Mutagenesis Advance Access originally published online on March 22, 2005
Mutagenesis 2005 20(2):81-92; doi:10.1093/mutage/gei017
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COMMENTARY |
The emperor wears no clothes in the field of carcinogen risk assessment: ignored concepts in cancer risk assessment
National Food Safety Toxicology Center, Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI 48824, USA
| Abstract |
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The following is a position paper challenging the paradigm that carcinogen = mutagen, and that the current rodent bioassay to predict risks to human cancers is relevant and useful. Specifically, we review current observations concerning carcinogenesis that might lead to another approach for assessing the identification of human carcinogenic hazards and the risk assessment that chemicals might pose. We give a brief review of the multistage and multimechanism process of cancer in a tissue that involves not only genotoxic but also epigenetic events, and the importance of stem and progenitor cells in the development of cancer. We focus on the often ignored epigenetic effects of carcinogens and the role of cell communication systems in epigenetically altering gene expression that leads to an imbalance of cell proliferation, differentiation and apoptosis in a tissue that can contribute to the cancer process. To draw attention to the fact that the current paradigm and policy to test toxic chemicals is often misleading and incorrect, we discuss how oxidative stress, in spite of the DNA damaging data, most probably contributes to cancer at the epigenetic level. Additionally, we briefly review how this mutagenic concept has greatly diverted attention away from doing research on the lower molecular weight, non-genotoxic, polycyclic aromatic hydrocarbons (PAHs), and how these low molecular weight PAHs are etiologically more relevant to the disease potential of environmental mixtures such as cigarette smoke.
| Introduction: Cancer is not a disease of a single celllimitations of a reductionalist's view |
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...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 also remember that the integration of normal cells with the welfare of the whole organisms is brought about entirely by molecular messages acting on molecular receptors (1
The goal of this rather iconoclastic challenge is to draw attention to the current paradigm and policy to test toxic chemicals that are correlated with cancer. Specifically, most still believe that cancer-causing chemicals are linked to DNA damage and mutations and that the current rodent bioassay to predict hazards and assess risks to human cancers is relevant and useful. While it could be argued that this carcinogen = mutagen paradigm has long been challenged, one needs only to examine the huge number of papers that are still published where this paradigm still drives the use of in vitro genotoxicity assays that are misinterpreted, and where both government regulatory agencies and pharmaceutical/industrial labs still require the use of these questionable assays. Specifically, we wish to review current observations concerning carcinogenesis that might lead to another approach for assessing the human carcinogenic risk that chemicals might pose.
Along with Potter's insight and the old adage, The whole is always greater than the sum of its parts, any best approach for assessing the human carcinogenic risk after chemical exposure will be confronted by no easy solutions. To begin with, the paradigm, carcinogens as mutagens (2
), illustrates the point we wish to make, namely, that carcinogenesis is more than mutagenesis! What would that more be? Clearly, when a cell (an adult stem cell; a progenitor cell or a terminally differentiated cell) is exposed to any chemical, there is a possibility of DNA damage and mutations in either or both nuclear and mitochondrial DNA. In addition, cell death by either necrosis or apoptosis could be the result. Finally, epigenetic alterations could ensue which, then, could alter the cell's commitment to cell division, cell differentiation, apoptosis, adaptive responses of differentiated cells or senescence.
In the recent past, the term, epigenetic mechanism, was a vague, non-universally accepted term. However, molecular demonstrations have now shown that genes in cancer cells can be abnormally expressed or that certain toxic/carcinogenic chemicals, without DNA-damaging capacity or mutagenic potential, can alter the patterns of gene expression by modifications of methylation and acetylation of DNA and histones (3
). Almost without reference to the introduction of DNA micro-array technology, which measures altered gene expression, much attention has been unconsciously focused on the role of epigenetic changes occurring during the carcinogenic process. It is as though a kind of risk assessment schizophrenia occurs in that the combination of using so-called in vitro genotoxicity assays, rodent bioassays, detection of DNA lesions and mutations in cells of tumors and the monitoring of altered gene expression, 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 prevailing paradigm shaping the risk assessment of potentially cancer-causing chemicals?
At the same time that all these cancer-associated chemicals presumably inducing lesions in DNA of various cell types in tissues of exposed animals, they induce intracellular signaling and alter gap junctional intercellular communication (GJIC) (4
). The broadest definition of an epigenetic change induced in a cell is that which alters the expression of the information of the genome at the transcriptional, translational or post-translational levels. It could occur as a heritable transcriptional change in proliferating cells or as a change in the expression of a stem cell to terminally differentiate or apoptose. However, altered gene expression is usually preceded by changes at the cell signaling level that governs transcriptional changes, as well as alterations in cellcell signaling within tissues. While epigenetic alterations are sometimes defined as inherited non-mutagenic alterations found in the expression of genes, it must be recalled that in vivo, chemicals affect the few stem cells, the progenitor and terminally differentiated cells. Stem cells induced to differentiate or apoptose by toxic chemicals, rather than proliferate, do so by epigenetic mechanisms when the cells do not proliferate. Moreover, when a terminally differentiated cell responds to a potentially toxic chemical, it might express stress genes. In addition, proliferating cells in Go phase can be induced to alter its gene expression transiently, to express genes for initiating cell cycle entrance but which return to the original transcription state after proliferation. Therefore, we chose to include the broader definition of epigenetic changes, which can occur in both proliferating and non-proliferating cells. The fact is that many chemicals can contribute to the carcinogenic process without inducing mutations or cell necrotic death. DDT, phenobarbital, saccharin, peroxisome proliferators, etc. are all experimentally known to contribute to the carcinogenic process in rodents.
Therefore, the real challenge is: How can one assess the epigenetic potential of a chemical's contribution to human cancer when it must be measured either in vitro or in experimental animals? No 2D in vitro assay, using either normal, primary rodent or human cells, or any immortalized normal or cancer cell line, can mimic the in vivo, human in situ condition of complex interactions between stem cells, proliferating progenitor cells and terminally differentiated cells within a tissue and between tissues (i.e. stromalepithelial interactions) (5
). Moreover, historic evidence has demonstrated the limitations of using experimental animals (6
) and epidemiological studies for a number of obvious reasons.
The fundamental issues raised by this challenge seem to include: (i) carcinogenesis in animals and human beings is a multistage, multimechanism process (i.e. the initiation/promotion/progress model) (7
); (ii) while experimental animals and human beings are fundamentally and biologically alike, in principle, they differ in many ways that could affect any one or all those stages; (iii) in both animals and human beings, individual genetic-, developmental stage-, gender-, dietary-, life style- and environmental-factors can, and do, influence each of these stages; (iv) since the experimental animal and human being consist of a hierarchical/cybernetic organization of multicell types, each interacting with cells within and between tissues (8
), use of cell-free, molecular targets, and 2D cell cultures, using either pure animal or human primary or immortalized/tumor cells, will never mimic the in vivo situation (9
). This is not to suggest that the use of molecular/biochemical or 2D cell cultures of primary or immortalized rodent or human cells will not generate a much needed mechanistic understanding of the carcinogenic process, as has experimental rodent carcinogenesis studies, particularly using the initiation/promotion/progress models, as well as transgenic and specific gene knock-outs/knock-ins; (v) delineation of mechanisms of each stage of carcinogenesis will be needed, particularly the potential of thresholds for epigenetic agents; (vi) the study of the in vitro mechanisms of carcinogenesis that might be identical to the carcinogenesis process in human beings; and (vii) the role of mixtures of carcinogenic factors (both endogenous and exogenous) that could influence any of the three steps of carcinogenesis to be additive, synergistic or antagonistic. As wonderful as these approaches have been and will continue to be, more will be needed.
The irony of this analysis is that, while we argue that current in vitro assays to measure mutagenesis, cytotoxicity or even epigenetic potential of chemicals are not adequate for accurate extrapolation to humans, the use of current animal bioassays will also fall far short of adequacy. Since space will not permit an attempt for rationalizing the use of 3D, human adult stem cells (9
) to attempt to breach this impossible task, the bottom line is that the use of human stem cells is where we believe that this field must aim for.
| Limitations of animal bioassays |
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While not wedded to using animal bioassays for the pre-testing of new chemicals for their potential risk in contributing to human carcinogenesis, one has to remember that today's bioassay protocol assumes that the induction of cancer in rodents, after an exposure to sublethal concentrations for the lifetime of the animal, generates a meaningful risk extrapolation information to human beings. However, such assumptions are overly simplistic. Since carcinogenesis is a multistage, multimechanism process, one would have to either assume that (i) the high dose-exposure of the single chemical being tested can induce all the mechanisms by itself; or (ii) the high dose of the chemical upsets the normal homeostatic control of the physiological state of the animal, which, in turn, affects one aspect of the multistage process.
Given that the cells of a tumor appear to be monoclonally derived from a single cell (10
), and in spite of their heterogeneity and genomic instability, and given that initiators seem to induce a stable, irreversible event, both mutagenesis (a irreversible change in the qualitative or quantitative nature of the genome) and a stable epigenetic mechanism (an altered expression of a gene) can contribute to the initiation phase of carcinogenesis. Short of directly testing changes in DNA sequences after exposure to an agent that is or could be an initiator, the problem is that the use of short-term in vitro assays to detect phenotypic changes, which are used as surrogates for mutations, will always generate too many false positives (11
,12
). Even measuring mutations in the cells of tumors created in rodents after exposure to a carcinogenic agent could be interpreted incorrectly if the agent acted to select a spontaneously generated mutation in an oncogene or tumor suppressor gene (13
).
Agents that can promote tumors appear to act epigenetically, i.e. the process of promotion can be interrupted; appear to have threshold levels of action; and can be ameliorated by antipromoters (14
). In addition to being species-, gender- and organ-specific, including developmental stage-specificity, promoting agents can act to select, clonally, the initiated cell by mitogenesis and/or blockage of apoptosis. Irritation or cell death or cell removal by surgery, can also act as a promoting condition of the initiated cell (14
).
Those agents, that, after the terminal stages of carcinogenesis, impart invasive, metastatic and angiogenic properties (progression phase of carcinogenesis), seem to do so in a stable manner. Both irreversible mutagenic and stable epigenetic events could contribute to this phase.
| Blinded by the carcinogen as mutagen paradigm, epigenetic mechanisms have been ignored! |
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As these reviewers look back at this complex problem, we see where Hanahan and Weinberg's comment is extremely relevant to the challenge: those researching the cancer problem will be practicing a dramatically different type of science than we have experienced over the past 25 years. Surely much of this change will be apparent at the technical level. But ultimately, the more fundamental change will be conceptual (15
| Promoters as inducers of oxidative stress do not make them genotoxic |
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Even when the promotion process appeared to involve a mitogenic 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 mutations needed to be fixed during the promotion process. While this hypothesis is, in part, true, it does not explain the mechanisms of agents that stimulate mitogenesis or inhibit apoptosis, both biological processes involved in the promotion phase (16
In order that it might not be missed, we suggest that chemicals, associated with carcinogenesis, might indeed induce DNA damage at high concentrations in certain kinds of cells, which probably die or will never divide (dead cells do not give rise to cancers), and which might induce DNA lesions in mitochondrial DNA, are probably epigenetic in nature, not mutagenic or genotoxic. If these concentrations are needed in vitro to induce DNA lesions and the recovery of phenotypic changes called mutations, the same concentrations in vivo would probably induce acute tissue damage, if not the death of the organism. To demonstrate that electrophiles of metabolized chemicals can interact with naked DNA or finding DNA lesions in extracted DNA from animal organs containing cells, most of which are probably not the target cells for cancer, do not constitute rigorous proof of the chemical's mechanism of action as an animal carcinogen. In other words, it would be highly unlikely that these so-called genotoxic chemical carcinogens do contribute to carcinogenesis as the mutagens of the mutations found in the oncogenes or tumor suppressor genes of the tumor found in the chemically treated animal. The latter statement should justify the claim that this review should be rather 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-existing oncogenic Hras1 gene mutations. While this and other similar studies have been swept under the regulatory rug, it also demonstrates that a chemical, interpreted as a mutagen through in vitro assays and shown to be a carcinogen in a bioassay, was assumed to induce mutations found in oncogenes in the tumors of the chemically exposed animal. The chemical was most likely an epigenetic agent that promoted a pre-existing spontaneously initiated cell. Even several in vitro transformation assay studies, which again seem to be ignored, have provided data that challenge the prevailing paradigm that a chemical, which induces rodent transformation in vitro, must be a mutagen. Brookes et al. (19
) and Mass and Austin (20
) showed that 7,12-dimethylbenz[a]anthracene (DMBA), the quintessential chemical mutagenic/carcinogen, did not mutate the Ki-ras and Ha-ras oncogene of the DMBA-transformed cells.
Apparently, many chronic diseases affiliated with oxidative stress, such as cancer, are not always a consequence of tissue necrosis, DNA mutations, or protein damage but rather, owing to an altered gene expression through epigenetic mechanisms (16
,21
). Another example that oxidative reactions do not contribute primarily to the genotoxic, initiating phase of cancer, are the results of rodents treated with organic and hydrogen peroxides in the twostage cancer model systems, in which these compounds exhibited tumor promoting and not initiating activity (22![]()
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25
), indicating that these oxidants are not mutagens but rather epigenetic toxicants. In the past 15 years, considerable research in oxidative stress has shifted from understanding how oxidations lead to macromolecular damage, to comprehending how reactive oxygen species (ROS) reversibly control the expression of genes at non-cytotoxic doses (26
). In this respect, at least 127 genes and signal transducing proteins have been reported to be sensitive to reductive and oxidative (redox) states in the cell (26
).
Although many intracellular signaling pathways are known to be redox-sensitive, the most studied signal transduction factors are mitogen-activated protein kinases (MAPK) and nuclear factor-kB (NF-kB) (26![]()
28
). These two pathways either directly or indirectly transduce most redox responses (26
). MAPK is not only activated by ROS (29
) but actually requires the presence of endogenously produced H2O2 (30
). This is one of several studies demonstrating that endogenous growth factors (extracellular ligands) generate ROS, which are then required downstream in intracellular signaling to successfully transmit their signals to the nucleus (31
). As mentioned above, the successful transmission of an extracellular signal from the membrane to the nucleus through intracellular signaling pathways in solid tissue cell types is also dependent upon intercellular signals through gap junctions (16
,21
). Not surprisingly, ROS have also been demonstrated to reversibly inhibit GJIC at non-cytotoxic levels (32
). If gap junctions were not closed, then the H2O2 generated by extracellular ligands could escape through gap junctions into neighboring cells, thereby potentially diluting to a subthreshold level that would be insufficient for MAPK-dependent activation of transcription factors. These examples demonstrate how extra-, intra- and inter-cellular signaling pathways might interact to coordinate the epigenetic expression of genes in response to ROS.
Antioxidants have also been demonstrated to serve as subcellular messengers for normal cell function (26
). For example, a major H2O2-scavenging pathway is the two-electron reduction of H2O2 catalyzed by glutathione peroxidase, which clearly serves as a protective role against peroxide-dependent oxidative injury. However, depletion of intracellular pools of glutathione (GSH), by inhibiting the rate-limiting step of its biosynthesis, paradoxically reverses the biological effect of H2O2 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 systems were depleted of GSH, which indicates that these signaling pathways not only required H2O2 but also GSH. Inhibition of GJIC and the induction of early-response genes are hallmarks of tumor promotion and in the results just described, a reduction in the natural antioxidant GSH could also potentially protect a cell from proliferative responses to extracellular ligands. Apparently, assessment of the true risk that oxidative stress poses to human health will need to move beyond the genotoxic, DNA-damaging paradigm, and incorporate an understanding of how oxidative reactions contribute to the epigenetic expression of genes.
| Polycyclic aromatic hydrocarbons: the mutagenic concept falls short of a comprehensive understanding of carcinogenic potential |
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In many cases, it has been argued that tumor-promoting chemicals can induce oxidative stress. Ergo, by definition by some, they are mutagens, yet when tested in in vitro assays, they prove not to be DNA damaging agents or mutagens (TCDD, TPA, DDT, phenobarbital, peroxisome proliferators, etc.). An excellent example is pentachlorophenol, which has been shown to induce oxidative stress (35
|
Considering that the fraction of cigarette smoke containing the three- and four-ringed PAHs is highly co-carcinogenic when applied to the skin of mice treated with benzo[a]pyrene (Figure 1) (39
|
|
To date, we have successfully demonstrated that many PAHs inhibit GJIC in pluripotent mammalian epithelial cells (46
|
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 form bay-like regions inhibit GJIC as well as phenanthrene that contains a bay region, whereas the 2-methyl or 2-chloroanthracene (2-meA, 2-Cl-A), which have linear configurations, do not inhibit GJIC (50
Although inhibition of GJIC may contribute to the mitogenic events of a promoter by removing an initiated cell from growth suppression, other epigenetic events such as the actual activation of a mitogenic-signaling pathway are also required. We have published results showing that GJIC-active (inhibitory) PAHs activate MAPK signaling pathways, while the GJIC-inactive PAHs do not induce MAPK (50
). The kinetic results also indicate that GJIC activity was affected before MAPK induction (a difference of 5 versus 15 min). These results are also consistent with the hypothesis that a quiescent cell must first be removed from growth suppression through inhibition of GJIC and at the same time or before the onset of mitogenic events.
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 signaling that favors proliferation; (ii) ignoring these molecular signaling events greatly underestimates the potential risk of these compounds to human health, particularly cancer; (iii) they are more consistent with the in vivo results of rodent systems exposed to tobacco smoke, indicating that combusted tobacco mixtures are strong tumor promoters but very weak initiators and complete carcinogens; (iv) they are more consistent with the reversible nature of tobacco smoke as indicated by the drastic decrease in cancer risk after cessation of smoking; (v) they offer novel molecular targets for the design of new chemopreventative and therapeutic strategies. Traditionally, the risk of these lower molecular weight PAHs to human health have been ignored precisely because of their non-genotoxic properties, and is a classic case of how the carcinogens = mutagens paradigm has greatly contributed to the underestimation of the true risk that environmental and food borne compounds pose to human health.
| The need for a mechanistic, biological cancer-risk assessment model |
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One major implication of knowing the mechanism of action of a potential chemical (Is it a mutagen, cytotoxicant or epigenetic toxicant?) is for epidemiological studies and risk analyses. Ignoring for the moment the contentious debate on chemical mutageniccarcinogenic initiators (e.g. Do they exist?; Are there threshold levels of exposure?; Do they cause the mutations in oncogenes and tumor suppressor genes in tumors of animals exposed to the chemical, etc.?), cytotoxicants can lead to cancers by their ability to induce compensatory hyperplasia, an indirect tumor promoting stimuli for surviving initiated cells. More importantly, it is the measured opinion of these commentators that most chemicals associated with tumors in exposed animals or human populations are epigenetic in character, by selectively cloning out of the target tissue pre-existing initiated cells. As tumor promoters, these chemicals do work at threshold levels (51
| Good newsbad news as the conundrum of chemical carcinogenesis |
|---|
To these commentators, the emperor wears no clothes in the field of chemical carcinogenesis when it comes to the selective view of using chemical carcinogen data to support one's assessment of the dangers for a particular chemical's ability to cause cancer (55
Phenobarbital appears to act as a promoter when it inhibits gap junction intercellular communication (58
). The liver tumors of the pre-weaned, initiated rat were embryonic-like (basophilic), suggesting that they might have originated from liver stem cells, which do not seem to have expressed connexin genes or functional gap junctions, similar to other adult stem cells (54
). Therefore, these pre-weaned rat tumors would not be promoted by phenobarbital, since they have no gap junctions. Agents that cause mitogenesis of stem cells probably stimulated the promotion of these basophilic tumors. It seems to be a fact that tumors can be characterized by a lack of functional heterologous or homologous GJIC (54
). In fact, the classic HeLa and MCF-7 cancer cell lines are cells that do not express their connexin genes (59
,60
), whereas many other cancer cells do have expressed connexins that are rendered non-functional because of some expressed oncogene.
Since phenobarbital was used as the example, it is interesting to note that, had regulations been in effect during the drug development and safety evaluation of this drug, it might never have been given human use approval because it is one of the classic promoters for rodent liver tumors. Phenobarbital can induce liver tumors in non-initiated animals at high doses and with chronic treatment. After decades of human use, there has been no epidemiological evidence of liver or head/oral cancers in human beings.
| Stem cells and cell communication as ignored hallmarks of cancer |
|---|
Guided by the insight of V.R.Potter in the aforementioned quote, it should be clear that carcinogenesis occurs in a complex in vivo environment where the single target cell exists in a tissue where there are cellmatrix interactions, stromalepithelial interactions (5
|
While the problem is yet to be resolved, the list of cancer hallmarks (11
Recently, the well-studied human carcinogen, benzene, which most, including the authors of this study, have thought was a mutagen (directly or via its metabolites) has been shown to differentially induce the cell death of human bone marrow CD34 hematopoietic progenitor cells (64
). Yet, Reddy et al. (99
) had already shown that benzene-treated mice did not exhibit any DNA adduct formation in the bone marrow. In fact, in the previous study, apoptosis was observed, suggesting a benzene-induced signal transduction-altered induction of the apoptosis signaling-gene system.
| The best approach to be used |
|---|
Assuming the aforementioned concepts that have been used to criticize our past and current approach to evaluate the risk to cancer after chemical exposure suggests the standard animal bioassay, and along the current short-term tests for mutagenicity, are, in large part, misinterpreted, we would recommend that their use in the future be discontinued. A recent series of invited papers has focused on assessing the current protocols used to determine the carcinogenic risk of chemicals (65
Ultimately, we recommend that a new emphasis be placed on the development and validation of several normal human stem cell 3D in vitro assays (human lung, liver, breast, prostate, kidney, brain, hematopoeitic, etc.) to test for cytotoxic and epigenetic endpoints (altered cell proliferation, differentiation, apoptosis, methylation changes, cellcell communication) at non-cytotoxic levels (3
,69
). These should be used to identify if any threshold levels of change are seen for these endpoints at non-cytotoxic levels.
| Three dimension organoid and differential sensitivity of 2D and 3D cultures |
|---|
Cells in vivo exist in a dynamic, interactive 3D milieu. Jacks and Weinberg have stated: The notion that cellular transformation and tumor progression involve the cooperative effects of proliferative signaling pathways and antiapoptotic pathways has been well studied in standard monolayer culture and in some in vivo models. However, the 3D culture system has the distinct advantage that it takes into account physiologically relevant interactions while being amenable to facile manipulation and biochemical analyses (9
In the last decade, more innovative techniques (70![]()
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75
) are being used to reconstruct some of the 3D dynamics seen in vivo, such as organotypic cultures (76
), co-culture of several cell types (77
), formation of 3D organoids (61
), insert wells to detect soluble factors that might identify stromalepithelial communication (78
), and embryonic and adult stem cells for testing toxicants (69
,79
). In all tissues, there exist three basic cell types, the few adult stem cells, the majority committed progenitor or transit cells with a finite life span and the terminally differentiated cells. The niche in which the adult stem cell resides controls its behavior, in addition to feedback signals from the terminally differentiated daughter cells (80
), in addition to environmental factors, such as the oxygen tension and calcium levels (81
), and other nutrients. Most in vitro assays used to detect the toxicity of chemicals are done at log phase and high oxygen levels, in media with high calcium levels and complex growth factors, such as serum. All these factors create conditions in the tested cells that do not mimic the physiological state of the potential target cells in vivo. Many studies have shown that the same cells tested in 2D log phase conditions, confluent 2D conditions or 3D systems do not yield the same toxic result (82
). No one has performed such an in vitro study with a proper mixture of human stem cells, their differentiated progenitor daughter cells and terminally differentiated progeny. This ought to be the ultimate goal for future risk assessment after chemical (or radiation) exposure from the in vitro to in vivo human situation. Such a study can be the closest experimental risk assessment that can be performed in humans. Even in this case, the limitations of individual genetic background, and other complex interacting factors will never be mimicked for extrapolation to a particular individual. It might be the best for which one can strive.
| Stromalepithelial interactions as one example of extracellular communication linked to GJIC: hormones, growth factors, cytokines, extracellular matrix modulation of gap junctions |
|---|
With the use of co-culture conditions and the development of new growth conditions for epithelial cell cultures, clear evidence of stromalepithelial interactions have been observed, which influences the biological control of cell behavior (83
Modulating cellcell communication through GJIC plays an essential role in the development of most epithelial and fibroblasts progenitor cells in vivo (85
). Endogenous factors that can modulate (increase or decrease GJIC) include growth factors (86
), various cytokines and hormones (87
), as well as extracellular matrices (88
). Modulation of GJIC between coupled cells can influence cell proliferation, cell differentiation and apoptosis, 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 the expression and function of the gap junction proteins, as well as the state of differentiation of cells.
| Redox disturbances of homeostatic control of cell proliferation/differentiation/apoptosis |
|---|
Inhibition of GJIC and apoptosis, altered differentiation, and stimulation of cell proliferation of these cells in the 3D cultures of normal human epithelial cells should be the closest we can get to mimicking the in vitro condition of the in vivo human situation. Any chemical testing positive at a given non-cytotoxic dose could be tested in a rodent system as a tumor promoter of initiated breast, liver, skin systems during a validation and species comparison phase. If a chemical is tested rigorously as being negative for any of the cytotoxic and epigenetic 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 on its potential biological and health effects will depend on a number of factors (genetic, gender, developmental state, target organ, cell type, cell cycle status, destination, endogenous metabolic fate, interaction with other endogenous/exogenous factors, concentration at target site, acute or chronic exposure patterns). The human being is a complex hierarchy of a homeostaticcybernetic system of interacting negative and positive signals from stem, progenitor and terminally differentiated cells in and between various organ systems. When chemicals in our food, medications, environment and life-style choices interfere with this delicate orchestration of homeostatic control of cell proliferation, differentiation, apoptosis and adaptive responses of our terminally differentiated cells, there is the potential of either a biological and a health effect or both. Disruption of this complex cellular communication system during embryonic and fetal development could lead to lethality or birth defects, impair neonatal and adolescent development, and could lead to maturation arrest and reproductive/neurological/behavioral dysfunctions, as well as diabetes, and after initiation of single cells could lead to cancer and atherogenesis (90![]()
92
). Although it must be stressed that while all health effects caused by exposures to chemicals have an underlying biological basis, not all biological responses as a result of these chemical exposures lead to health effects.
One lesson from these mechanistic studies that might apply to understanding epidemiological interpretations of human intervention studies, particularly with potential chemo-preventive agents correlated with reduced diseases in animals or humans consuming certain nutrients or diets, is that adding supplements to individuals who are deficient might be beneficial. However, adding these supplements to individuals who are sufficient for these supplements might not show any improvements. In fact, if the supplements are added at doses that are pharmacological rather than physiological, there might even be detrimental health effects. The real tasks here are to determine the amount of the sufficient levels that confer normal health in each individual (developmental stage, gender, genetic polymorphism, etc.).
The recent perception in various disease studies has detected a potential role of the inflammatory process with various chronic diseases, such as cancer, atherosclerosis, diabetes, etc. (93![]()
95
). Inflammation is a quintessential example of an extracellular communication process, involving secreted factors from one cell type to another cell type, evolutionarily designed for adaptive purposes, but which, if sustained in a chronic fashion, can be very maladaptive. Triggering this inflammatory process or chemically mimicking it in a sustained manner could lead to various health consequences. It should be noted that the classic tumor promoter, TPA, was shown to be an inflammatory inducer, induce oxidative stress in cells (96
), yet not shown to be a mutagen. In addition, many of the antitumor promoters have antioxidant activity.
This suggests that there might be a shared underlying component to many chronic diseases associated with sustained chronic inflammation that probably influences the tumor promotion phase of carcinogenesis. This, again, suggests that the most efficacious intervention strategy for chemoprevention is the use of antioxidants during the promotion phase of carcinogenesis to prevent or delay the initiated cells from accruing the necessary hallmarks of cancer.
| Summary as providing mechanistic insights for a biological approach to cancer risk assessment of chemicals |
|---|
A story is told of Albert Einstein's lecture to a lay audience about his recent Theory of Relativity. After his lecture, a reporter in the lecture hall came up to him and said, Professor Einstein, now that you physicists understand the workings of the universe, don't you think it is complicated? Einstein thought for a moment and replied, Young man, when you know nothing of the universe, it is, indeed, complicated. However, when you begin to understand, it is MERELY COMPLEX! The same could be said today of our understanding of the carcinogenic process. In the face of ignorance, it is, indeed, complicated. However, now that we are beginning to understand the process, it is merely complex. With that as the backdrop of our challenge, and with the statement by the late Robert Good, the cancer immunologist, It does not matter whether a hypothesis is right or wrong, but rather does it stimulate good experiments?, we feel it is time to critically re-examine the hypothesized role of chemical carcinogens as DNA damaging agents/mutagens and the carcinogen-induced effects 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 different kinds of cells, the few adult stem cells, the finite-limited progenitor cells and the terminally differentiated cells. These cells interact with each other through both extra- and gap junctional intercellular communication mechanisms. While some chemicals are metabolized to electrophiles and these chemicals can damage DNA, as well as other cellular molecules, they do not necessarily damage the three types of cells in an equivalent fashion. Evaluating tissues that have but a few adult stem cells in the tissue and finding DNA lesions, one cannot conclude that these lesions were in the cells that led to the cancers, let alone to the mutations found in any tumor in that tissue. Even the use of sensitive DNA micro-array technology to detect gene expression changes found in affected tissues, actually detects mostly primary changes in the non-stem or speculated target cell for cancer or the secondary or tertiary changes found in the different cell types owing to an upset in homeostatic regulation of the communication mechanisms among the different cell types.
One assumption is that the few adult stem cells are the cancer-target cells. If these cells do not have metabolizing enzymes, or if they do or do not repair their DNA the same way as the other type of cells, any conclusions, concerning the identification of that DNA lesion, or the mutation found in any oncogene or tumor suppression gene of the tumor from that tissue, is a real intellectual stretch.
It cannot be argued that chemicals do not influence cancers formed in animals exposed to chemicals or cancers found in individuals of a population exposed to a chemical. Clearly, the cancer-causing chemical did contribute in some way to the formation of that tumor. If the mechanism of action is not through mutagenesis, and only in those cases exposed to high concentrations of a chemical or microbial or parasitic toxins, which could kill cells in a sustained fashion leading to chronic inflammation and cytotoxicity (alcoholism or hepatitis, as examples), then some epigenetic mechanism must be attributed to its carcinogenicity. Given that the tumor promotion phase in human beings is the result of a sustained exposure to a non-genotoxic chemical at a threshold level and the absence of antitumor promoters, this phase of carcinogenesis must be considered as the most efficacious place of chemopreventive intervention. If, at least, the inflammatory process plays a role in some tumor promoting chemicals, anti-inflammatory agents could play a role in the antipromotion phase of carcinogenesis. However, even if a little bit of these antioxidant/anti-inflammatory agents can be beneficial, more might not be better because its potential of assisting individuals deficient in anti-oxidants might not assist those that are sufficient. In fact, the antioxidant properties of these agents might become pro-oxidants under different conditions found in the individual.
These chemicals, which prove to be carcinogenic through some epigenetic mechanism, must be viewed as having properties of all tumor promoters, having the hallmarks of species, gender, tissue and cell-type specificity; must work at threshold levels or above these levels; must be used in a sustained, chronic fashion; and must be found in individuals absence or deficient in antioxidants. Moreover, these chemicals do not affect cells in a vacuum. They interact with other exogenous and endogenous chemicals that could be additive, synergistic or antagonistic to the chemical of interests. This explains, in large part, why two genetically identical individuals, exposed to the same amount and duration of a specific chemical, will not exhibit the same risk to cancer.
Finally, any epidemiological or risk assessment analysis of a chemical's potential to induce cancer must take into account the epigenetic mechanisms of action of chemicals. While it is beyond the scope of this analysis to translate the implications of chemical carcinogens acting as epigenetic toxicants in risk assessment models, the future of cancer risk assessment modeling must integrate the characteristics of epigenetic toxicants on initiated stem cells in tissues. Some of these factors include: (i) threshold concentrations by which these chemicals work as promoters; (ii) sustained and chronic exposures; (iii) absence of antipromoters; and (iv) species, gender and developmental stage-specificity. Recognition of potential differential responses of the different cell types to the chemical of interest must be taken into account. Moreover, this is a complex or systems view of how these chemicals not only alter complex signaling within cells, but also, the complex signaling between different cell types within an organ and the interaction of signals between organs within an organism. Finally, the concepts of the role of adult stem cells and of secreted- and gap junctional intercellular communication must be considered in viewing the pathogenesis of cancer (100
).
| Acknowledgments |
|---|
Supported by an NIEHS Superfund Basic Science Program grant to JET (PA42 ES04911).
| Notes |
|---|
* To whom correspondence should be addressed. Tel: +1 517 432 3100, Ext. 188; Fax: +1 517 432 6340; Email: james.trosko{at}ht.msu.edu
| References |
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Received on January 4, 2005; revised on February 15, 2005; accepted on February 16, 2005.
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I. P. Pogribny, V. P. Tryndyak, L. Muskhelishvili, I. Rusyn, and S. A. Ross Methyl Deficiency, Alterations in Global Histone Modifications, and Carcinogenesis J. Nutr., January 1, 2007; 137(1): 216S - 222S. [Abstract] [Full Text] [PDF] |
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