicant. There are 3 basic end points of toxicity—mutagenesis (genotoxicity), cell death (cytotoxicity by necrosis, apoptosis, autophagy, or anoikis), and alteration of the expression of genes at the transcriptional, translational, or posttranslational levels (toxico-epigenomics).

Although physical, chemical, and biological agents have the potential to induce 1 or more of these end points, it will be assumed that, in contradiction to the current paradigm that chemicals can induce mutations, these toxic chemicals actually are working by epigenetic mechanisms. Understanding the risk to exposures to chemicals requires (a) use of appropriate in vitro systems to understand their mechanisms of action and (b) understanding how that mechanism could affect the pathogenesis of any disease. Although any and all in vitro assays are limited by the fact that they can never mimic the dynamic in vivo individual human situation during chemical exposure (individual genetic background of the individual, developmental state of the individual, gender, diet, life style factors, medication, nutritional state, not all normal cells being tested respond equally to exposure, etc), clear extrapolation of results of studies on 2-dimensional (2D) cultures, grown under artificial atmospheric conditions, on plastic/glass, with either primary mixture of cells, immortalized or neoplastically transformed cells, will not be relevant for accurate risk assessment for either drug efficacy or toxicity assessment for risk. The use of normal specific organ adult stem cells (eg, brain, liver, lung, prostate, etc) grown in 3D, under niche conditions, using markers for both universal and organ-specific stem cell markers and markers of differentiation of that stem cell lineage might be the only reasonable approach for the future. ¹⁸

When chemicals interact with an organism at the cellular level, only 4 things can potentially happen (a) nothing happens, (b) cell dies by necrosis, apoptosis, autophagy, or anoikis (cytotoxicity), (c) an error of DNA repair of damaged nuclear DNA or an error of replication without a lesion in the template DNA and a mutation occurs (genotoxicity), or (d) there is an alteration in gene expression at the transcriptional, translational, or posttranslational levels (an epigenetic event). ¹⁹ Note that a chemical or its metabolite could damage mitochondrial DNA leading to a mutation in the mitochondria, this is distinctly different from a mutation in the nuclear genome of a potentially dividing stem or progenitor cell. These potential effects of chemicals (mutagenesis, cytotoxicity, and altered gene expression) are believed to contribute to diseases. The current paradigm in chemical genetic toxicology is founded on the fundamental assumption that if a chemical causes any disease, it most likely involves DNA damage/mutations. This idea stems from (a) genotoxicity assays, which generate positive results for chemicals linked to various diseases; (b) mutations in oncogenes and tumor-suppressor genes found in tumor cells of animals exposed to a chemical; (c) these chemicals’ ability to induce oxidative stress; (d) experiments demonstrating DNA adducts in toxic chemical-exposed tissues; and (e) heredity diseases that are associated with both gene and chromosomal germ-line mutations.

The lack of data on what are the target cells in exposed tissues contributes to the genotoxic paradigm. This will become clearer when one understands that most, if not all, data on chemicals’ genotoxicity come from cells that were not the target cells for the start of the carcinogenic process. In addition, because the genotoxicity assays, themselves, are subject to many artifacts as well as legitimate disagreements in the interpretations of the positive and negative results, the results from these assays have to be viewed in conjunction with other toxicity end points to make an overall judgment on the disease-causing effects of these chemicals.

At this point, challenging the role of toxic chemicals’ ability to induce mutations that could lead to these human diseases would seem foolhardy. After all, one can observe real demonstrated mutations associated with various diseases. Clearly, mutations do exist in the germ line that are directly associated with many hereditary human diseases. Indeed, mutations in genes, classified as either oncogenes or tumor-suppressor genes, have been clearly shown to exist in tumor cells that appeared after an animal or human being was exposed to a given chemical. A legitimate question is “Did those mutations in oncogenes or tumor-suppressor genes arise because the chemical associated with the production of the cancer, actually induce DNA damage that generated the mutations?” or “Did the chemical only select out cells that were previously mutated by some other factor?” To reiterate, the challenge of this commentary is not that mutations do not exist or that mutations do not contribute to diseases such as cancer, but rather, the mutations, found associated with chemically associated cancers or with phenotypic changes seen in many short-term genotoxicity assays, are not caused by nuclear DNA damage of the chemical.

How Did This Paradigm of Chemical Genotoxicity Get Perpetuated: Putting Chemical “Square Pegs” Into Theoretical “Round” Holes

Although there might be differing answers to this question, it had been shown, decades before, even before chemicals were on the radar screen, that ionizing and nonionizing, UV radiation, could induce (a oxidative damage (particularly at high doses), (b both gene and chromosomal mutations (point mutations, deletions, chromosome aberrations and rearrangements, chromosome duplications, etc), and (c reactive oxygen species (ROS) and oxidative stress, which paralleled changes in nuclear DNA, cell death, inherited mutant cells, and subsequent health effects. Research on oxidative stress focused primarily on determining how ROS damage cells by indiscriminate reactions with their macromolecular machinery, particularly lipids, proteins, and DNA. However, many chronic diseases are not always a consequence of tissue necrosis, DNA or protein damage but rather due to altered gene expression. Gene expression is highly regulated by the coordination of cell signaling systems that maintain issue homeostasis. Therefore, much research has shifted to the understanding of how ROS reversibly control gene expression through cell signaling mechanisms. ²⁰

With the advent of Rachel Carson’s book, The Silent Spring, ²¹ all of a sudden, the public, the politicians and, therefore, the money, for which scientists could chase, shifted the focus away from radiation genetics/biology to chemical toxicity. As early as 1973, the term, genotoxicity, was born. ²² Although the demonstration that the lack of genomic DNA repair of ultraviolet light-induced pyrimidine dimers was associated with cell death, cell mutation, and skin cancer in the xeroderma pigmentosum syndrome ^23,24 seemed very solid, it was demonstrated that a chemical, acetyl amino fluoride, could damage DNA. ²⁵ By causing a large adduct, in a manner similar to UV light, which could not be repaired in cells of xeroderma pigmentosum, it was quickly assumed that all chemicals that were associated with cancers were detected in experimental animal studies, by in vitro assays to detect genotoxicants and by epidemiological associations, were genotoxic.

The concept of genotoxicity was further reinforced by the sophisticated analytical biochemistry of James and Betty Miller. ²⁶ Those chemicals, which were associated with the appearance of cancers in animals, were often metabolized into electrophiles that could damage DNA, either directly in naked DNA or in the tissues of animals exposed to the chemical. In years that passed, more sophisticated analytical detection of many kinds of lesions were found in DNA at extremely low levels of adducts. ²⁷ It has to be emphasized that these lesions are found in DNA, derived from the various cell types of the tissue (eg, few stem cells and the many progenitor and differentiated cells) and from both mitochondrial and nuclear DNA).

This ability to detect lesions in DNA with great precision should lead to studies where the amount of DNA lesions should be delineated between the mitochondrial versus nuclear DNA, and be directly associated with some biological effect, such as altered cell proliferation, differentiation, apoptosis, or necrosis, as well as to true genomic mutations in the daughter cells. In particular, these kinds of studies should be performed in human adult stem cells (see later discussion).

In the case where DNA lesions, such as those induced in skin cells by UV light, ²⁸ which are very strongly associated with specific mutations in the tumor-suppressor gene, p53, support the idea that any DNA lesion shown to be induced by a toxic chemical reinforces the opinion that these chemical-induced lesions caused genomic mutations found in the cells that led to the diseased tissue. It is true that many toxic chemicals have been shown to induce (a) DNA lesions in many cell types, in vitro and in vivo, particularly at cytotoxic concentrations; and (b) positive results in 1 type of in vitro and in vivo genotoxicity assay (eg, Ames; sister-chromatid exchange (SCE), micronucleus; hypoxanthine guanine-phosphoribosyl transferase (HG-PRT), thymidine kinase (TK); chromosome aberration assays, etc) have been demonstrated. However, it has to be remembered that all in vitro assays, designed to measure mutations of the genome, are really surrogates for the real thing, namely real changes in the quality and quantity of the genomic DNA. Equally important, these assay results should reflect realistic exposure levels to which any tissue in the human body will be exposed. As will be discussed later, possibly the most important aspect of measuring mutations in human cells will be the use of the ultimate target cell of any human tissue, namely the adult stem cell. Consequently, a concerted effort must be done to determine whether DNA repair, mutation induction and other forms of toxicity are different for stem, progenitor, and terminally differentiated cells.

A major criticism of these in vitro assays (and even the use of the transgenic mutation-detecting mice) is that they all have limitations and potential artifacts built into them. ^2,29 . Far too many positives can be false positives; for example, both the TK) assay and the HG-PRT assay, designed to identify cells that are unable to metabolize toxic drugs because of mutations, could also detect cells in which the genes were transcriptionally suppressed. In other words, true gene deletions and point mutations render these 2 genes nonfunctional, thus giving rise to true mutant cells, so, too, could an epigenetic alteration of these 2 genes give rise to the same phenotypes (ie, resistance to toxic drugs). The Ames assay, sister chromatid assay, comet assays, and so on have different mechanistic bases for their false positives. Chromosome aberrations and micronuclei could be the result of nucleases released in cells after high cytotoxic levels are released from their internal sequestered areas. ³⁰

Because in vitro genotoxicity results are the result of errors in DNA repair that always results in some cytotoxicity, risk assessors must be careful not to over-extrapolate those results, especially when concentrations, used to induce some measureable positive effects, would cause massive pathological effects in the human individual if the same amount of cytotoxicity occurred in vivo.

When chemicals are given at levels that induce massive oxidative damage, which can, in principle, damage all macromolecules, nucleic acids, proteins, lipids, and so on, ³¹ there will be membrane damage not only to all cellular membranes, such as the plasma membrane, mitochondrial membrane, endoplasmic reticulum, and nuclear membrane but also to both mitochondrial DNA and nuclear DNA. Under those conditions, one might ask, “What if you find lesions in nuclear DNA?” That cell will never give rise to a mutant cell or to a cancer because dead cells do not give rise to cancers, directly. Necrotic cell death could trigger compensatory hyperplasia, thus making cell death a potential indirect tumor promoter. ³²

One additional fact must be kept in mind regarding a chemical’s ability to induce genomic mutations. Every time a chemical encounters a cell, the first roadblock it encounters is the cell membrane. When that happens, the chemical binds to a receptor, as would an endogenous chemical, mimicking the actions of the endogenous chemical or inhibiting the endogenous chemical’s ability to work. If the chemical could interfere with ion channels (inhibits or enhances), it will alter the ion’s normal function in the cell. Lastly, if the chemical just interacts with the membrane (either via membrane fluidity changes, general membrane permeability effects by being lipophilic, or by acting as a chelator of critical ions needed for membrane stability), it has the ability to trigger signal-transducing systems, such as STAT-3, Frizzle, protein kinase C (PKC), or mitogen activated protein kinase (MAPK). In general, no chemical is going to get into a cell without triggering some signaling mechanism.

The cell membrane was designed to be an exquisite sensor of external ions and molecules. Thus, even if a chemical could damage genomic DNA, it will always trigger membrane-associated signaling. Membrane-associated signaling is the means by which cells can adapt to environmental changes by epigenetically altering gene expression at the posttranslational level (phosphorylation of signaling molecules), translational modulation of the message (messenger RNA [mRNA] splicing or half-life modulation), and transcriptional modulation of genes (induction of apoptosis genes, induction of ornithine decarboxylation gene needed for DNA replication, etc)

Finally, what about in vitro transformation assays? All too often, these assays, such as the Syrian hamster embryo transformation assay, have been equated with the paradigm of mutagen as carcinogen, because it has been assumed that a neoplastic-transformed colony in this assay has been caused by genotoxicants. Yet, when one carefully looks at many of these chemical carcinogen mutagens, they test as tumor promoters not tumor initiators (see below). ³³ Take for example, phenobarbital. Why has not this raised some eyebrows in those who continue to use this assay in a blind fashion?

Molecular Genomic DNA Damage in Cells and the Whole Biology of Human Cancer: Are They the Same?

In all due respect, the concept, carcinogen as mutagen while describing a very creative technology that was designed to solve a major problem of chemically associated toxicities, ³⁴ gave rise to a chemical Frankenstein. After this Ames assay was developed, with all its current permutations, and the myriad of new genotoxicity in vitro and in vivo assays to detect chemical mutagens, a false idea was created as to how mutations do play a real role in carcinogenesis. In point of fact, the processes of mutagenesis are not equivalent to the processes of carcinogenesis.

Although the topic of what are the various mechanisms of true gene and chromosomal mutations is not the purpose of this commentary, processes leading to errors in replication, which could alter the genomic information, or to errors in repair, do, in fact, generate mutations that could contribute to the carcinogenic process. The question is, “Can chemicals really damage genomic DNA, such that errors in repair might cause a mutation?” (Here we leave out any discussion of how chemicals might lead to errors in replication by stimulating cells to divide, thereby, increasing the finite probability that a mutation might occur). However, deference is made to the article by the late Weinstein ³⁵ who argues that this is not a likely explanation. Although it is probably true that mutations are formed only during DNA replication, which should not be immediately equated to mean that chemicals stimulating mitogenesis are weak mutagens causing errors in replication. If the adult stem cell, once released from the niche microenvironment, resting in its niche, is not replicating, it might become prone to these errors of replication once it commits itself to become a progenitor or transit amplifying cell.

Because of the discovery of oncogenes and tumor-suppressor genes, sub-disciplines in molecular biology, such as molecular oncology and molecular epidemiology, were born. Although probably not intended, just by studying genes (eg, DNA lesions, DNA repair, mutations in cancer cells, in oncogenes/tumor-suppressor genes, and mutagenic changes in the human genome) one can understand and explain the complex process of carcinogenesis. The formation of a cancer in experimental animal or in human beings involves a myriad of complex interactions, not only at the level of a mutated genomic oncogene or tumor-suppressor gene, but at the cellular, intercellular, physiological (and even at the psychosocial/cultural levels). VR Potter has correctly stated that:

The cancer problem is not merely a cell problem; it is a problem of cell interaction, not only within tissues, but also with distal cells in other tissues. But in stressing the whole organism, we must remember that the integration of normal cells with the welfare of the whole organism is brought about by molecular messages acting on molecular receptors. ³⁶

Unfortunately, since the birth of molecular oncology, few have examined the insights gained by the study of coal tar experiments on mouse skin ³⁷ and rabbit ears. ³⁸ Even fewer in the fields of molecular epidemiology have used the valuable concepts learned from the so-called initiation/promotion/progression experimental animal carcinogenesis studies.

In brief, the aforementioned studies gave rise to the multistage/multimechanism concept of carcinogenesis. ³⁹ It was shown that initiation was a distinct process that was triggered when a sub-carcinogenic dose of a chemical was applied to an animal, such that no or few tumors were seen above the control, nontreated animals. Additionally, if these treated animals were then exposed to a chemical, which, alone, did not bring about any increase in the background frequency of tumors but did bring about an increase in both the earlier appearance and frequency of tumors, then it was determined that the initiation exposure induced an irreversible stable change in a single cell. These initiated cells could remain quiescent in the body for a long time, unless it was followed by a noninitiator, whereby the single initiated cell was clonally expanded. (This was termed as the promotion phase caused by promoters.) The expansion mechanism could be interrupted or even reversed, either by stoppage of exposure to the promoting agents or by interference of the promoters by co-exposures to antipromoters. ⁴⁰ Even more striking, but usually ignored, is the observation of promoters having threshold levels of action. ^41,42 If on further exposure to another agent that could induce a stable change in 1 of the expanded cells of the benign lesion, conversion of this initiated cell into an invasive, metastatic cell was done at the progression stage.

Although, even to this day, the underlying molecular mechanisms for each of these 3 distinct phases of cancer formation in an experimental animal is not universally accepted, there are a few accepted facts: (a) initiation is irreversible on an operational level; (b) promotion is a clonal expansion of a single cell, which can be interrupted or even reversed; and (c) the progression phase, which involves the conversion of the premalignant cell to the invasive/metastatic stage, also seems irreversible. ⁴³

Most of these initiation/promotion/progression experimental animal studies, which have been done on skin, liver, bladder, and mammary tissues, were done with chemicals which were classified as initiators or promoters. Because initiators, operationally, induce irreversible effects in a single cell, it has been universally accepted that initiation must be the result of the molecular process of mutagenesis, because a mutagenic event is irreversible for all intensive purposes in an organism. This, together with all the in vitro and molecular studies cited above, the beginning of a circular reasoning has emerged that proposes a chemical is carcinogenic because it is mutagenic and the chemical is mutagenic because it is associated with cancers with mutations in oncogenes or tumor-suppressor genes in tissues having DNA lesions.

In addition, because the promotion process appears to bring about the clonal expansion of the initiated cell, operationally, in the experimental animal, it has been assumed to be a mitogenic, not a mutagenic process. Although in a some cases, these promoting chemicals have been interpreted as being mutagenic in some in vitro assay to measure genotoxicity, in the majority of cases, tumor promoters, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), dichlorodiphenyltrichloroethane (DDT), 2,3,7,8-Tetrachlorodibenzodioxin (TCDD), polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), pentachlorophenol (PCP), phthalates, phenobarbital, and so on, do not induce DNA lesions, do not induce positive results via in vitro genotoxicity assays, but they can induce oxidative stress and mitogenesis of initiated cells without killing cells. Furthermore, these promoting chemicals inhibit cell killing by apoptosis. ⁴⁴ No true mutagen inhibits apoptosis because by inducing DNA damage to cause a mutation, many of the DNA lesions can cause cell death by necrosis. Mutagens do not inhibit cell death by blocking apoptosis (which requires altered gene expression, an epigenetic process).

Both of these processes (mitogenesis and apoptosis) seem to require inhibited gap junctional intercellular communication (GJIC), ^45,46 and these cellular processes appear to be the cellular mechanism of tumor promotion (see multiple reviews of this). ^47,48 Although various tumor promoters and many tested oncogenes inhibit GJIC, reversibly or stably, respectively, they do so via multiple biochemical mechanism at threshold levels. ⁴⁹ In addition, the promotion process can be brought about by surgery, solid particles, or growth hormones, and cell death inducing compensatory hyperplasia (or chronic inflammation). ⁵⁰

Therefore, if a chemical, such as diethylnitrosamine (DEN) or 7, 12-dimethylbenz[a]anthracene (DMBA), is used as an initiator, followed by a promoter, such as TPA, phenobarbital, or DDT, it seems very convincing that the initiation event, caused by the chemical, must be the result of a mutagenic process. Furthermore, when one examines the tissues of the tumor site and finds DNA lesions, there can be no doubt to those who believe that chemicals can be mutagenic and cause lesions in genomic DNA. Furthermore, on molecular examination of an oncogene, such as ras, in cells of the tumors and one finds mutations, the evidence seems irrefutable within this paradigm. It now must seem to go beyond question that the chemical initiator must be mutagenic. Need one even consider the unthinkable that, maybe, DMBA or DEN did not induce the mutation found in the cancer cells of the tumor found in the animal exposed to the chemical? It seems that, in science, alternative explanations should be considered for the testing of the various hypotheses.

Observations Swept Under the Paradigm’s Rug

One of the first of these observations came from a major believer in the chemical carcinogen as mutagen ranks. Brooks ⁵¹ had shown that tumors, induced in animals by many chemicals, were found to have mutations in the oncogenes, but the mutations in those oncogenes did not correspond to the known chemical lesion of the chemical’s interaction with DNA. In addition, there are so many conflicting published observations, which go beyond this commentary, about chemicals that can cause cancer in experimental animals that failed genotoxicity assays or vice versa, or about chemicals that were positive in 1 or more genotoxicity assays that did not cause cancers in experimental animals. A few deserve some attention. The first is the observation by Thilly. ⁵² In his studies of mutations found in human lung cancers’ ras gene of nonsmokers and cigarette smokers, the molecular mutation spectra for both were essentially the same. This suggests that the mutations in the oncogene of the smokers’ lung cancers were not caused by the chemicals in cigarette smoke. Furthermore, the observation, that cancers associated with cigarette smoking are most likely the result of smoking acting as a tumor promoter rather than as an initiator of lung cancer, must be seriously considered. ^53,54

Probably, one of the very early challenges to the short-term in vitro assays to detect genotoxicants was the claim that nitrofluorobenzene, which was shown to be extremely genotoxic in the Ames assay, would be a powerful carcinogen mutagen or initiator. ⁵⁵ In point of fact, it was not a mutagen in mammalian assays but a powerful inhibitor of GJIC at nontoxic concentrations, in a manner similar to TPA. Furthermore, it had been shown to be a tumor promoter. ⁵⁶ Now, how does one explain the mechanism of action of chemicals, such as TPA, DDT, phenobarbital, pentachlorophenol, and TCDD, all of which can induce ROS, oxidative stress, and even some DNA lesions, but, at noncell killing doses, they do not induce mutations, do not initiate carcinogenesis, but do induce cell signaling, alter gene expression, inhibit apoptosis, and act as tumor promoters?

In a very exhaustive and important survey of 472 marketed pharmaceuticals, ⁵⁷ the reported mutagenicity and/or carcinogenicity (as determined by a wide variety of mutation and carcinogenicity assays) demonstrated a wide range of results, demonstrating, again, the lack of any meaningful insights into the mechanism of toxicity of these drugs. In addition, no data were presented that might have demonstrated any epigenetic mechanism of action for the pharmacological action of the drug, let alone any potential risk to human health via the potential epigenetic mechanism of action. Although it was pointed out that a large fraction of these drugs were developed and marketed prior to current regulations, even those that were tested with a genotoxic, carcinogenic, or both assays, the disconnect between what was interpreted as a positive in any of these assays did not necessarily correlate to the presumed mutagenicity with its potential to be carcinogenic in a bioassay. Clearly, little data would exist for determining whether the drug was also carcinogenic in the human patient. So while the authors suggested it would be prudent to do a reevaluation of these drugs, in view of the NAS report, and our commentary, we would only add that the reevaluation not be with the existing assays.

How long can the fields of mutation research, chemical carcinogenesis, molecular oncology, and molecular epidemiology continue to ignore the volumes of published literature that question the interpretation related to chemicals causing cancer by inducing the mutations found in the oncogenes and tumor-suppressor genes of the tumors of chemically exposed animals or people? As indicated by the Toxicity Testing in the 21st Century Report, ¹ it is clear that current concepts and technologies in the field of chemical toxicity testing are seriously inadequate. Could there not be an alternative explanation that fits all the data published of the last 50 years? Could these chemicals have selected out of the exposed animals and human beings preexisting spontaneously existing mutated or initiated cells? This idea should not be rejected out of hand just because one believes that the prevailing paradigm is so convincingly satisfying. Today, our observations, related to chemicals’ mechanisms of action and to the mechanisms of carcinogenesis, seem to call into question the carcinogen as mutagen paradigm. The carcinogen as mutagen paradigm will not be useful for cancer risk assessment. The time is called for a dramatic revision or rejection of the paradigm. When that will happen, both science and society should move ahead.

The Role of Mutations and Epigenetic Events in the Paradigm of Stem Cells as Targets for Carcinogenesis

Although the concept of stem cells in biological systems has been considered in embryology, developmental biology, and cancer research for many decades, it has recently jumped into the headlines of modern biology because of the isolation of human embryonic stem cells ^58,59 and the cloning of Dolly. ⁶⁰ From a holistic viewpoint, all multicellular organisms started from a totipotent stem cell or fertilized egg (it gives rise to all the 200+ cell types of the human body) to a whole organism, which, then gives rise to a pluripotent (embryonic) stem cell, which in turn can give rise to most of the cells of the body. In turn, multipotent stem cells, derived from the pluripotent stem cell, can give birth to the cell types within an organ. On further restriction of their ability to differentiate, these multipotent stem cells give rise to bipolar stem cells, which, as the name implies, can differentiate into 2 types of cells. The oval cells of the liver give rise to hepatocyte and bile ductal lineages. The unipolar stem cells are further restricted, in that they give rise to a single lineage of differentiated cells. The progenitor cells are those born from a stem cell. Unlike the stem cells, which are fundamentally immortal until they are induced to terminally differentiate (ie, to mortalize) or apoptose, the progenitor cells can proliferate but ultimately they senescence. Terminally differentiated cells are, by definition, unable to proliferate (eg, the red blood cells, lens cells, neurons). What makes a stem cell unique, as opposed to its progenitor daughter cells, is that it can divide symmetrically to produce 2 stem cells or asymmetrically to produce a progenitor cell that ultimately terminally differentiates, apoptosis, or senesces. ⁶¹

Within a tissue of any organ in a multicellular organism, the 3 main cell types, adult stem cells, progenitor daughter cells, and the terminally differentiated cells, coexist in a delicate, orchestrated homeostatic relationship. Regulation of their ability to proliferate differentiate, apoptose, or adaptively function is facilitated by communication mechanisms. Extracellular (hormones, cytokines, growth factors, neurotransmitter, extracellular matrices, adhesion molecules), intracellular (receptor-linked signaling systems, transcription factors, etc), and gap junctional intercellular communication (GJIC) mechanisms are intimately linked to each other to determine whether a cell proliferates, differentiates, die by apoptosis, or adaptively responds. ⁶² The recent hypothesis of associating stem cells to cancer stem cells, ^63,64 re-focuses attention to the stem cell theory of cancer, ^65,66 which has been recently supported by demonstrating that several normal adult human stem cells shared the same marker (Oct-4) as did the cancer cells. ^{67 –69} This evidence is consistent with the hypothesis of the adult stem cells as targets for initiating the carcinogenic process and are, most likely, the origin of the cancer stem cells, which also share the Oct-4 marker of the adult normal stem cells but not the cancer nonstem cells. ^67,70 There is now evidence that cancer stem-like cells have been isolated from benign tumors. ⁷¹

To put the stem cell issue into the context of chemical toxicology, one must realize that the gene expression of the 3 types of cells within tissues will shape a different physiological state for cell type. The terminally differentiated cells, such as tetraploid hepatocytes, will have drug metabolizing enzymes that are not expressed in the stem cells of the liver. The DNA repair systems of all the different cell types are yet unknown because no research has yet focused on this important question. However, from an evolutionary vantage point, as once was stated by Dobzhansky, “Nothing in biology makes sense except in the light of evolution,” ⁷² it would make no sense if both the stem cells of any organ had the same sensitivity to a toxin/toxicant as its differentiated progenitor daughter cells. One would imagine that the terminally differentiated cell would act more like a drone bee than the queen bee (or stem cell). The terminally differentiated cell would sacrifice its life so that the queen bee or stem cell could replace it. If both had the same chemical sensitivity, the organ and organisms would not survive. Clearly, there are agents, such as X-rays, that seem to affect stem cells. ⁷³ Moreover, there is evidence that the cancer stem cells within a tumor are resistant to radiation therapy. ⁷⁴

In addition, while many hypotheses have been offered to explain aging, most hypotheses tend to be of a reductionist character and are focused on the intrinsic genetic/molecular codes for aging or some cellular limiting factor, such as the Hayflick limitation of cell proliferation. ⁷⁵ Another way of viewing aging would be the depletion of a stem cell pool. ^65,61 Because the human body does not age uniformly, environmental agents that could deplete the adult stem cell pool could affect the aging process of a particular organ. Most recently, the discovery that lamin 1 in the Hutchenson-Gilford (Progeria), pre-mature aging syndrome, ^76,77 seems to affect the stem cell pools of these individuals supports this hypothesis. ⁷⁸

The main reason of bringing in the role of stem cells into the understanding of chemically induced toxicity is because the recent use of expression-array technology to study biological effects of chemicals cannot be used to study the underlying mechanisms. The reason is simple; when an organism or in vitro cell system is exposed to a given toxicant, there are multiple cell types expressing different genes (eg, the few stem cells, the many potentially dividing progenitor cells, and the terminally differentiated cells). In addition, some cells are stressed; others are going through the cell cycle, whereas some are dying by apoptosis or necrosis. Grinding up that tissue would be a summation for all these different gene expression profiles. If the ultimate disease, such as cancer, is the result of 1 stem cell being affected by a given chemical (cancer is known to be monoclonal derived ⁷⁹ ), one would never see that effect, because the gene expression of that single stem cell would be masked by all the noise of the gene expression of the chemical’s differential effects on the majority cell types.

What has all this to do with this challenge to the chemical genotoxicity hypothesis? The most important point is that none of the in vitro genotoxicity assays have utilized adult human stem cells for their end points. Looking at DNA lesions in tissues does not identify those specifically in the stem cell population, nor in the mitochondrial versus nuclear DNA of the stem cells. Emerging on the in vitro scene is the role of oxidative stress on stem cells. By using any in vitro assay to study the effect of any chemical on cells’ behaviors when grown in 20% oxygen does not in any way mimic what that chemical might be doing in vivo, where a stem cell exists in much lower oxygen microenvironments. ⁸⁰

The issue of the widespread use of DNA microarray toxicogenomics seems very relevant to a paradoxical or schizophrenic manner of interpreting these data. The technique is being used to measure changes in gene expression (an epigenetic) event after exposure to chemicals, suspected or known to have toxic and negative health effects. Many investigators still interpret these changes in gene expression seen in exposed or diseased tissues as being triggered initially by the chemical’s ability to damage nuclear DNA. Because mutagens are believed to act stoichiometrically, one would not expect to see regular, reproducible gene expression changes after repeated exposures to a given chemical. However, those regular gene exchanges would be exactly what one would expect if the chemical acted epigenetically or deterministically.

Systems Biology in Toxicology: Another Fad Without a Biological Foundation

The recent buzz word in organizing symposia, requesting grant proposals, and writing review articles is that of systems biology. ⁸¹ Molecular epidemiologists, molecular biologists, risk assessors, ecologists, and so on have recently created an explosion of systems ideas. However, these commentators have viewed these attempts as ignoring the old systems views articulated by Weiner and Schade, ⁸² Von Bertalanffy and Novikoff, ⁸³ Brody, ⁸⁴ and many others. Of course, a multicellular organism, such as a human being, is a hierarchical, cybernetic being that is interacting with the multiple environments every moment of its existence (physical, chemical, dietary, social, psychological, cultural, etc). ⁸⁵ All of our higher order attributes that have emerged are an ordered organization of all the levels below it.

It is here where classic biologists, such as Markert, ⁸⁶ saw that, in a systems sense, exposure of an organism to any chemical that could interfere with the delicate communication that has to be going on, not only during embryogenesis, fetal, neonatal and adolescent development, but through life, could lead to homeostatic disruption.

Cells interact and communicate during embryonic development and through inductive stimuli mutually direct the divergent courses of their differentiation. Very little cell differentiation is truly autonomous in vertebrate organisms. The myriad cell phenotypes present in mammals, for example, must reflect a corresponding complexity in the timing, nature, and amount of inductive interactions. Whatever the nature of inductive stimuli may be, they emerge as a consequence of specific sequential interactions of cells during embryonic development.

The first embryonic cells, blastomeres, of mice and other mammals are all totipotent. During cleavage and early morphogenesis, these cells come to occupy different positions in the 3D embryo. Some cells are on the outside, some inside. The different environments of these cells cause the cells to express different patterns of metabolism in accordance with their own developing programs of gene function. These patterns of metabolism create new chemical environments for nearby cells and these changed environments induce yet new programs of gene function in responding cells.… The genome seems no longer responsible to the signals that were effective earlier in development. ⁸⁶

Of course, changes can occur in adult cells that lead to renewed cell proliferation and altered differentiation as seen in neoplasms, both benign and malignant, but such changes are very rare indeed when one considers the number of cells potentially available for neoplastic transformation.… However, most genetic changes in adult cells can probably lead to cell death since random changes in patterns of gene activity are not likely to be beneficial.

In the delicate and critical struggle to find efficacy of drug or of chemical usage and its potential toxicity and pathogenesis of diseases, currently, the chemical’s potential efficacy must now be assessed against the conditions of inducing epigenetic toxicity. ⁵³ Even worse, the public is now exposed to new chemicals that were deemed negative, based on these imperfect assays. The distinction between phenobarbital-like or thalidomide-like pharmaceutical chemicals, depending on the context of exposure, can be either beneficial or detrimental to humans. This demands an understanding that they work as epigenetic agents in a biological system.

It addition, it is as critical for pharmaceutical companies and chemical/food companies that there are means to screen for potential mechanisms of toxicities of chemicals to which human, animal and the ecosystem will be exposed. ⁸⁷ Even in the field of anticancer (or of any disease) treatment, ⁸⁸ understanding the mechanism of toxicity must integrate new concepts and experimental technology/findings in biology, and biotechnology. Part of the recommendation will be the use of human cells, together with computational systems. ^{3 –5}

In addition to these recommendations, recognizing the hierarchical and cybernetic system model of a developing human being (from embryo, fetus, neonate, adolescent, adult, and geriatric stages of both genders, each being genetically unique, and no one being exposed to the same environmental exposures, as well as the fact that toxicity tests cannot be done, ethically or practically, on each individual at each stage of development), we must strive to test in human cells in a situation which might best simulate some aspects of the in vivo situation. Clearly, many physiological factors cannot be accurately reproduced in vitro. In the cutting-edge area of in vitro biology, the use of 3D dimensional culture conditions, are being proposed. ^18,89,90 Moreover, trying to mimic niche conditions of adult stem cells, together with critical stromal−epithelial interactions ^{91 –93} for each adult organ stem cell (lung, liver, skin, brain, prostate, breast, etc) will have to be performed.

The use of rodent or abnormal human cells, just because these cells that are easy to grow in vitro, will not give extrapolatable results to the in vivo human situation. Recognizing that each organ, in vivo, has adult stem cells, in their niches, interacting with their daughter progenitor and lineage-derived terminally differentiated daughters, and that each cell type has different expressions of their genome and different cellular physiologies, they will all differentially react to a chemical. Therefore, within any 3D organoid in vitro assay, using adult stem cells, they will create their own niche by proliferating and differentiating. Under low-oxygen conditions with media to approach a static physiological state, perhaps with interacting stromal cocultured cells, these in vitro cultures might be used to screen for chemicals that might alter cell proliferation, differentiation, apoptosis, senescence, and necrosis in a dose-dependent fashion. ¹⁸ The importance of using normal human adult stem cells in future toxicity testing must be considered, because the germinal and adult somatic stem cell must be viewed as critical targets for exposures to chemicals.

Prenatal Modulation of the Quality and Quantity of Adult Stem Cells on Postnatal Diseases: A New View of the Barker Hypothesis

The Barker hypothesis suggested that adult diseases could be linked to prenatal and early postnatal life. More recently, 1 explanation of exposure to epigenetic toxicants during pregnancy is that gene expression in the developing cells could be altered. ⁹⁴ However, another explanation might explain these observations. If the epigenetic toxicants could alter (increase or decrease) the numbers of stem cells in the developing embryo or fetus, then the resultant altered phenotype later in life could provide support for the Barker hypothesis. An example of this might be the observation by Strohsnitter et al, ⁹⁵ which indicated the correlation of the levels of umbilical cord blood hematopoietic stem and progenitor cells with birth weight and they speculated on its influence on cancer risk.

Breast and prostate cancers (as are all cancers) consist of both cancer stem cells and cancer nonstem cells. A concept has been offered that environmental/dietary factors might influence the cancer frequencies by influencing (a) the normal stem cell pool as potential targets for carcinogenic initiation and (b) the promotion/antipromotion or expansion/suppression of the initiated breast or prostate stem cell. ⁷⁰ In addition, alteration of the ratio of cancer nonstem cells to cancer stem cells could influence cancer therapy.

From the atomic bomb survivor studies, radiation seems to have increased the frequency of breast cancers. ⁹⁶ However, one of the reasons radiation-associated breast cancers were detected in this study was because the background frequency of breast cancers in Japan at that time was so low. The question is, “Was it low because the pool of breast stem cells was low in the Japanese women (control population, as well as in the atomic bomb radiation-exposed population)?” Since the demonstration of the isolation/characterization of human breast stem cells, ⁹⁷ dietary factors, such as genistein (a chemical in soy products which were staples in the Japanese diet), were shown to induce differentiation of the normal breast stem cells. ⁹⁸ By reducing the breast stem cell pool, the target size for inducing breast cancers from the stem cells would be reduced. However, once initiated, the breast stem cell, which has been shown to express the estrogen receptor, ⁹⁹ could be either promoted by estrogen or suppressed by agents that induced partial differentiation of the breast cancer stem cell (ER+; connexin 43−; Oct-4+) to become ER−, Cx43+, and Oct-4−. Therefore, these cells would be promoted or suppressed by antipromotion agents that either inhibited GJIC or prevented the inhibition of GJIC by promoters. ⁶⁶ To complicate the epidemiological test of this hypothesis is the fact that caloric restriction, a well-documented factor in reducing chronic diseases in animals, has not been seriously considered a factor influencing breast carcinogenesis in the atomic bomb survivor study. ¹⁰⁰ The fact that the frequency of osteoporosis is very high in the old Japanese females might also be related to a similar effect of the components of the soy diet on the differentiation of bone stem cells. Clearly, this will have to be experimentally determined.

Using the same logic as above, bisphenol-A, an epigenetic chemical that alters the expression of genes, was shown to induce prostate cancer in male rats which were exposed in utero, ¹⁰¹ a hypothesis could be envisioned where bisphenol-A, in utero, might cause the prostate stem cell population in the developing male rat fetus to increase, but not mutated, by this chemical. This could increase the probability that a stem cell in this increased stem cell pool might incur a mutation via an error of replication postnatally. However, in the same study, in utero bisphenol-A-exposed rats, treated with genistein, reduced, dramatically, the frequency of prostate cancers in the male offspring later in life. In this case, the genistein might have reduced either the prostate stem cell pool by causing them to differentiate or by preventing the promotion of initiated prostate cancer stem cells. This idea can be tested with markers for both the normal prostate stem cells and cancer stem cells. The implications of this observation for the differences seen in human prostate frequencies throughout the world seem obvious.

If this interpretation of prenatal environmental or dietary exposures is correct, this can be seen as an epigenetic effect, not only by altering the expression of genes within a cell but also by altering the stem cell pool in a tissue (effectively, altering the expression of genes in a toxicogenomic microarray analysis). This starts to bring a potential mechanistic interpretation of the Barker hypothesis.

Even the suggestion that transgenerational effects can occur via epigenetic mechanisms has been raised. ¹⁰² This issue, again, points to the need of creating new in vitro normal human cell systems to screen for these types of agents that could bring about alterations in gene expression in germinal stem cells that would be expressed in both germ cells and adult somatic stem cells of transgenerational offspring.

Are Cancer Therapeutic Chemicals Genotoxic and Do They Induce Mutations to Cause Multidrug Resistance Cells to Survive Therapy?

One area where chemicals are deliberatively used as toxicants is cancer chemotherapy. The basic strategy has been to kill cancer cells in a tumor in a manner that spares the normal cells of the patient. One fundamental assumption that has been made is that all the cells within the tumor were essentially of equal sensitivity, although it has been recognized that microenvironmental differences within the tumor could affect the sensitivity to the therapeutic agent. With the demonstration of cancer stem cells, a new conceptual approach has been offered to how cancer therapeutic drugs must work to eliminate the cancer stem cells. ^{63,64,103 –108} It also raises new challenges as to how chemicals must act to kill cancer stem cells. Early hypotheses to explain the mechanism of action of cancer therapeutic drugs, such as cisplatin, were that they were DNA-damaging agents, such as X rays. Therefore, the concerns have always been that the same agent, which was used to kill cancer cells, could induce cancer in the same patient. However, given the risk/benefit reasoning, it was always concluded that the short-term benefit to the patient by reducing the risk of death by the cancer outweighs the long-term risk of inducing new cancers to the patient. Again, many short-term genotoxicity tests, including the demonstration that these chemotherapeutic agents could interact with naked DNA, form DNA lesions in tissues, and, in some cases, inducing cancers in animals, seemed to support the hypothesis that these chemicals were genotoxic.

Pulling together diverse observations from several fields, it now appears that many of these cancer therapeutic chemicals, while toxic to some cells within a tumor, do seem to either induce cells within the tumor to become resistant to the drug. This has led to much research on the multidrug resistance mechanisms and multidrug-resistant genes. However, in view of the emerging stem cell and cancer stem cell research findings, another interpretation is possible. That is, both the normal and cancer stem cells are naturally resistant to many of the cancer chemotherapeutic chemicals because they constitutively express the multidrug-resistant gene/genes. The relatively recent demonstrations that side-population cells, found in studies in which toxic fluorescent chemicals were given to all the cells within a tumor, were found when all the cells were sorted. ^109,110 In some of these cases, those side-population cells were found to have the ability to differentiate. ^{111 –113} This implies that they had stem-cell-like attributes.

Again, going back to the quote by Dobzhansky, it makes sense that stem cells, both normal and cancer stem cells, have their multidrug-resistant genes expressed. From an evolutionary viewpoint, if all the cells within a tissue were equally sensitive to a toxic chemical, the stem cells, which are needed for replenishing damaged, dying, and lost progenitor and terminally differentiated cells, the organ, and hence the organism would not survive. Therefore, cancer therapeutic agents were thought to be genotoxic when they kill normal and some cancer nonstem cells within a tumor because some tumor cells become resistant to the therapeutic toxicant. More likely, the toxic chemical eliminates the cancer nonstem cells (whose multidrug-resistant genes have been transcriptionally suppressed in some partially differentiated cancer stem cells, due to the changing microenvironments within a growing tumor) and selects out the surviving cancer stem cells, which normally express these genes. This hypothesis is currently being tested.

A point not to be ignored is the observation that chemicals, judged to have human toxicities, have potential beneficial pharmaceutical effects. This is, also noted in the many, if not all, pharmaceutical drugs that provide therapeutic effects, while having many toxic side effects. One example is that of thalidomide. It is a well-known human teratogen. ¹¹⁴ Currently, its mechanism of action includes its ability to inhibit angiogenesis. ¹¹⁵ It is also not genotoxic but can induce GJIC. ¹¹⁶ Today, this drug is being used in cancer therapy by virtue of its ability to inhibit angiogenesis to the growing tumor. This should be a lesson in toxicity testing, namely, by understanding the mechanism by which a chemical alters a fundamental biological function allows one to use the chemical under defined conditions (eg, not to use thalidomide in pregnant women at specific window periods in pregnancy but to use it under conditions were inhibition of angiogenesis might benefit a patient).

Paradigm Shift in the Field of Chemical Toxicity?

There comes a time in every field of science when useful paradigms accumulate many observations that simply cannot be ignored because they are not neatly encompassed by the prevailing theories. Moreover, sometimes a new challenge to a prevailing paradigm only helps to modify the paradigm, making it stronger. However, in a few cases, the challenge not only forces a new paradigm to emerge, but the new paradigm helps to enlarge the scope of its range of explanatory power, which includes explaining previously unexplained observations, but generating more testable hypothesis that expands the area of science.

This attempt to challenge the idea that chemicals work as toxicants (eg, teratogens, carcinogens, neurotoxins, cancer chemotherapeutic agents, etc) was to draw attention to many observations that have, for far too long, been summarily ignored (eg, the shortcomings of all in vitro genotoxic assays; the real discrepancies linking molecular lesion studies to biological end points, such as cancers; agents that are nongenotoxic that induce real toxic end points; emerging stem cell and cancer stem cell biological studies; etc). Obviously, while this challenge does not have the weight of the years of extensive or expensive research that supports the toxic chemicals as mutagen paradigm, the recent emerging research of epigenetic effects of toxic chemicals ^{19,32,49,94,117} and the role of epigenetic mechanisms in the field of carcinogenesis, ^{118 –120} this new potential paradigm must now be seriously examined.

Care and critical thinking must be put into the new sophisticated technologies to examine epigenetic changes, either via DNA microarray analyses of whole tissue gene expression (a mixture of cell types and physiological states) and molecular analyses of DNA methylation and histone modifications. In the latter case, changes seen in methylation/histone modifications might reflect true effects of an epigenetic chemical on a given cell or it might reflect the fact that the chemical selected a class of cells (ie, a stem cell), in which these methylation/histone patterns are the normal state of their differentiation. An urgent need of a biologically and mechanistically based, risk assessment strategy is needed for the development of new chemicals and drugs, for safety assessment of their use, and for risk assessment of potential health consequences after exposures. This challenge, in no way, implies that mutagenesis and cytotoxicity are not important toxic end points in human disease processes but only that chemicals probably do not play a role in these toxicities for genomic DNA in the critical target cells of the human body (ie, the stem cells) under normal real-life exposures.