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Nongenotoxic carcinogens in the regulatory environment
REGULATORY
TOXICOLOGY
AND
Nongenotoxic
PHARMACOLOGY
Carcinogens
9,244-256
( 1989)
in the Regulatory
Environment’
BYRON E. BUTTERWORTH Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received February I, I989
The biological activity of many carcinogens is to directly induce mutational events, thereby altering the information encoded in the DNA. Short-term tests for potential carcinogens and risk assessment models generally rely on the assumption that the agent in question will operate through a genotoxic mechanism. However, carcinogenesis is a multistep process, and it is increasingly clear that the primary biological effect for many carcinogenic chemicals involves events other than direct DNA reactivity. For many experimental rodent models as well as human cancers, nongenotoxic mechanisms appear to be the driving force in the formation of tumors. Many of these nongenotoxic mechanisms are highly species-specific. Thus, it is increasingly important to ask if the rodent model applies to the human situation at all, in addition to the examination of appropriate, hypothetical, mathematical risk assessment models. More research is now being focused to better define the mechanisms by which the many distinctly different classes of nongenotoxic carcinogens are acting. This understanding will become the basis for new predictive assaysand more realistic risk assessment models. If specific conditions are met, then a no observed effect level with a safety factor may be the most appropriate risk model for some carcinogens. 0 1989 Academic Rw., Inc.
GENOTOXIC
VS NONGENOTOXIC
CARCINOGENS
A genotoxic agent is one capable of altering the information encoded in the DNA either directly or through the formation of a reactive metabolite. These alterations can be point mutations, insertions, deletions, or changes in chromosome structure or number. This activity usually can be detected by assays that measure reactivity with the DNA, induction of mutations, induction of DNA repair, or cytogenetic effects in bacterial or mammalian cell culture assays as well as in the whole animal (McCann et al., 1975; Hsie et al., 1979; Bridges et al., 1982). Indeed, the basis of short-term tests for potential carcinogens, such as the Ames test, is the observation that genotoxic agents represent a major class of chemical carcinogens. Examples of genotoxic carcin’ Presented at the 4th Annual Meeting of the International macology, October 17- 18,1988, Baltimore, MD. 244 0273-23OQ/89 $3.00 CDpyriBht 1331989 by Academic Pres. Inc. Au rights of reproductionin any fmm reserved.
Society of Regulatory Toxicology and Phar-
NONGENOTOXIC
CARCINOGENS
245
ogens are 2-acetylaminofluorene (ZAAF), dimethylnitrosamine (DMN), and benzo[a]pyrene (Miller and Miller, 198 1). Nongenotoxic chemicals are those that lack genotoxicity as a primary biological activity in currently used assays. While these agents may yield genotoxic events as a secondary result of other induced toxicity, such as forced cellular growth, their primary action does not involve reactivity with the DNA, particularly when observed in the target organ at doses and by the route of administration that produced tumors in the animal study. Examples of nongenotoxic carcinogens are saccharin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Ashby, 1985; Poland and Rimbrough, 1984). CLASSES
OF NONGENOTOXIC
CARCINOGENS
A dual classification scheme of genetic and epigenetic (nongenotoxic) carcinogens has been proposed and many of the different subclasses of agents defined (Weisburger and Williams, 198 1; Williams and Weisburger, 1983). These classifications need to be continually expanded to include all of the many distinctly different groups of nongenotoxic carcinogens as they are identified. Often the scientific distinction between genotoxic and nongenotoxic agents is unclear because there can be a gradation of different activities between compounds. In some cases a chemical may be placed in more than one category. Nevertheless, it is misleading to refer to the nongenotoxic carcinogens as though they were a single class. For example, considerations of risk assessment models, mechanisms of action, and predictive assays for the potent hormone-like promoter TCDD are not the same as for saccharin that appears to be exhibiting its effects by inducing bladder hyperplasia. Herewith are some of the major classes of chemical carcinogens that have been identified.
Genotoxicants For these chemicals the weight of evidence indicates a consistent and reproducible pattern of activity in bacterial, cell culture, and/or whole animal assays for genotoxicity (McCann et al., 1975; Hsie et al., 1979; Bridges et al., 1982). It is valuable to confirm evidence of genotoxic activity in the target tissue of the treated animal. DNA adducts, induced DNA repair, or cytogenetic effects can be measured in animals treated with the test chemical in the manner that produced tumors.
Promoters This is a very large and diverse group of chemicals that promote development of tumors from initiated cells (Slaga, 1983-1984). In general, the action of tumor promoters is reversible and continuous application over extended periods are required to exhibit carcinogenic activity. A high degree of species and target organ-specificity are often seen with these agents. The classical definition of promoters is agents that are not themselves carcinogenic, but promote the formation of tumors from initiated cells. Since there are many mutagens in the natural environment, practically, the promoters must be considered carcinogenic agents.
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As mechanisms become better understood, many promoters may be placed in more defined categories according to their mechanism of action. For example, 12U-tetradecanoylphorbol13-acetate (TPA) is a potent promoter in the mouse skin painting model. Elegant research has now identified protein kinase C as the receptor for the phorbol ester tumor promoters (Blumberg, 1988). As an illustration of how difficult it can be to place agents into a specific category, some favor the hypothesis that TPA is acting through the generation of DNA-damaging oxygen radicals (Cerutti, 1987). In contrast, other skin tumor promoters, such as the anthrones, are acting by mechanisms of action that would place them in a different category than TPA (DiGiovanni, 1987). Assays have also been defined to assess promotion and progression in the liver (Goldsworthy et al., 1986). An example of a potent liver tumor promoter that appears to be acting through a specific receptor is TCDD (Poland and Kimbrough, 1984).
Mitogenic Liver Carcinogens For chemicals in this group, no chemically induced cytotoxicity is observed, as measured by the lack of liver-specific enzymes in the serum. Hyperplasia is characterized by mitogenic stimulation of cell replication and increased organ size. Examples are phenobarbital, a-hexachlorocyclohexane, polybrominated biphenyls, butylhydroxytoluene, ethinylestradiol, and cyproterone acetate (Schulte-Hermann et al., 1982; Schulte-Hermann et al., 1983; Schroter et al. 1987; Schulte-Hermann et al., 1988).
Peroxisomal Proliferating/Mitogenic Liver Carcinogens The peroxisomal proliferating carcinogens are a diverse group of chemical agents that induce an initial growth in liver size, hepatic peroxisomal proliferation, lipofustin accumulation, and continual increased cell turnover for the more active carcinogens (Reddy and Lalwani, 1983; Butterworth, 1987; Marsman et al., 1988; Conway et al., 1989). Examples range from very weak agents such as di(2-ethylhexyl)phthalate (DEHP) to potent carcinogens such as Wy-14,643, methyl clofenapate, and ciprofibrate (Conway et al., 1989). There exists a high degree of species specificity in the induction of liver peroxisomal enzymes, with rodent hepatocytes responding to a much greater extent than human hepatocytes (Elcombe and Mitchell, 1986; Butterworth et al., 1989).
Cytotoxic Liver Carcinogens These chemicals exhibit cytotoxicity and produce necrosis. Liver-specific enzymes are seen in the serum as an indication of cellular injury. Cell division is observed as regenerative cell growth to replace damaged tissue. An example of such a carcinogen would be carbon tetrachloride (Doolittle et al., 1987; IARC, 1979a; Loury et al., 1987a).
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CARCINOGENS
247
Cytotoxic Kidney Carcinogens A variety of nongenotoxic nephrotoxicants yield kidney tumors, including unleaded gasoline, nitrilotriacetate, chloroform, perchloroethylene, and pentachloroethane (Anderson, 1987; Kluwe et al., 1984; Loury et al., 1987a; MacFarland et al., 1984; Trump et al., 1985; Weisburger, 1977). For many of these kidney carcinogens, the pattern of activity is the induction of tumors in the kidneys of male rats but not female rats or mice of either sex. The primary mechanism of action for these agents would appear to be chemically-induced accumulation of the male rat-specific protein a&obulin in the form of protein droplets in the kidney tubule cells, resulting in cell death and regenerative hyperplasia (Short et al., 1986; Loury et al., 1987b; Swenberg and Short, 1987; Wachsmuth, 1987; Goldsworthy et al., 1988). It is this continual cell proliferation that would appear to be the driving force in the induction of tumors.
Cytotoxic Bladder Carcinogens/Promoters Three classes of nongenotoxic bladder promoters have been identified (Cohen et al., 1987). The first includes sodium salts of weak acids such as saccharin and ascorbate (Cohen, 1985; Imaida et al., 1984). The second class of promoters includes uracil and terephthalic acid and yield urinary calculi (Heck, 1987). These calculi produce a foreign body reaction and result in hyperplasia. The third class includes the antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisol and the amino acids isoleucine and leucine (Imaida et al., 1984; Nishio et al., 1986). For these substances, massive doses of over 1% in the diet must be administered for long periods of time to produce papillomas or cancer. The primary action of these compounds appears to be enhancement of bladder hyperplasia at the site that eventually yields the tumors.
Cytotoxic Stomach Carcinogens Many compounds have been shown to induce forestomach tumors in rodent models (Kroes and Wester, 1986). While this is a common site for genotoxic carcinogens, some of these agents appear to be acting through nongenotoxic mechanisms. For example, the antioxidant butylated hydroxyanisole (BHA) induces rat forestomach tumors at very high doses (Ito et al., 1983). However, BHA is not genotoxic and the tumors appear to be related to the induction of hyperplasia (Williams, 1986; Clayson et al., 1986). In fact, BHA has actually been shown to inhibit the action of a broad spectrum of carcinogens, mutagens and tumor promoters (Wattenberg, 1986). Similarly, ethyl acrylate causes malignant neoplasms in the forestomach of Fischer 344 rats when administered chronically by gavage (National Toxicology Program, 1983). In the course of healing the forestomach lesions produced by ethyl acrylate, hair and food particles become entrapped. Evidence is convincing that this tumor formation by ethyl acrylate is secondary to foreign body reactions and induced epithelial cell proliferation (Ghanayem et al., 1986a,b).
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Hormones As regulators of cell proliferation and function, many hormones have been implicated in the development and progression of cancer (IARC, 1979b). For example, diethylstilbestrol (DES) is carcinogenic in a variety of rodent models and is associated with an increased incidence of endometrial carcinoma (IARC, 1979~). DES has been causally associated with an increase in vaginal and cervical clear-cell adenocarcinomas in daughters of women who were administered the hormone during pregnancy (IARC, 1979~). While there are conflicting reports as to whether DES is mutagenic, evidence from cell transformation assays and studies of lesions in patients exposed in utero suggest that induction of aneuploidy may be mechanistically related to tumor formation, which would be viewed as a genotoxic event (Barrett et al., 1981; Fu et al., 1979; Tsutsui et al., 1986; Degen and Metzler, 1987). However, because of its profound effects on gene expression and cellular function, we must remain open to the possibility that DES may play several mechanistic roles in the process of carcinogenesis.
Thyroid Carcinogens Thyroid gland follicular tumors can be produced by a variety of conditions, natural products or chemical agents, that result in hormone imbalance with resulting hyperplasia of the thyroid (Paynter et al., 1988). For example, low iodine diets or subtotal thyroidectomy decrease circulating levels of thyroid hormones in the blood. To compensate, the anterior pituitary gland releases increased amounts of thyroid stimulating hormone, which induces thyroid hypertrophy and hyperplasia. Because there is no resulting increase in thyroid hormone levels, this hyperplasia is sustained by the prolonged stimulation of the thyroid and pituitary glands and may progress to neoplasia. Restoration of the normal hormone balance before tumor formation results in a return to the normal state. This is clearly an example of a nongenotoxic mechanism because no chemicals were administered to the animal at all. Several groups of chemical agents have been identified that yield thyroid tumors by mechanisms that appear to be analogous to that just described (Paynter et al., 1988). Thiocarbonyl compounds such as thiouracil, propylthiouracil, and methimazole are potent thyroid function inhibitors and are used in the treatment of hyperthyroidism even though they have been shown to produce thyroid tumors in rodent models (Willis, 196 1; Woo et al., 1985).
Solid-State Carcinogens This group includes mineral fibers such as asbestos and is of particular concern because of the indications that people can be susceptible to cancer resulting from exposure to specific types of asbestos (Lee, 1985; Zurer, 1985). Many of the properties of asbestos, such as fiber size and shape, the ability to produce aneuploidy, and induce local cell proliferation are being correlated with its carcinogenic activity (Bhatt et al., 1984; Hesterberg and Barrett, 1984; Moalli et al., 1987). However, much more information needs to be gathered before we will know exactly which properties to
NONGENOTOXIC
249
CARCINOGENS
avoid in selecting a safe substitute for asbestos and the many other types of manmade mineral fibers that are now in widespread use. CHEMICALLY
INDUCED
CELL
PROLIFERATION
The most common biological activity for many of these nongenotoxic carcinogens is that of chemically induced cell division. There are numerous roles that cell proliferation might play in the process of carcinogenesis. Enhanced replication may increase the frequency of spontaneous mutations (Stott et al., 1981). Increased rates of cell turnover increases the probability of converting DNA adducts from natural sources into mutations before they can be repaired. Tumor promotion is associated with a sustained induction of cell replication (Slaga, 1983-l 984). The multistep process of carcinogenesis involves the clonal expansion of initiated or altered cells (Farber, 1980; Pitot and Sirica, 1980). Finally, the mutation or altered expression of oncogenes and/or growth control proteins are strongly implicated in cancer (Land et al., 1983; Stewart et al., 1984; Adams et al., 1985; Reynolds et al., 1987). The chance for an initiating event is enhanced during replication because many of these genes are being transcribed, during which time that segment of the DNA is exposed and may experience greater susceptibility to environmental carcinogen induced DNAdamage. In a National Toxicology Program/National Cancer Institute study to compare mutagenic activity with carcinogenic activity for 73 chemicals, there was not a single chemical that required over 500 mg/kg/day to produce tumors that was mutagenic in the Ames test (Tennant et al., 1987). This raises the concern that the tumors produced by such chemicals may arise by a secondary mechanism, such as organ-specific toxicity and resultant cell turnover, that would not occur at lower doses of the agent. If the extent and duration of cell turnover can be correlated with carcinogenic potential, then this endpoint might be used as a predictive assay (Loury et al., 1987a). Unfortunately, cell proliferation studies are labor intensive and the data base needed to establish this correlation does not exist at this time. A typical example of a chemical that appears to be acting by this mechanism is saccharin. This artificial sweetener yielded bladder tumors when administered at a dose of 5% saccharin in the diet, about 3,000 mg/kg body weight/day (mg/kg/day), in a two generation study (Arnold et al., 1980). Whereas sodium saccharin in the diet at 5% produces bladder hyperplasia, acid saccharin, interestingly, does not. One might also predict on this basis that acid saccharin would not be a bladder carcinogen (Cohen et al., 1987; Schoening et al., 1985). MAXIMUM
TOLERATED
DOSE
A developmental drug or new product that is nontoxic presents a problem for industry. Such massive doses are required in order to reach the mandatory maximum tolerated dose that some adverse effect often appears in the animals in the course of what many consider an unrealistic study. One concern when conducting a cancer bioassay is to avoid or at least be aware of situations where high doses of a chemical compromise the health or lifespan of the animal, overwhelm natural detoxification mechanisms, or yield tumors secondary to excessive organ-specific toxicity. On the
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E. BUTTERWORTH
other hand, with so few animals to test, it is important to choose doses as high as possible to maximize the ability to detect carcinogenic effects of the chemical. The most common biological activity of many classes of nongenotoxic carcinogens would appear to be the induction of hyperplasia (Loury et al., 1987a). Gathering chemically induced cell proliferation data as part of the ninety day study that precedes a cancer bioassay could provide valuable information to aid in setting rational doses for longterm studies. RISK
ASSESSMENT
Currently, mathematical extrapolation models are predominantly used to estimate theoretical tumor incidence rates at low doses by extrapolating from high doses where tumors were actually observed. As an illustration of how variant these models can be, the maximum likelihood estimates of cancer risk for the probit and multistage models for exposure to 0.1 ppm formaldehyde differ by nineteen orders of magnitude (Starr and Buck, 1984). Yet, in the face of all the uncertainty associated with cancer studies, risk estimates for regulatory purposes usually rely only on the upper 95% confidence bound of the multistage model and sometimes have been given to three significant figures (National Research Council, 1983). Further, there has been considerable resistance to deviate at all from this regulatory posture (Perera, 1984). There is substantial evidence that the genotoxic rodent carcinogens present a carcinogenic risk to human beings. Genotoxic agents tend to produce tumors in multiple tissues and species (Ashby and Tennant, 1988). Comparative studies show that those agents that induce DNA repair in rat hepatocytes also induce DNA repair in human hepatocytes (Butterworth et al., 1989). Analysis of the known human carcinogens reveals that the majority are overtly genotoxic (Shelby, 1988). In contrast, concerns with the inappropriateness of the rodent model for carcinogenesis loom particularly large with nongenotoxic carcinogens because of the high degree of species specificity often associated with these agents. Key mechanisms of action allowing meaningful risk assessment often remain unknown and there are serious questions as to whether some rodent responses can be extrapolated to people at all. It would also be prudent to address the issue as to whether the rodent model is applicable to the human situation, in addition to employing a hypothetical mathematical risk assessment model. For example, TCDD is 10,000 times more potent than TPA as a promoter in the C3H 10T l/2 initiation/promotion assay, 100 times more potent than TPA as a skin tumor promoter in the hairless mouse, and is one of the most potent rat liver tumor promoters ever described (Pitot et al., 1980; Poland et al., 1982; Abernethy et al., 1985). This activity stands in stark contrast to the human situation. In 1949 an industrial accident at a Monsanto Company facility in Nitro, West Virginia, resulted in the exposure of 121 workers to TCDD in large enough amounts to produce chloracne and many related health problems, such as liver enlargement. These symptoms persisted for longer than 4 years, consistent with the observation that TCDD is retained in the body for long periods. A follow-up study 30 years later that was 100% complete revealed no apparent excess in total mortality or in deaths from malignant neoplasms (Zack and Suskind, 1980). The most important question with TCDD is: Why is this extremely potent rodent carcinogen not an obvious human carcinogen? It is hoped that mechanistic studies with TCDD will
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CARCINOGENS
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provide the answers to this question (Poland and Kimbrough, 1984; Greenlee et al., 1987). The unsettling companion question is: Are there potent human carcinogens that are inactive in rodents? Similar concerns are raised with the male rat-specific cytotoxic kidney carcinogens that appear to be acting through a*,-globulin mediated toxicity and subsequent cell proliferation. Since a&obulin is a male rat-specific protein, one must question whether the response produced by these nongenotoxic agents can be extrapolated to people (Short et al., 1986; Loury et al., 1987b; Charbonneau et al., 1987; Lock et al., 1987). The first priority with the many nongenotoxic chemicals that exhibit this pattern of activity is to conduct comparative mechanistic studies contrasting the male rat and the human, rather than apply a rigid mathematical model to extrapolate from rodent to man. There have been 18 long-term cancer studies with saccharin (Oser, 1985). Some of which have involved periods of saccharin intake of over 12,000 mg/kg/day (Oser, 1985). The latest study involved 2,500 rats and examined seven doses between 1 and 7.5% saccharin (Schoening et al., 1985). Rather than do more cancer bioassays, perhaps it is time to increase our research efforts to understand why saccharin yields tumors and if those events are likely to occur in people. Nongenotoxic thyroid carcinogens that act by prolonged stimulation of the pituitary/thyroid axis would not be expected to exert any carcinogenic effects at doses that do not induce thyroid hyperplasia (Hill et al., 1988). Available evidence confirms that humans are less sensitive to the carcinogenic effects of long-term thyroid stimulation than animals (Hill et al., 1988). Different risk models may be appropriate for different classes of carcinogens. The most commonly used cancer risk models rely on assumptions of direct mutagenicity and assume that no matter how low the dose, there remains the possibility that the critical target gene may be hit. The formulas used allow the calculation of a theoretical risk at any dose. These models suffer because they consider only tumor incidence and do not account for chemically-induced cell proliferation as it applies either to the expansion of initiated cells or the direct role that cell turnover may have in the carcinogenic process. If one accepts the concept from experimental evidence that tumor formation by some nongenotoxic carcinogens is secondary to organ-specific toxicity and subsequent cell proliferation, then fundamental assumptions for risk models will be different. Many times the action of a toxicant is on a specific cellular protein. There may be thousands of copies of this protein in the cell. If the toxicant in question removes only one copy of the protein, such that its level in the cell stays within normal limits, then there will be neither cell death nor resulting regenerative cell division. There would be a threshold to the carcinogenic effects of such an agent. If one had assurances that induced tumors were secondary to organ-specific toxicity, then a no observed effect level (NOEL) with a safety factor would be a rational risk assessment model. Using more than one model helps remind us that we are dealing with an inexact science. In either case, one is extrapolating to low doses where the induction of tumors cannot practically be measured. If one assumes that chemically-induced cell proliferation is the driving force for tumor formation, then the NOEL approach for risk assessment could be employed. As a prerequisite to this approach, the experimental carcinogen would have to exhibit all of the following properties to assure that no direct genotoxic mechanism was involved:
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(1) A weight of evidence analysis indicates that the test chemical is not genotoxic in a battery of short-term tests. [These assays are sensitive and reliable indicators of genotoxic activity (Hsie et al., 1979; Bridges et al., 1982)]. (2) Over 500 mg/kg/day was required to produce tumors. [None of the compounds in the NTP/NCI study that required over 500 mg/kg/day to produce tumors was mutagenic in the Ames test (Tennant et al., 1987). While there could be flexibility of this cutoff, one should be cautious with any chemical that produces tumors at very low doses, because of the possibility that it may be acting through an unknown mechanism]. (3) Tumors appeared only near the end of the study. [This precludes potent promoters that may be working through specific receptors such as TCDD (Poland and Kimbrough, 1984)]. (4) Hyperplasia appeared in the target organ prior to tumor development. [The assumption is that it is this chemically-induced cell turnover that is the driving force for tumor formation)]. (5) Tumors appeared only in that single target organ. [Genotoxic agents tend to produce tumors in multiple tissues and species (Ashby and Tennant, 1988)]. The basis for selecting the safety factor would include considerations of mechanism of action, the dose at which hyperplasia was evident, species differences in susceptibility, the shape of the dose-response curve, and human experience with the chemical. The advantages to this approach are that the model conforms to the perception of the mechanism of action of the chemical and permits the use of additional relevant information. An example of a chemical for which this risk approach might be used is saccharin. A credible public cancer policy must not only identify potential human carcinogens, but also must distinguish between chemicals and usage patterns that represent a clear and present danger and those that present risks that are trivial in nature. Tobacco smoking is the cause of 30 to 40% of deaths from cancer (Loeb et al., 1984; National Research Council, 1986). Age-adjusted lung cancer mortality rates for American women increased 337% between 1950 and 1980 and will soon exceed breast cancer as the leading cause of cancer death in women (Loeb et al., 1984). It is doubtful that anyone has ever contracted cancer related to saccharin consumption (Morgan and Wong, 1985). Yet, the word “cancer” appears as a warning on both a pack of cigarettes and a packet of saccharin, with no guidance to the average person as to their relative risks. The rodent model for carcinogenesis is central to public cancer policy. In the exercise of assessing risk, scientific honesty requires a discussion of the strengths and weaknesses of the bioassay and a comparison of the theoretical risk calculated from several mathematical models. The uncertainty in the risk estimates should be acknowledged and the data presented to only one significant figure. Merits of different models, safety factors, mechanistic information and risk vs benefit considerations should be openly debated. Then the difficult political decision of setting regulatory limits to exposure could be made without relying on the illusion that science had produced an infallible number. REFERENCES ABERNETHY,
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GHANAYEM, B. I., MARONPOT, R. R., AND MATTHEWS, H. B. (1986a). Association ofchemically induced forestomach cell proliferation and carcinogenesis. Cancer Lett. 32,27 l-278. GHANAYEM, B. I., MARONPOT, R. R., AND MATTHEWS, H. B. (1986b). Ethyl acrylate-induced gastric toxicity. III. Development and recovery of lesions. Toxicol. Appl. Pharmacol. 83,576-583. GOLDSWORTHY, T. L., HANIGAN, M. H., AND PITOT, H. C. (1986). Models of hepatocarcinogenesis in the rat-contrasts and comparisons. CRC Crit. Rev. ToxicoI. 17,6 l-89. GOLDSWORTHY, T. L., LYGHT, O., BURNETT, V. L., AND POPP, J. A. (1988). Potential role of (Y-~Pglobulin, protein droplet accumulation, and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene, and pentachloroethane. Toxicol. Appl. Pharmacol. 96, 367379. GREENLEE, W. F., OSBORNE, R., AND DOLD, K. M. (1987). Altered regulation of epidermal cell proliferation and differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In Reviews in Biochemical Toxicology. (E. Hodgson, Bend, J. M. Philpot and J. R. Bend, Eds.), Vol. 8, l-35. Elsevier, NY. HECK, H. d’A. (1987). Bladder stones and bladder cancer: A review of the toxicology of terephthalic acid. In Nongenotoxic Mechanisms in Carcinogenesis (B. E. Butterworth and T. J. Slaga, Eds.), Banbury Report 25, pp. 233-243. Cold Spring Harbor Laboratory, NY. HESTERBERG, T. W., AND BARRETT, J. C. (1984). Dependence of asbestos- and mineral dust-induced transformation of mammalian cells in culture on fiber dimension. Cancer Res. 44,2 170-2 180. HILL, R. N., ERDERICH, L., PAYNTER, 0. E., ROBERTS, P. A., ROSENTHAL, S. L., AND WILKINSON, C. F. (1988). Thyroid Follicular Ceil Carcinogenesis: Mechanistic and Science Policy Considerations. U.S. Environmental Protection Agency, Washington, DC. HSIE, A. W., O’NEILL, J. P., AND MCELHENY, V. K., (Eds.) (1979). Mammalian Cell Mutagenesis: The Maturation of Test Systems, Banbury Report 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. IARC (1979a). IARC Monographs on the Evaluation of the Carcinogenic Risk ofchemicals to Humans pp. 37 l-388. IARC Sci. Publ. 20. IARC (1979b). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans Sex Hormones (II). pp. 139-55 1. IARC Sci. Publ. 2 1. IARC (1979c). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans Sex Hormones (II). pp. 173-23 1. IARC Sci. Publ. 2 1. IMAIDA, K., F~JKUSHIMA, S., SHIRAI, T., MASUI, T., OGISO, T., AND ITO, N. (1984). Promoting activity of butylated hydroxyanisole butylated hydroxytoluene and sodium L-ascorbate on forestomach and urinary bladder carcinogenesis initated with methylnitrosourea in F344 male rats. Gann 75,769. ITO, N., FUKUSHIMA, S., HAGIWARA, A., SHIBATA, M. AND GGISO T. (1983). Carcinogenicity ofbutylated hydroxyanisole in F344 rats. J. Natl. Cancer Inst. 70,343. KLUWE, W. M., ABJX, K. M., AND HUFF, J. (1984). Chronic kidney disease and organic chemical exposures: Evaluations of causal relationships in humans and experimental animals. Fundam. Appl. Toxicol 4,889-90 1. KROES, R., AND WESTER, P. W. (1986). Forestomach carcinogens: possible mechanism of action. Food Chem. Toxicol. 24,1083-1089. LAND, H., PARADA, L. F., AND WEINBERG, R. (1983). Cellular oncogenes and multistep carcinogenesis. Science222,771-778. LEE, K. P. (1985). Lung response to particulates with emphasis on asbestos and other fibrous dusts. CRC Crit. Rev. Toxicol. 14,33-86. LOCK, E. A., CHARBONNEAU, M., STRASSER,J., SWENBERG,J. A., AND Bus, J. S. (1987). 2,2,4-trimethylpcntane-induced nephrotoxicity. II. The reversible binding of a TMP metabolite to a renal protein Ii-action containing q,-globulin. Toxicol. Appl. Pharmacol. 91,182- 192. LOEB, L. A., ERNSTER, V. L., WARNER, K. E., ABBOTTS, J. AND LASZLO, J. (1984). Smoking and lung cancer: An overview. Cancer Res. 44,5940-5958. LOURY, D. J., GOLDSWORTHY, T. L., AND BUTTERWORTH, B. E. (1987a). The value of measuring cell replication as a predictive index of tissue-specific tumorigenic potential. In Nongenotoxic Mechanisms in Carcinogenesis (B. E. Butterworth and T. J. Slaga, Eds.), Banbury Report 25, pp. 119-136. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. LOURY, D. J., SMITH-OLIVER, T., AND BUIIZRWORTH, B. E. (1987b). Assessment of unscheduled and replicative DNA synthesis in rat kidney cells exposed in vitro or in vivo to unleaded gasoline. Toxic&. Appl. Pharmacol. 87,127- 140.
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CARCINOGENS
255
MACFARLAND, H. N., ULRICH, C. E., HOLDSWORTH, C. E., KITCHEN, D. N., HALLIWELL, W. H., AND BLUM, S. C. (1984). A chronic inhalation study with unleaded gasoline vapor. J. Amer. College Toxicol. 3,23 l-248. MARSMAN, D. S., CATTLEY, R. C., CONWAY, J. G., AND Pope, J. A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,643) in rats. Cancer Res. 48,6739-6744. MCCANN, J., CBOI, E., YAMASAKI, E., AND AMES, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Nat. Acad. Sci. USA 72,5 135-5 139. MILLER, E. C., AND MILLER, J. A. (198 1). Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules. Cancer 47,2327-2345. MOALLI, P. A., MACDONALD, J. L., GOOIXLICK, L. A., AND KANE, A. B. (1987). Acute injury and regeneration of the mesothelium in response to asbestos fibers. Amer. J. Pathol. 128,426-445. MORGAN, R. W., AND WONG, 0. (1985). A review of epidemiological studies on artificial sweeteners and bladder cancer. Food Chem. Toxicol. 23,529-533. National Research Council (1983). Drinking Water and Health. Vol. 5. National Academy Press, Washington, DC. National Research Council (1986). Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects. National Academy Press, Washington, DC. National Toxicology Program (NTP) (1983). Carcinogenesis Bioassay ofEthyl Acrylate. NTP Technical Report Series No. 259. National Institutes of Health, Bethesda, MD. NIH Publication No. (NIH)822515. NISHIO, Y ., KAKIZOE, T., OHTANI, M., SATO, S., SUGIMURA, T., AND FUKUSHIMA, S. ( 1986). L-isoleucine and L-leucine: Tumor promoters of bladder cancer in rats. Science 231,843. OSER, B. L. (1985). Highlights in the history of saccharin toxicology. Food. Chem. Toxicol. 23,535-542. PAYNTER, 0. E., BURIN, G. J., JAEGER, R. B., AND GRECORIO, C. A. (1988). Goitrogens and thyroid follucular cell neoplasia: Evidence for a threshold process. Regul. Toxicol. Pharmaco/. 8, 102- 119. PERERA, F. P. (1984). The genotoxic/epigenetic distinction: Relevance to cancer policy. Environ. Res. 34, 175-191. PITOT, H. C., GOLDSWORTHY, T., CAMPBELL, H. A., AND POLAND, A. (1980). Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-pdioxin of hepatocarcinogenesis from diethylnitrosamine. CancerRes. 40,3616-3620. PITOT, H. C., AND SIRICA, A. E. (1980). The stages of initiation and promotion in hepatic carcinogenesis. Biochem. Biophys. Acta. 605,19 1. POLAND, A., PALEN, D., AND CLOVER, E. (1982). Tumor promotion by TCDD in skin of HRS/J hairless mice. Nature (London) 300,27 l-273. POLAND, A., AND K~MBROUGH, R. D., (Eds.) (1984). Biologica/ Mechanisms of Dioxin Action, Banbury Report 18, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. REDDY, J. K., AND LALWANI, N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12, l-58. REYNOLDS, S. H., STOWERS,J. S., PA~RSON, R. M., MARONPOT, R. R., AARONSON, S. A., AND ANDERSON, M. (1987). Activated oncogenes in B6C3Fl mouse liver tumors: Implications for risk assessment. Science 237,1309- 13 16. SCHOENING, G. P., GOLDENTHAL, E. I., CEIL, R. G., FRITH, C. H., RICHTER, W. R., AND CARLBORG, F. W. (1985). Evaluation of the dose response and in utero exposure to saccharin in the rat. Food Chem. Toxicol. 23,475-490. SCHROTER, C., PARZEFALL, W., SCHROTER, H., AND SCHULTE-HERMANN R. (1987). Dose-response studies on the effects of QI-,/3-, and -r-hexachlorocyclohexane on putative preneoplastic foci, monooxygenases, and growth in rat liver. Cancer Res. 47,80-88. SCHIJLTE-HERMANN, R., OCHS, H., BURSCH, W., AND PARZEFALL, W. (1988). Quantitative structureactivity studies on effectsof sixteen different steroids on growth and monooxygenases of rat liver. Cancer Res. 48,2462-2468. SCHULTE-HERMANN, R., SCHUPPLER, J., TIMMERMAN-TROSIENER, I., OHDE, G., BUR~CH, W., AND BERGER, H. (1983). The role of growth of normal and preneoplastic cell populations for tumor promotion in rat liver. Environ. Health Perspect. 50,185- 194.
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3-15. SHORT,
B. G., BURNETT, V. L., AND SWENBERG, J. A. (1986). Histopathology and cell proliferation induced by 2,2,4-trimethylpentane in the male rat kidney. Toxicol. Pathol. 14,194-203. SLAGA, T. J., (series Ed.) (1983-1984). Mechanisms ofTumor Promotion, Vols. 1-4, CRC Press, Inc., Boca Raton, FL. STARR, T. B., AND BUCK, R. D. (1984). The importance of delivered dose in estimating low-dose cancer risk from inhalation exposure to formaldehyde. Fundam. Appl. Toxicol. 4,740-753. STEWART, T. A., PA~ENGALE, P. K., AND LEDER, P. (1984). Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes. Cell 38,627-637. STOW, W. T., REITZ, R. H., SCHUMANN, A. M., AND WATANABE, P. G. (I 98 1). Genetic and nongenetic events in neoplasia. Food Cosmet. Toxicol. 19,567-576. SWENBERG, J. A. AND SHORT, B. G. (1987). The influence of cytotoxicity on the induction of tumors. In Nongenotoxic Mechanisms in Carcinogenesis (B. E. Butterworth and T. J. Slaga, Bds.), Banbury Report 25, pp. 15 I- 164. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. TENNANT, R. W., MARGOLIN, B. H., SHELBY, M. D., ZEIGER, E., HASMAN, J. K., SPALDING, J., CASPARY, W., RESNICK, M., STASIEWICZ,S., ANDERSON, B., AND MINOR, R. (1987). Prediction ofchemical carcinogenicity in rodents from in vitro genetic toxicity assays.Science 236,933-941. TRUMP, B. F., JONEST. W., AND LIPSKY, M. M. (1985). Light hydrocarbon nephropathy. In Renal Hetere geneity and Target Cell Toxicity (P. H. Bach and E. A. Lock, Eds.), pp. 493-504. John Wiley and Sons, New York. TSUTXJI, T., SUZUKI, N., MAIZUMI, H., MCLACHLAN, J. A., AND BARRETT, C. J. (1986). Alteration in diethylstilbestrol-induced mutagenicity and cell transformation by exogenous metabolic activation. Carcinogenesis7, 1415-1418. WACHSMUTH, E. D. ( 1987). Chemically induced cell turnover in the kidney and its possible role in carcinogenesis. In Nongenotoxic Mechanisms in Carcinogenesis (B. E. Butterworth and T. J. Slaga, Eds.), Banbury Report 25, pp. 137- 150. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. WA-ITENBERG, L. W. (1986). Protective effects of 2(3)-tert-butyl+hydroxyanisole on chemical carcinogenesis. Food Chem. Toxicol. 24,1099-l 102. WEISBURGER, E. K. ( 1977). Carcinogenicity studies on halogenated hydrocarbons. Environ. Health Perspect. 21,7-16. WEISBURGER, J. H., AND WILLIAMS, G. M. (198 1). Carcinogen testing: Current problems and new ap proaches. Science 214,40 l-407. WILLIAMS, G. M. ( 1986). Epigenetic promoting effects of butylated hydroxyanisole. Food Chem. Toxicol. 24,1163-1166. WILLIAMS, G. M., AND WEISBURGER, J. H. (1983). Carcinogen risk assessment. Science 221, p. 6. WILLIS, J. ( 196 1). The induction of malignant neoplasms in the thyroid gland of the rat. J. Puthol. Bacteriol. 82,23-27. Woo, Y., LAI, D. Y., ARCOS, J. C., AND ARGUS, M. F. (1985). Aliphatic and polyhalogenated carcinogens. In Chemical Induction of Cancer: Structural Bases and Biological Mechanisms, (J. Amos, Ed.), Vol. 3B, pp. 357-394. Academic Press, New York, New York. ZACK, J. A., AND SUSKIND, R. R. ( 1980). The mortality experience ofworkers exposed to tetrachlorodibenzodioxin in a trichlorophenol process accident. J. Occup. Med. 22,l I-14. ZURER, P. S. (1985). Asbestos. pp. 28-41 Chem. Eng. News.
TOXICOLOGY
AND
Nongenotoxic
PHARMACOLOGY
Carcinogens
9,244-256
( 1989)
in the Regulatory
Environment’
BYRON E. BUTTERWORTH Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received February I, I989
The biological activity of many carcinogens is to directly induce mutational events, thereby altering the information encoded in the DNA. Short-term tests for potential carcinogens and risk assessment models generally rely on the assumption that the agent in question will operate through a genotoxic mechanism. However, carcinogenesis is a multistep process, and it is increasingly clear that the primary biological effect for many carcinogenic chemicals involves events other than direct DNA reactivity. For many experimental rodent models as well as human cancers, nongenotoxic mechanisms appear to be the driving force in the formation of tumors. Many of these nongenotoxic mechanisms are highly species-specific. Thus, it is increasingly important to ask if the rodent model applies to the human situation at all, in addition to the examination of appropriate, hypothetical, mathematical risk assessment models. More research is now being focused to better define the mechanisms by which the many distinctly different classes of nongenotoxic carcinogens are acting. This understanding will become the basis for new predictive assaysand more realistic risk assessment models. If specific conditions are met, then a no observed effect level with a safety factor may be the most appropriate risk model for some carcinogens. 0 1989 Academic Rw., Inc.
GENOTOXIC
VS NONGENOTOXIC
CARCINOGENS
A genotoxic agent is one capable of altering the information encoded in the DNA either directly or through the formation of a reactive metabolite. These alterations can be point mutations, insertions, deletions, or changes in chromosome structure or number. This activity usually can be detected by assays that measure reactivity with the DNA, induction of mutations, induction of DNA repair, or cytogenetic effects in bacterial or mammalian cell culture assays as well as in the whole animal (McCann et al., 1975; Hsie et al., 1979; Bridges et al., 1982). Indeed, the basis of short-term tests for potential carcinogens, such as the Ames test, is the observation that genotoxic agents represent a major class of chemical carcinogens. Examples of genotoxic carcin’ Presented at the 4th Annual Meeting of the International macology, October 17- 18,1988, Baltimore, MD. 244 0273-23OQ/89 $3.00 CDpyriBht 1331989 by Academic Pres. Inc. Au rights of reproductionin any fmm reserved.
Society of Regulatory Toxicology and Phar-
NONGENOTOXIC
CARCINOGENS
245
ogens are 2-acetylaminofluorene (ZAAF), dimethylnitrosamine (DMN), and benzo[a]pyrene (Miller and Miller, 198 1). Nongenotoxic chemicals are those that lack genotoxicity as a primary biological activity in currently used assays. While these agents may yield genotoxic events as a secondary result of other induced toxicity, such as forced cellular growth, their primary action does not involve reactivity with the DNA, particularly when observed in the target organ at doses and by the route of administration that produced tumors in the animal study. Examples of nongenotoxic carcinogens are saccharin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Ashby, 1985; Poland and Rimbrough, 1984). CLASSES
OF NONGENOTOXIC
CARCINOGENS
A dual classification scheme of genetic and epigenetic (nongenotoxic) carcinogens has been proposed and many of the different subclasses of agents defined (Weisburger and Williams, 198 1; Williams and Weisburger, 1983). These classifications need to be continually expanded to include all of the many distinctly different groups of nongenotoxic carcinogens as they are identified. Often the scientific distinction between genotoxic and nongenotoxic agents is unclear because there can be a gradation of different activities between compounds. In some cases a chemical may be placed in more than one category. Nevertheless, it is misleading to refer to the nongenotoxic carcinogens as though they were a single class. For example, considerations of risk assessment models, mechanisms of action, and predictive assays for the potent hormone-like promoter TCDD are not the same as for saccharin that appears to be exhibiting its effects by inducing bladder hyperplasia. Herewith are some of the major classes of chemical carcinogens that have been identified.
Genotoxicants For these chemicals the weight of evidence indicates a consistent and reproducible pattern of activity in bacterial, cell culture, and/or whole animal assays for genotoxicity (McCann et al., 1975; Hsie et al., 1979; Bridges et al., 1982). It is valuable to confirm evidence of genotoxic activity in the target tissue of the treated animal. DNA adducts, induced DNA repair, or cytogenetic effects can be measured in animals treated with the test chemical in the manner that produced tumors.
Promoters This is a very large and diverse group of chemicals that promote development of tumors from initiated cells (Slaga, 1983-1984). In general, the action of tumor promoters is reversible and continuous application over extended periods are required to exhibit carcinogenic activity. A high degree of species and target organ-specificity are often seen with these agents. The classical definition of promoters is agents that are not themselves carcinogenic, but promote the formation of tumors from initiated cells. Since there are many mutagens in the natural environment, practically, the promoters must be considered carcinogenic agents.
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As mechanisms become better understood, many promoters may be placed in more defined categories according to their mechanism of action. For example, 12U-tetradecanoylphorbol13-acetate (TPA) is a potent promoter in the mouse skin painting model. Elegant research has now identified protein kinase C as the receptor for the phorbol ester tumor promoters (Blumberg, 1988). As an illustration of how difficult it can be to place agents into a specific category, some favor the hypothesis that TPA is acting through the generation of DNA-damaging oxygen radicals (Cerutti, 1987). In contrast, other skin tumor promoters, such as the anthrones, are acting by mechanisms of action that would place them in a different category than TPA (DiGiovanni, 1987). Assays have also been defined to assess promotion and progression in the liver (Goldsworthy et al., 1986). An example of a potent liver tumor promoter that appears to be acting through a specific receptor is TCDD (Poland and Kimbrough, 1984).
Mitogenic Liver Carcinogens For chemicals in this group, no chemically induced cytotoxicity is observed, as measured by the lack of liver-specific enzymes in the serum. Hyperplasia is characterized by mitogenic stimulation of cell replication and increased organ size. Examples are phenobarbital, a-hexachlorocyclohexane, polybrominated biphenyls, butylhydroxytoluene, ethinylestradiol, and cyproterone acetate (Schulte-Hermann et al., 1982; Schulte-Hermann et al., 1983; Schroter et al. 1987; Schulte-Hermann et al., 1988).
Peroxisomal Proliferating/Mitogenic Liver Carcinogens The peroxisomal proliferating carcinogens are a diverse group of chemical agents that induce an initial growth in liver size, hepatic peroxisomal proliferation, lipofustin accumulation, and continual increased cell turnover for the more active carcinogens (Reddy and Lalwani, 1983; Butterworth, 1987; Marsman et al., 1988; Conway et al., 1989). Examples range from very weak agents such as di(2-ethylhexyl)phthalate (DEHP) to potent carcinogens such as Wy-14,643, methyl clofenapate, and ciprofibrate (Conway et al., 1989). There exists a high degree of species specificity in the induction of liver peroxisomal enzymes, with rodent hepatocytes responding to a much greater extent than human hepatocytes (Elcombe and Mitchell, 1986; Butterworth et al., 1989).
Cytotoxic Liver Carcinogens These chemicals exhibit cytotoxicity and produce necrosis. Liver-specific enzymes are seen in the serum as an indication of cellular injury. Cell division is observed as regenerative cell growth to replace damaged tissue. An example of such a carcinogen would be carbon tetrachloride (Doolittle et al., 1987; IARC, 1979a; Loury et al., 1987a).
NONGENOTOXIC
CARCINOGENS
247
Cytotoxic Kidney Carcinogens A variety of nongenotoxic nephrotoxicants yield kidney tumors, including unleaded gasoline, nitrilotriacetate, chloroform, perchloroethylene, and pentachloroethane (Anderson, 1987; Kluwe et al., 1984; Loury et al., 1987a; MacFarland et al., 1984; Trump et al., 1985; Weisburger, 1977). For many of these kidney carcinogens, the pattern of activity is the induction of tumors in the kidneys of male rats but not female rats or mice of either sex. The primary mechanism of action for these agents would appear to be chemically-induced accumulation of the male rat-specific protein a&obulin in the form of protein droplets in the kidney tubule cells, resulting in cell death and regenerative hyperplasia (Short et al., 1986; Loury et al., 1987b; Swenberg and Short, 1987; Wachsmuth, 1987; Goldsworthy et al., 1988). It is this continual cell proliferation that would appear to be the driving force in the induction of tumors.
Cytotoxic Bladder Carcinogens/Promoters Three classes of nongenotoxic bladder promoters have been identified (Cohen et al., 1987). The first includes sodium salts of weak acids such as saccharin and ascorbate (Cohen, 1985; Imaida et al., 1984). The second class of promoters includes uracil and terephthalic acid and yield urinary calculi (Heck, 1987). These calculi produce a foreign body reaction and result in hyperplasia. The third class includes the antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisol and the amino acids isoleucine and leucine (Imaida et al., 1984; Nishio et al., 1986). For these substances, massive doses of over 1% in the diet must be administered for long periods of time to produce papillomas or cancer. The primary action of these compounds appears to be enhancement of bladder hyperplasia at the site that eventually yields the tumors.
Cytotoxic Stomach Carcinogens Many compounds have been shown to induce forestomach tumors in rodent models (Kroes and Wester, 1986). While this is a common site for genotoxic carcinogens, some of these agents appear to be acting through nongenotoxic mechanisms. For example, the antioxidant butylated hydroxyanisole (BHA) induces rat forestomach tumors at very high doses (Ito et al., 1983). However, BHA is not genotoxic and the tumors appear to be related to the induction of hyperplasia (Williams, 1986; Clayson et al., 1986). In fact, BHA has actually been shown to inhibit the action of a broad spectrum of carcinogens, mutagens and tumor promoters (Wattenberg, 1986). Similarly, ethyl acrylate causes malignant neoplasms in the forestomach of Fischer 344 rats when administered chronically by gavage (National Toxicology Program, 1983). In the course of healing the forestomach lesions produced by ethyl acrylate, hair and food particles become entrapped. Evidence is convincing that this tumor formation by ethyl acrylate is secondary to foreign body reactions and induced epithelial cell proliferation (Ghanayem et al., 1986a,b).
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Hormones As regulators of cell proliferation and function, many hormones have been implicated in the development and progression of cancer (IARC, 1979b). For example, diethylstilbestrol (DES) is carcinogenic in a variety of rodent models and is associated with an increased incidence of endometrial carcinoma (IARC, 1979~). DES has been causally associated with an increase in vaginal and cervical clear-cell adenocarcinomas in daughters of women who were administered the hormone during pregnancy (IARC, 1979~). While there are conflicting reports as to whether DES is mutagenic, evidence from cell transformation assays and studies of lesions in patients exposed in utero suggest that induction of aneuploidy may be mechanistically related to tumor formation, which would be viewed as a genotoxic event (Barrett et al., 1981; Fu et al., 1979; Tsutsui et al., 1986; Degen and Metzler, 1987). However, because of its profound effects on gene expression and cellular function, we must remain open to the possibility that DES may play several mechanistic roles in the process of carcinogenesis.
Thyroid Carcinogens Thyroid gland follicular tumors can be produced by a variety of conditions, natural products or chemical agents, that result in hormone imbalance with resulting hyperplasia of the thyroid (Paynter et al., 1988). For example, low iodine diets or subtotal thyroidectomy decrease circulating levels of thyroid hormones in the blood. To compensate, the anterior pituitary gland releases increased amounts of thyroid stimulating hormone, which induces thyroid hypertrophy and hyperplasia. Because there is no resulting increase in thyroid hormone levels, this hyperplasia is sustained by the prolonged stimulation of the thyroid and pituitary glands and may progress to neoplasia. Restoration of the normal hormone balance before tumor formation results in a return to the normal state. This is clearly an example of a nongenotoxic mechanism because no chemicals were administered to the animal at all. Several groups of chemical agents have been identified that yield thyroid tumors by mechanisms that appear to be analogous to that just described (Paynter et al., 1988). Thiocarbonyl compounds such as thiouracil, propylthiouracil, and methimazole are potent thyroid function inhibitors and are used in the treatment of hyperthyroidism even though they have been shown to produce thyroid tumors in rodent models (Willis, 196 1; Woo et al., 1985).
Solid-State Carcinogens This group includes mineral fibers such as asbestos and is of particular concern because of the indications that people can be susceptible to cancer resulting from exposure to specific types of asbestos (Lee, 1985; Zurer, 1985). Many of the properties of asbestos, such as fiber size and shape, the ability to produce aneuploidy, and induce local cell proliferation are being correlated with its carcinogenic activity (Bhatt et al., 1984; Hesterberg and Barrett, 1984; Moalli et al., 1987). However, much more information needs to be gathered before we will know exactly which properties to
NONGENOTOXIC
249
CARCINOGENS
avoid in selecting a safe substitute for asbestos and the many other types of manmade mineral fibers that are now in widespread use. CHEMICALLY
INDUCED
CELL
PROLIFERATION
The most common biological activity for many of these nongenotoxic carcinogens is that of chemically induced cell division. There are numerous roles that cell proliferation might play in the process of carcinogenesis. Enhanced replication may increase the frequency of spontaneous mutations (Stott et al., 1981). Increased rates of cell turnover increases the probability of converting DNA adducts from natural sources into mutations before they can be repaired. Tumor promotion is associated with a sustained induction of cell replication (Slaga, 1983-l 984). The multistep process of carcinogenesis involves the clonal expansion of initiated or altered cells (Farber, 1980; Pitot and Sirica, 1980). Finally, the mutation or altered expression of oncogenes and/or growth control proteins are strongly implicated in cancer (Land et al., 1983; Stewart et al., 1984; Adams et al., 1985; Reynolds et al., 1987). The chance for an initiating event is enhanced during replication because many of these genes are being transcribed, during which time that segment of the DNA is exposed and may experience greater susceptibility to environmental carcinogen induced DNAdamage. In a National Toxicology Program/National Cancer Institute study to compare mutagenic activity with carcinogenic activity for 73 chemicals, there was not a single chemical that required over 500 mg/kg/day to produce tumors that was mutagenic in the Ames test (Tennant et al., 1987). This raises the concern that the tumors produced by such chemicals may arise by a secondary mechanism, such as organ-specific toxicity and resultant cell turnover, that would not occur at lower doses of the agent. If the extent and duration of cell turnover can be correlated with carcinogenic potential, then this endpoint might be used as a predictive assay (Loury et al., 1987a). Unfortunately, cell proliferation studies are labor intensive and the data base needed to establish this correlation does not exist at this time. A typical example of a chemical that appears to be acting by this mechanism is saccharin. This artificial sweetener yielded bladder tumors when administered at a dose of 5% saccharin in the diet, about 3,000 mg/kg body weight/day (mg/kg/day), in a two generation study (Arnold et al., 1980). Whereas sodium saccharin in the diet at 5% produces bladder hyperplasia, acid saccharin, interestingly, does not. One might also predict on this basis that acid saccharin would not be a bladder carcinogen (Cohen et al., 1987; Schoening et al., 1985). MAXIMUM
TOLERATED
DOSE
A developmental drug or new product that is nontoxic presents a problem for industry. Such massive doses are required in order to reach the mandatory maximum tolerated dose that some adverse effect often appears in the animals in the course of what many consider an unrealistic study. One concern when conducting a cancer bioassay is to avoid or at least be aware of situations where high doses of a chemical compromise the health or lifespan of the animal, overwhelm natural detoxification mechanisms, or yield tumors secondary to excessive organ-specific toxicity. On the
250
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E. BUTTERWORTH
other hand, with so few animals to test, it is important to choose doses as high as possible to maximize the ability to detect carcinogenic effects of the chemical. The most common biological activity of many classes of nongenotoxic carcinogens would appear to be the induction of hyperplasia (Loury et al., 1987a). Gathering chemically induced cell proliferation data as part of the ninety day study that precedes a cancer bioassay could provide valuable information to aid in setting rational doses for longterm studies. RISK
ASSESSMENT
Currently, mathematical extrapolation models are predominantly used to estimate theoretical tumor incidence rates at low doses by extrapolating from high doses where tumors were actually observed. As an illustration of how variant these models can be, the maximum likelihood estimates of cancer risk for the probit and multistage models for exposure to 0.1 ppm formaldehyde differ by nineteen orders of magnitude (Starr and Buck, 1984). Yet, in the face of all the uncertainty associated with cancer studies, risk estimates for regulatory purposes usually rely only on the upper 95% confidence bound of the multistage model and sometimes have been given to three significant figures (National Research Council, 1983). Further, there has been considerable resistance to deviate at all from this regulatory posture (Perera, 1984). There is substantial evidence that the genotoxic rodent carcinogens present a carcinogenic risk to human beings. Genotoxic agents tend to produce tumors in multiple tissues and species (Ashby and Tennant, 1988). Comparative studies show that those agents that induce DNA repair in rat hepatocytes also induce DNA repair in human hepatocytes (Butterworth et al., 1989). Analysis of the known human carcinogens reveals that the majority are overtly genotoxic (Shelby, 1988). In contrast, concerns with the inappropriateness of the rodent model for carcinogenesis loom particularly large with nongenotoxic carcinogens because of the high degree of species specificity often associated with these agents. Key mechanisms of action allowing meaningful risk assessment often remain unknown and there are serious questions as to whether some rodent responses can be extrapolated to people at all. It would also be prudent to address the issue as to whether the rodent model is applicable to the human situation, in addition to employing a hypothetical mathematical risk assessment model. For example, TCDD is 10,000 times more potent than TPA as a promoter in the C3H 10T l/2 initiation/promotion assay, 100 times more potent than TPA as a skin tumor promoter in the hairless mouse, and is one of the most potent rat liver tumor promoters ever described (Pitot et al., 1980; Poland et al., 1982; Abernethy et al., 1985). This activity stands in stark contrast to the human situation. In 1949 an industrial accident at a Monsanto Company facility in Nitro, West Virginia, resulted in the exposure of 121 workers to TCDD in large enough amounts to produce chloracne and many related health problems, such as liver enlargement. These symptoms persisted for longer than 4 years, consistent with the observation that TCDD is retained in the body for long periods. A follow-up study 30 years later that was 100% complete revealed no apparent excess in total mortality or in deaths from malignant neoplasms (Zack and Suskind, 1980). The most important question with TCDD is: Why is this extremely potent rodent carcinogen not an obvious human carcinogen? It is hoped that mechanistic studies with TCDD will
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provide the answers to this question (Poland and Kimbrough, 1984; Greenlee et al., 1987). The unsettling companion question is: Are there potent human carcinogens that are inactive in rodents? Similar concerns are raised with the male rat-specific cytotoxic kidney carcinogens that appear to be acting through a*,-globulin mediated toxicity and subsequent cell proliferation. Since a&obulin is a male rat-specific protein, one must question whether the response produced by these nongenotoxic agents can be extrapolated to people (Short et al., 1986; Loury et al., 1987b; Charbonneau et al., 1987; Lock et al., 1987). The first priority with the many nongenotoxic chemicals that exhibit this pattern of activity is to conduct comparative mechanistic studies contrasting the male rat and the human, rather than apply a rigid mathematical model to extrapolate from rodent to man. There have been 18 long-term cancer studies with saccharin (Oser, 1985). Some of which have involved periods of saccharin intake of over 12,000 mg/kg/day (Oser, 1985). The latest study involved 2,500 rats and examined seven doses between 1 and 7.5% saccharin (Schoening et al., 1985). Rather than do more cancer bioassays, perhaps it is time to increase our research efforts to understand why saccharin yields tumors and if those events are likely to occur in people. Nongenotoxic thyroid carcinogens that act by prolonged stimulation of the pituitary/thyroid axis would not be expected to exert any carcinogenic effects at doses that do not induce thyroid hyperplasia (Hill et al., 1988). Available evidence confirms that humans are less sensitive to the carcinogenic effects of long-term thyroid stimulation than animals (Hill et al., 1988). Different risk models may be appropriate for different classes of carcinogens. The most commonly used cancer risk models rely on assumptions of direct mutagenicity and assume that no matter how low the dose, there remains the possibility that the critical target gene may be hit. The formulas used allow the calculation of a theoretical risk at any dose. These models suffer because they consider only tumor incidence and do not account for chemically-induced cell proliferation as it applies either to the expansion of initiated cells or the direct role that cell turnover may have in the carcinogenic process. If one accepts the concept from experimental evidence that tumor formation by some nongenotoxic carcinogens is secondary to organ-specific toxicity and subsequent cell proliferation, then fundamental assumptions for risk models will be different. Many times the action of a toxicant is on a specific cellular protein. There may be thousands of copies of this protein in the cell. If the toxicant in question removes only one copy of the protein, such that its level in the cell stays within normal limits, then there will be neither cell death nor resulting regenerative cell division. There would be a threshold to the carcinogenic effects of such an agent. If one had assurances that induced tumors were secondary to organ-specific toxicity, then a no observed effect level (NOEL) with a safety factor would be a rational risk assessment model. Using more than one model helps remind us that we are dealing with an inexact science. In either case, one is extrapolating to low doses where the induction of tumors cannot practically be measured. If one assumes that chemically-induced cell proliferation is the driving force for tumor formation, then the NOEL approach for risk assessment could be employed. As a prerequisite to this approach, the experimental carcinogen would have to exhibit all of the following properties to assure that no direct genotoxic mechanism was involved:
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(1) A weight of evidence analysis indicates that the test chemical is not genotoxic in a battery of short-term tests. [These assays are sensitive and reliable indicators of genotoxic activity (Hsie et al., 1979; Bridges et al., 1982)]. (2) Over 500 mg/kg/day was required to produce tumors. [None of the compounds in the NTP/NCI study that required over 500 mg/kg/day to produce tumors was mutagenic in the Ames test (Tennant et al., 1987). While there could be flexibility of this cutoff, one should be cautious with any chemical that produces tumors at very low doses, because of the possibility that it may be acting through an unknown mechanism]. (3) Tumors appeared only near the end of the study. [This precludes potent promoters that may be working through specific receptors such as TCDD (Poland and Kimbrough, 1984)]. (4) Hyperplasia appeared in the target organ prior to tumor development. [The assumption is that it is this chemically-induced cell turnover that is the driving force for tumor formation)]. (5) Tumors appeared only in that single target organ. [Genotoxic agents tend to produce tumors in multiple tissues and species (Ashby and Tennant, 1988)]. The basis for selecting the safety factor would include considerations of mechanism of action, the dose at which hyperplasia was evident, species differences in susceptibility, the shape of the dose-response curve, and human experience with the chemical. The advantages to this approach are that the model conforms to the perception of the mechanism of action of the chemical and permits the use of additional relevant information. An example of a chemical for which this risk approach might be used is saccharin. A credible public cancer policy must not only identify potential human carcinogens, but also must distinguish between chemicals and usage patterns that represent a clear and present danger and those that present risks that are trivial in nature. Tobacco smoking is the cause of 30 to 40% of deaths from cancer (Loeb et al., 1984; National Research Council, 1986). Age-adjusted lung cancer mortality rates for American women increased 337% between 1950 and 1980 and will soon exceed breast cancer as the leading cause of cancer death in women (Loeb et al., 1984). It is doubtful that anyone has ever contracted cancer related to saccharin consumption (Morgan and Wong, 1985). Yet, the word “cancer” appears as a warning on both a pack of cigarettes and a packet of saccharin, with no guidance to the average person as to their relative risks. The rodent model for carcinogenesis is central to public cancer policy. In the exercise of assessing risk, scientific honesty requires a discussion of the strengths and weaknesses of the bioassay and a comparison of the theoretical risk calculated from several mathematical models. The uncertainty in the risk estimates should be acknowledged and the data presented to only one significant figure. Merits of different models, safety factors, mechanistic information and risk vs benefit considerations should be openly debated. Then the difficult political decision of setting regulatory limits to exposure could be made without relying on the illusion that science had produced an infallible number. REFERENCES ABERNETHY,
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