Chem. Res. Toxicol. 1992,5, 742-748
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Risk Assessment Based on High-Dose Animal Exposure Experiments Samuel M. Cohen*J and Leon B. Ellweid Department of Pathology and Microbiology and Eppley Institute for Cancer Research, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-3135, and National Eye Institute, National Institutes of Health, Building 31, Room 6A08, Bethesda, Maryland 20892 Received July 8, 1992
Introduction Chemicals and mixtures have been known for more than two centuries to cause cancer in humans, and the list of suspect agents continues to expand as our ability to detect carcinogenic effectsin experimentaltest systems improves. Ultimately, a causal relationshp to human cancer requires epidemiological evidence, which is generally insensitive to less than strong relationships and not specific in the face of exposure to a wide variety of chemicals. Since chemicals which cause cancer in humans also cause cancer in animal model systems, particularly in rats and mice, considerable effort has gone into developing and utilizing experimental models to screen chemicals for the purpose of controlling human exposure (1-3). In the United States, this has given rise to the National Toxicology Program and development of standard bioassay protocolsfor the evaluation of chemicals for carcinogenicity in rata and mice; similar protocols are utilized worldwide in the evaluation of chemicals. Two fundamental assumptions are inherent in the use of bioassay results in assessing human risk: (1)If a chemical causes cancer in rats and/or mice, it has a high probability of causing cancer in humans (species extrapolation). (2) If a chemical causes a significant increase in cancer incidence when administered at a high dose, it will also cause cancer, albeit at a lower incidence, at low doses (dose extrapolation). Chemicalsare tested at the maximum tolerated dose,which is variably defined but generally corresponds to the dose in a 28- or 90-day study that produces clear evidence of toxicity, for example, a 10% retardation in growth (4). The development of the bioassay and its use in human risk assessment were based on evaluation of agents such as radiation or potent, genotoxic chemicals such as diethylnitrosamine (DEN): AAF, BP, and aflatoxin. There is considerable experimental evidence that species and dose extrapolations are appropriate for these agents (58). Administration of these chemicals, even at low doses, results in a significant increase in tumor incidence. More importantly,even at doses at which there is not a detectable incidence of tumorigenicity, there is evidence of DNA adduct formation and/or DNA damage, implying the possibility of an increased risk of tumor induction at these low doses (9, 10).
* To whom reprint requesta and correspondence should be addressed.
t University of Nebraska Medical Center.
National Institutes of Health. Abbreviations: AAF, 2-(acetylamino)fluorene;BP, benzo[alpyrene; DEN, diethylnitrosamine; DMBA, 7J2-dimethylbenzanthracene; 3-MC, 3-methylcholanthrene; MUP, mouse urinary protein; TPA, 12-O-tetradecanoylphorbol 13-acetate. 8
The fact that radiation and genotoxic chemicals have the potential of causing genetic damage at very low doses is consistent with the hypotheais that exposure to low doses leads to an increased risk of developing cancer, even though the increased incidence may not be detectable with present bioassay or epidemiological techniques. Evidence in further support of the species and dose extrapolation assumptions comes from findings that chemicals known to be carcinogensin humans are also carcinogenicin animal models; nearly all of these are genotoxic chemicals (1). Dose-response relationships may be affected by pharmacokinetic and cellular response differences between species (11). Nevertheless, these are quantitative rather than qualitative issues. The above two assumptions underpinning the use of the bioassay in human risk assessment are reasonable and can govern the establishment of estimates of potential risks to humansfor radiation and genotoxic chemicals (12-14). Nongenotoxic compounds are another matter.
Nongenotoxic Carcinogens Distinction between genotoxic and nongenotoxic carcinogens was not considered before the 19705, primarily because it was not until the late 1960s that it became clear that the former compounds interacted with DNA leading to genetic damage, whether point mutation or other types of DNA changes. The advent of short-term mutagenicity studies, such as the Ames’ assay utilizing Salmonella typhimurium, gave rise to the evaluation of numerous known human and animal carcinogens. Most of the chemicals known to be carcinogenicin animals and/or man tended to be mutagenic (15). However, it soon became evident that there were a significant number of chemicals which were negative in short-term screens for genotoxicity, yet produced an increased incidence of various types of cancers in rodent bioassays (16). In addition, a few known human carcinogens,such as estrogen, were also considered to be nongenotoxic. This led Williams and Weisburger (17)to suggest that there were two types of carcinogens, with markedly different biologicalproperties, which they classified as genotoxic and nongenotoxic. Compounds within the nongenotoxic class of carcinogens have also been referred to by other names, such as epigenetic, promoter, indirect, and carcinogens involving secondary mechanisms. Based on theoretical considerations and modeling of epidemiologicaland experimental data, a two-event model of carcinogenesis, taking into account not only genotoxic events but also spontaneous genetic damage arising during cell division (DNA replication), was proposed by Moolgavkar et al. (based on epidemiological data) (18) and
0893-228~/92/2705-0742$03.00/00 1992 American Chemical Society
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Forum Target cell replication
leading to canar
Cell Death
Differentiation
Figure 1. Competing events that can alter the number of cell
divisionsin atarget cellpopulation. Under normal circumstances, tissues maintain a balance in cell number by generating new cells (k,) sufficient to replace cell loss (kzand k3). If kl is increased and/or kz or ka decreased, the result is to increase the number of cell divisions per unit time. k4 represents the probability of a critical genetic error necessary for cancer production occurring during a cell division. Under normal circumstances, kd is extremely small. An agent can increase the possibility of cancer by increasing the rate of genetic damage (k4)and/or increasing the number of cell divisions per unit time (increasing kl and/or decreasing kz + k3).
Greenfield et al. (19)(based on experimentalcarcinogenesis in the rat). This biologically-based model quantifies the potentially pivotal influence of cell proliferation in carcinogenesis, not only as an independent factor, but in its contribution to the dose-response of genotoxic chemicals (12-14,18, 19).
This model is based on several assumptions: (1)Cancer arises due to alterations in various genes. (2) More than one genetic error (two) is necessary. (3) Genetic errors can occur probabilistically, albeit at a very low rate, every time DNA replicates (each cell division). (4) The genetic errors must occur in a pleuripotential (’stem”) cell. On the basis of these assumptions, a chemical, or, for that matter, any agent, can alter the risk of developing cancer by either increasing the change of genetic damage during each cell division or increasingthe number of cell divisions subjected to a spontaneous risk of genetic damage. An agent, of course, also can do both. Obviously, those agents that interact with DNA and affect the rate of genetic damage per cell division are genotoxic carcinogens. For most such agents, they also have an effect on cell proliferation at high doses, which markedly increases the slope of the tumor-response curve (5,12). At doses below the increase in cell proliferation, only the genotoxic effect is present. As discussed above, this genotoxicity effect probably extends to very low doses, essentially representing a nonthreshold phenomenon. In contrast are agents (Figure l),which are not genotoxic, that only increase cell proliferation. We have further subdivided such agents into those which act via a specific cell receptor and those which do not (13,14). In general, chemicals that act via a specific cell receptor are operative at low doses, which is in contrast to those nongenotoxic chemicals that do not involve a specific cell receptor. It is likely that there are chemicals with a cell proliferative effect via a cell receptor and yet, also, which can be metabolicallyactivated to intermediates that damage DNA directly. This is possibly the case with agents such as
tamoxifen,which clearly interacts with estrogen receptors, but also is metabolically activated to agents that form DNA adducts (20). It is essential to note that the initial response to the chemical is critical. For example, an agent might interfere with the metabolism of thyroxine, ultimately leading to an activation of TSH secretion from the pituitary, which in turn leads to increased proliferation of the thyroid follicular cells and ultimately thyroid adenomas and carcinomas (21). Excess TSH by itself has been shown to cause thyroid tumors. However, the chemical‘s initial effect is on thyroxine metabolism, not likely a receptormediated phenomenon, whereas TSH interaction with thyroid follicular cells is clearly a receptor-mediated phenomenon. Agents that act via specific cell receptors may or may not have doseresponse thresholds as discussed below. On the other hand, nongenotoxic agents that do not act via a cell receptor are likely to have specificdose thresholds with respect to proliferative response and, therefore, the same threshold with respect to carcinogenic effect (13, 14). Because a dose-responsivethreshold occurs with such chemicals, and because the response may be tissue as well as species specific, the two fundamental assumptions underlying extrapolation from the rodent bioassay to humans may not hold. It is this critical differencebetween nongenotoxic chemicals, particularly those not acting through a specific cell receptor, and genotoxic chemicals that requires that their evaluation with respect to potential human risk be approached differently. That is not to say that every nongenotoxic,non-receptor-mediated chemical is without risk to humans, but specific understanding of doseresponse relationships and mechanism in different species must be understood to properly evaluate potential risk to humans. This information requirement goes beyond that available from the traditional rodent bioassay. Illustrative examples will be described, as well as a discussion of possible exceptions to the general message being conveyed.
Calculi and Urinary Bladder Carcinogenesis Extensive proliferation of the urothelium can be produced by the presence of a foreign body in the lumen of the urinary bladder of rats or mice (22). Rata appear to be considerably more susceptible to both the proliferative and ultimate tumorigenic response. The foreign body can be surgically implanted into the lumen or it can be generated by the formation of a calculus,through alteration of physiological processes, through administration of exogenous chemicals, or even by a variety of surgical manipulations to other portions of the body (14,22-24). Regardless of how the foreign body is generated, extensive proliferation occurs, ultimately resulting in the development of tumors. The increase in cancer risk is tied qualitativelyto the presence or absence of the foreign body. The time sequence of this process has been most clearly demonstrated by Jull (25)utilizing surgically implanted paraffin wax pellets into the lumen of the mouse urinary bladder. An incidence of 10.6 9% was present after 40-50 wks, 26.8% after 70-80wks, and 53.8% after 150-160 wks. Other substanceshave been surgically implanted, including cholesterolpellets, glass beads, and stainless steel (23,24). The major feature which determines the extensiveness of the proliferationand ultimate tumorigenic activity appears to be the degree of coarseness of the surface; the smoother
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the surface, the less the proliferation and the less the tumorigenic activity (26). It should be noted that the extent of mitotic rate enhancement and the increase in number of cells (because of the appearance of a papillary proliferation) are greater in rats than in mice. The mouse tends to have a more nodular proliferation, which involves adding a smaller number of cells (27). In either case, the proliferative activity of the physically stimulated bladder is considerablygreater than the slow turnover rate present in normal urothelium. Studies examining the addition of other substances within these pellets suggest that several factors can contribute to the increased rate of proliferation and ultimate development of tumors (23-26). The speed at which a chemical is leached from the pellet influences the coarseness of the surface. The addition of genotoxic chemicals within the pellet greatly increases tumorigenicity. Thus, 3-MC, a known carcinogen, mixed into the paraffin wax or cholesterolpellet greatly increasesthe rate of tumor development compared to the pellet without added 3-MC. The urothelium has the enzymatic complement necessary for activation of the carcinogen to its ultimate metabolite with binding to DNA. Calculi in the urine can have the same effect. Calculi can form from a variety of substances (14,22-24,28,29), including those that are administered exogenously, such as uracil, melamine, or calcium oxalate, they can appear secondaryto the metabolism of exogenously administered compounds, such as diethylene glycol (which results in oxalate stones), or they can arise by endogenous physiological alterations secondary to metabolic changes, such as the production of uric acid or calcium- or phosphatecontaining stones secondary to hypercalcemia and/or hyperphosphatemia. Regardless of the chemicalinvolved, or whether due to exogenous or endogenous mechanisms, the resulting calculi produce extensive cell proliferation and ultimately tumors. Most of these chemicals are nongenotoxic in in vivo circumstances. To estimate potential risk to humans, it is necessary to determine the amount of chemical necessary before a calculus can form in the urine. This requires knowledge not only of the solubility of the chemical but also of its metabolism, pharmacokinetics, and physical-chemical influences of urinary constituents, such as mucopolysaccharides and protein, that may enhance or inhibit calculi formation. Coarseness of the calculi is important in producing cell proliferationas is the competing necrotizing influence of the calculus and resulting inflammation. As mentioned above, rats are considerably more susceptible than mice: Implanted paraffm wax pellets produce a 100% incidence of bladder tumors in rats in less than a year, whereas the incidence in mice is less even after a period as long as 2 years. Humans appear to be even less susceptible than mice to the tumorigenic effects of calculi. It is unclear from epidemiological studies whether calculi by themselves can produce tumors in humans, or whether tumors that do occur are related to the usually associated bacterial infections, cystitis, and urinary obstruction and stagnation (30). Clearly,chemicals that produce calculi have a threshold effect, and the dose extrapolation assumption of the bioassay is inappropriate. The threshold amount to which humans can be exposed can be reasonably well estimated on the basis of physiologic,pharmacokinetic,and physicalchemical determinations (14, 28, 29).
Cohen and Ellwein
Sodium Saccharin and Related Salts Saccharin is a widely consumed chemical by humans, being utilized in a variety of food, cosmetic, and pharmaceutical products (31). In the 19705,it was shown to be a bladder carcinogen when administered as the sodium salt at high doses to rats, with a greater incidence in male rats compared to female rats. Subsequent research has demonstrated that administration beginning at 6 weeks of age or later carrieswith it little risk of developing bladder tumors compared to that beginning at birth or in the early neonatal time period. In addition, the mouse, hamster, and monkey appear to be without effect, and multiple epidemiological studies have found no indication of increased risk of bladder cancer secondary to exposure to artificial sweeteners, including saccharin. In general, saccharin is a nongenotoxic chemical in most short-term assays, but it increases proliferation of the urothelium when administered at high doses in the diet. Whether evaluated for carcinogenicity or for its tumorpromoting or hyperplasiagenic effects in the male rat, a threshold response appears to be involved: No effects are seen in the male rat bladder epithelium at doses below 1%,while effects are observed when the chemical is administered at doses of 2.5% or higher. These data suggest that the dose extrapolation assumption of the rodent bioassay may be inappropriate. More importantly, on the basis of mechanism, it would appear that interspecies extrapolation is also inappropriate. It has recently been demonstrated that the proliferativeresponse to saccharin occurs as a regenerative phenomenon due to mild toxicity to the urothelium, leading to erosion of the superficial bladder epithelial cells and consequent regenerative hyperplasia (31). This toxic effect appears to be contingent upon the presence of a precipitate in the urine (whichalso includes the formation of microcrystals); the formation of this precipitate is dependent upon high levels of protein in the urine, where different proteins appear to have varying potential for generating this precipitate (31, 32). azu-Globulin, a male rat-specific protein to which saccharin binds quite extensively, is present as the major protein in this precipitate (32). Albumin and other proteins also appear to be involved with the formation of this precipitate, but to a lesser extent. Although the female rat does not have large amounts of azu-globulin,its urine contains high levels of albumin. In contrast, although the mouse has a protein related to a2u-globulin,it does not generate a precipitate in response to sodium saccharin administration. Most importantly, the human does not have a protein analagous to a2,-globulin; also, the rat has 100-1O00 times the concentration of total protein in the urine compared to humans (33). Thus, on the basis of mechanistic considerations alone, humans are unlikely to develop bladder cancer as a consequence to exposure to saccharin, even if humans consumed levels as high as those consumed by the rat. The human urinary tract response to saccharin is like that in the mouse, hamster, and monkey and unlike the susceptible rat species. As with the formation of calculi, requirementsfor precipitate formation can be determined on the basis of chemical, physiologic, and pharmacokinetic parameters, with ascertainment of thresholds in the susceptible species.
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az,-Globulin-Associated Nephropathy Chemicals such as unleaded gasoline,trimethylpentane, and d-limonene produce kidney tumors only in male rats, with no effect in the female rat or in male or female mice (34). This response is related to the binding of these chemicals, or one of their metabolites, to the az,-globulin protein, present in large amounts in male rats of certain species. This protein is normally absorbed by proximal tubule cells and degraded in lysosomes. However, when bound to these specific chemicals, it cannot be easily digested by the cell, leading to toxicity, cell death, significant cell regeneration,and ultimately a low incidence of renal cell tumors. Administration of these chemicals to male NBR rats, which do not secrete a2,-globulin in the urine, does not result in renal tubular necrosis, regenerative proliferation, or kidney tumor formation (35).Also, although mice have a related protein excreted in large quantities in the urine, called MUP, it does not lead to the accumulation within tubular cells, or to cell necrosis; therefore, mice do not develop regenerativehyperplastic or tumorigenicresponse (34).
The critical question is whether the human situation can be related to the positive response in certain species of male rats. az,-Globulin is a member of a large gene family including several related proteins, such as retinoic acid binding protein, which are found in humans. However, none of these proteins behave like azu-globulin.To begin with, they are not present in the urine in large quantities. Also, it appears that they do not have the same potential for renal cell toxicity. Thus, the azUglobulin mechanism by which these nongenotoxic chemicals cause renal cell carcinomas in male rats is an inappropriate model for humans; the interspecies extrapolation assumption does not hold. Further experimental evidence strongly suggests that there is threshold response for these chemicals even in the male rat. If this is true, the dose extrapolation assumption is also incorrect.
Specific Target Cell Population A major assumption of the carcinogenesis model presented is that the critical genetic events must occur in a pluripotential, "stem" cell population. We realize that the term stem cell has numerous connotations depending on the organ system being evaluated (36). Nevertheless, it is clear that in every organ, except perhaps for the nervous, cardiacmuscle, and skeletal muscle systems,there is a group of cells that have the potential for repopulating a tissue if it is damaged. This excludes those cells committed to differentiation, which is a pathway to cell death. Thus, if cell damage, such as DNA adduct formation, occurs in differentiated cells, it will not have a tumorigenic effect. Similarly, if differentiated cells proliferate,this has no direct tumorigeniceffect; an indirect effect may take place if the action of differentiated cells influences the proliferation and replication of the stem cell population. A few examples are cited to illustrate the importance of focusing on the dynamics of the stem cell population in interpreting cancer incidence data. In rat mammary carcinogenesis, utilizing the DMBA model, administration at day 55 of age results in nearly 100% incidence of mammary tumors (37). If the same dose of chemical is administered at day 100 of age, the incidence is considerably lower. However, if all epithelial
cells are isolated from the breast tissue and the DNA is analyzed for DMBa adducts, the quantitative level of adduct formation is similar in the two groups. One interpretation is that these adducts are unrelated to carcinogenesis,but this is highly unlikely given the plethora of data supporting DNA adducts as a marker of mutations in various chemical carcinogenesis models. A second explanation, and the more likely, is based on the status of the cell populations at each of these ages. At the earlier age, the breast tissue is relatively undifferentiated and rapidly proliferating. In contrast, at the later age, the breast tissue has undergone considerable differentiation, with a decreased replication rate. This is especiallytrue if the rat has gone through pregnancy, which is a strong inducement for the breast tissue to undergo differentiation and ultimately to reduce cell proliferation. The differentiation status of the cell population within the breast at the time of treatment is obviously critical in interpreting the results of these carcinogenesis experiments. In mouse skin carcinogenesis, it has long been known that chemicals that produce hyperplasia have different abilities to act as so-called promoting agents (38,391.Thus, TPA is a strong hyperplasiagenic agent and is a strong tumor promoter in the mouse skin model. On the other hand, mezerein and turpentine produce hyperplasia and yet are only weak tumor-promoting chemicals. This contrast has been used as an argument against the relationship between increased cell proliferation (hyperplasia) and promoting activity in the mouse skin model. However, if one examines the situation closer, there are at least two major differences in the type of hyperplasia induced by TPA versus that produced by mezerein and turpentine (40). First, the hyperplastic response to TPA is persistent following continued administration of the chemical, whereas the proliferation associated with mezerein or turpentine treatment diminished over time. Second, the cells that proliferatein response to TPA administration appear to be in the stem cell compartment of the skin, a subset of the basal cell layer of the epidermis. A distinction between dark and light cell types has been described by Klein-Szanto (40) and others. The dark cells, which proliferate in response to TPA, appear to at least include the stem cell population of the skin, whereas the light cells, which respond more in response to mezerein, do not include the stem cell population. Distinction between types of proliferating cells in squamous epithelium is readily seen in hyperplasia of the forestomach followingadministration of various chemicals (41, 42). Two types of proliferation have been distinguished one being a more basaloid proliferation, whereas the other is a proliferation of the more mature keratinocytes in cell layers above the basal cell layer. It is the proliferation of the basal cell layer, or at least some component of it, which is the stem cell population of the squamous epithelium and which is required for development of cancer. In humans, the importance of the stem cell population is also evident (43). Adenomatous polyps of the colon, whether tubular or villous, are precursor lesions to the development of adenocarcinomas. These polyps reflect a proliferation of the crypt cells, which is the stem cell compartment of the colonicepithelium. This type of polyp occurs in familial polyposis coli, a genetic disorder with nearly a l O O L risk of developing colon cancer unless the
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colon is removed at an early age. In contrast, hyperplastic polyps of the colon are a proliferation of the more differentiated, mucus-containing cells of the colon; they are no more likely to evolve into cancer than the normal epithelium, whether there is a single hyperplastic polyp or whether there are several thousand as seen in the rare cases of multiple hyperplastic polyposis. Again, there is an important difference between proliferation occurring in a stem cell compartment compared to that in a differentiated cell compartment.
Toxicity and Cell Proliferation Increased cell proliferation in the defined target cell population results in an increased risk of cancer development because, with each cell DNA replication cycle, there is a small, but nonzero probability that a mistake can occur in a gene critical in the development of cancer (13,14,18,19). Itmustberealizedthattherearenumerous ways to increase cell proliferation, either directly or by blocking processes that compete with the growth of the stem cell population (Figure 1). Under normal circumstances, stem cells divide into two daughter cells: one to replace the stem cell itself, and the other committed to differentiation. Stem cells can also divide into two stem cells, with an overall increase in the stem cell population. The differentiation process competes with this proliferative growth of the stem cell population (Figure 1). Thus, increased cell proliferation can occur simply by partially or completely blocking the mechanism with which cells undergo differentiation after the stem cell divides. Another competing process is that of cell death, whether following a specific mechanism such as apoptosis or a nonspecific process such as cell injury. Obviously, decreasing the rate of either of these cell death processes will increase the population of cells available for continued proliferation. Proliferation of cells can occur through one of two basic ways. The first is a direct mitogenic stimulus to the cell resulting in cell division. The second, and the one more common with respect to chemical exposure, is that which occurs in the process of regeneration following cell injury. Within these two generally-defined processes are innumerable pathways for producing the proliferative effects. Obviously, there are numerous ways by which chemicals can injure cells, which is the basis for the field of toxicology. Increasingly, mechanisms are being discovered by which agents can have direct mitogenic effects on cells. This direct mitogenic effect frequently involves interactions with specific cell receptors, usually related to specific growth factors or inhibitors. A wide variety of enzymatic mechanisms are available in cells for metabolically activating exogenous chemicals to reactive genotoxic intermediates; in an analogous fashion, cells have numerous pathways by which they can directly or indirectly produce increased rates of proliferation upon exposure to chemical agents. A major source of confusion in the recent literature in understanding the role of cell proliferation in carcinogenesis has been brought about by a focus on toxicity itself rather than on increased cell proliferation (44-46). To begin with, there are numerous mechanisms of toxicity that do not involveincreased cell proliferation whatsoever, including many forms of neurotoxicity, cardiac toxicity, and behavioral toxicity. The major determinant for cell proliferation to affect carcinogenesis when it is produced
Cohen and Ellwein
through toxicity is an increase in the number of cell divisions per unit time in the susceptible cell population within the entire organ. Thus, measurement of labeling index by itself is not a proper determinant, since it conveys no information about the magnitude of the denominator (the total number of cells in the tissue). For example, monuron is a markedly toxic chemical in mice, producing severe growth retardation as well as some liver toxicity (14),but it is not a hepatocarcinogen in mice. Although a labeling index has not yet been reported with this chemical, it is essential to note that the size of the liver in the treated animals is approximately one-half that of the liver in controls. Thus, the labeling index would have to be double the normal rate just to maintain the same total number of cell divisions within the entire liver. A doubling of labeling index is quite substantial and frequently is not achieved even with known carcinogenic chemicals. The key difference in the monuron case is that the population to which the labeling index applies is markedly decreased compared to controls, and the total number of cell divisions per organ may be actually decreased rather than increased. Another aspect of toxicity is identification of the cell population in which it is occurring. Cell toxicity resulting in proliferation of differentiated or committed cells will not necessarily increase the rate of tumorigenesis. Evaluation of proliferative rates in intermediate cell populations, such as hyperplastic nodules in the liver, is also of critical importance; they may show increased proliferative rates leading to an increased risk of cancer, whereas the surrounding normal cells, such as normal liver, might not be affected (14). Persistence of the proliferative response is important. It is hardly surprising that there is little correlation between the general observation of toxicity and carcinogenesis. This lack of correlation with toxicity has been equated by some as a demonstration of the lack of a relationship between increased cell proliferation and carcinogenesis. Such a conclusion is blatantly inappropriate and only serves to confuse the actual issues involved. Toxicity does not equate to increased proliferation in a critical target cell population. In a similar vein, there is considerably less than a complete correlation between carcinogenesis and mutagenesis (16); nevertheless, mutagenesis is not discounted as a critical factor in carcinogenesis. Furthermore, there is not even an absolute correlation between DNA adduct formation and carcinogenicity (47). Adducts can be detected following administration of noncarcinogenic chemicals or in tissues not producing tumors by carcinogenic chemicals. It has also been questioned why there are not more tumors seen during in utero development, a time at which proliferative rates are extremely high. This is at best a naive interpretation of the carcinogenic process, which ignores the importance of time in producing rare genetic events. Even if cancer were to require only two critical genetic errors, the odds of this occurring in the same cell during cell cycles within the early period of an organ’s development are extremely remote. This has been described by several investigators (13,14,18,19)including the seminal contribution by Knudson inferring the existence of tumor suppressor genes (48).Even if a fully malignant cell was produced during the in utero period, a tumor of detectable size must still evolve, which requires alarge number of cell divisions and a considerable amount of time.
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Forum CHEMICAL CARCINOGEN
1 GENOTOXIC
AemC14” wrn DNA?
NON-GENOTOXIC Reaction with cell receptor?
1. Threshold unlikely 2. Dose-response may be affected by cell proliferation (usually toxicity related at highdoses). PROLIFERATIVE
PROLIFERATIVE
1. Threshold questionable
1. Threshold likely
2. Usually effective at low doses
2. Usually related to toxicity and regeneration
Figure 2. Proposed classification scheme for carcinogens. The effect of genotoxic chemicals can be accentuated if cell proliferative effects are also present. Nongenotoxic chemicals act by increasing cell proliferation directly or indirectly, either through interaction with a specific cell receptor or nonspecifically by (i) a direct mitogenic stimulus; (ii) causing toxicity or consequent regeneration; or (iii) interrupting physiological processes. Examples of the latter mechanism include TSH stimulation of thyroid cell proliferation after toxic damage to the thyroid, and viral stimulation of proliferation after immunosuppression. (Reprintedwith permission from ref 13. Copyright 1990 by the American Association for the Advancement of Science.)
Implications for Risk Assessment We have previously put forward a general classification of chemical carcinogens consistent with the above dis(Figure 2). Underlying this cussion and examples (13,14) classification is the separation of carcinogenic effects produced by genotoxic versus nongenotoxic, proliferative mechanisms. Genotoxic chemicals are unlikely to have a true threshold effect. A single molecule of a genotoxic chemical has a probability, albeit infinitesimal, of undergoing activation to a metabolite which interacts with the DNA of a gene critical for the development of cancer. ObGiously, the larger the number of molecules to which an organism is exposed, the more likely it is that one of them will get through the maze of activating and inactivating mechanisms available in the organism, and the more likely that interaction with a gene specific for the development of cancer will take place. Since the fixation of a genetic mistake appears to require cell division,or certainly is greatly enhanced by a cell in the active part of the cell cycle, increasing the number of target tissue cell divisions during exposure to the genotoxic carcinogen will increase the likelihood of occurrence of a cancer-related genetic effect. Thus, the slope of the cancer dose-response curve is markedly increased when a genotoxic chemical is administered at doses sufficient to produce increased cell proliferation, which interacts synergistically with the genotoxic influence of the chemical (12-14). In contrast, nongenotoxic chemicals do not interact directly with DNA, but exhibit their effects through increasing the number of cell divisions in a target tissue with a spontaneousrisk of genetic alteration during mitosis. For chemicals acting through a specific cell receptor, the possibility of a threshold exists, but it is dependent on a number of factors (14).From the study of certain receptormediated processes it is clear that occupancy of more than one receptor per cell is required to activate the cellular response, whether cell division or other response. The required receptor occupancy percentage ranges from as low as approximately 1% to a much greater percentage. Even a level of 1% represents a large number of molecules, since for any specific cell receptor there are generally
thousands of receptor sites per cell. Thus, on the basis of these initial considerations, one would presume that a threshold phenomenon would exist for nongenotoxic chemicals involving cell receptor-mediated mechanisms. However, because several of these chemicals act through cell receptors that are already being occupied by endogenous agents, this generalization does not hold. For example, estrogen is already present in the organism a t specific levels and frequently is well above the threshold necessary for stimulating a critical response, such as endometrial cell division. Similar to the chances of a critical mutation occurring following exposure to a single molecule of a genotoxic chemical, exposure to even one additional molecule of estrogen has a small, but nonzero chance of producing binding to a cell receptor and triggering one additional cell division, increasing the risk of developing cancer, albeit infinitesimally. If the receptormediated mechanism is not already above the threshold level, then exposure to the exogenous agent will likely involve a threshold phenomenon. For nongenotoxic carcinogens that do not act through a cell receptor mechanism, a threshold can be expected as described here for calculi, saccharin, and azu-globulinrelated nephropathy, and by others in several additional model systems. The determining factor is the mechanism by which these chemicals cause cell proliferation. It is necessary to determine whether the proliferation is a direct mitogenic response or whether it is secondary to toxicity and cell regeneration. The proliferative dose response can be determined and the necessary physiological and pharmacokinetic parameters estimated for extrapolation of rodent effects to the human. It is on this basis that a more realistic estimate of risk to humans can be made. For many nongenotoxic chemicals, the doses at which humans are exposed will be below the threshold level. For others, the mechanism involved in generating the tumor in the rodent bioassay will not occur in humans, such as that described above for saccharin and the ~~2~-globulin-related nephropathy chemicals.
Conclusions As stated at the beginning of this article, two fundamental assumptions involving dose and species extrapolation underlie the estimation of risk to humans for chemicals tested in the long-term rodent bioassay. If either of these is not appropriate for a given chemical, which is generally the case for nongenotoxic chemicals, then the extrapolation and risk assessment process must be different from what has been standard practice (13,14,28, 29,49). It is necessary to take into consideration both dose and mechanistic factors in estimating quantitatively the potential risk, if any, to humans at typically low-dose exposures. To blindly extrapolate from high dose to low dose on the basis of some mathematical formula is not only inadequate and inappropriate,but actually misleading (50). We have an obligation to society to incorporate today’stechnology and biological knowledge in the conduct of risk assessments. Over two decades of scientific exploration have provided the basis for going beyond almost exclusivereliance on the high-dose animal bioassay as the primary data source for this process. References (1) ZARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (1980) Long-term and short-term screening
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