Animal extrapolation A look inside the toxicologist’s black box
C.
Edward J. Calabrese
Universiry of Massachusetts Amherst. Mars. 01003 The process of estimating human cancer risks posed by exposure to chemical carcinogens has been a focus of controversy among toxicologists for at least the past decade. One issue that received much national attention was the Fond and DNg Administration (FDA) proposal to ban saccharin fromsoft drinks because it causes cancer in the bladders of male rats. The general public had 618 Environ. Sci. Technol.. MI. 21, No. 7, 1987
difficulty accepting the regulatory premise that it was possible to predict risk to humans-who at most are expected to drink several 12- cans of diet soft drinks a day--from studies on rats that received the equivalent of 800loo0 cans a day. Although public pressure led FDA to withdraw the proposed ban on saccharin, it did not end the debate over the role of animal data in reflatory matten. S p x i f i d y , the question remains of how the toxicological and regulatory communities should go about predicting risk posed by low or ambient levels of carcinogens on the basis of animal studies that involve far greater expo-
sum. Moreover, even though the public may have been skeptical about the abiity of FDA’s researchers to e x t r a p late -from the equivalent of 800-loo0 cans of soda per day down to 1-2 per day, the situation often is more extreme. In the case.of carcinogenic solvents that are not acutely toxic, such as tricldomthylene (TCE) and perchloroethylene WE), the doses used in the cancer bioassays exceed those consumed by humans by a factor of more than a million. Thus, in the case of these solvents, agencies such as EF’A are extrapolating over six orders of magnitude of dose as compared with “only” three orders of magnitude of
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dose for saccharin. It is seldom possible to estimate lowdose cancer risk on the basis of epidemiological studies. To predict human responses, regulatory agencies usually must use the results of lifetime animalmodel studies performed on rats and mice at maximum tolerated doses and fractions thereof. To estimate human low-dose cancer risk, regulatory agencies must make at least three major extrapolations: animal to human, normal-risk to high-risk segment of the population, and high dose to low dose. Attempts at estimating human risk from exposure to environmental carcinogens are fraught with uncertainties. Often the demands made by society fir exceed the capacity of modern toxicology to provide accurate estimates of cancer risk. ’TWenty or more years ago the issue concerning carcinogens was considered more qualitative in nature, that is, “Is the chemical agent carcinogenic or not?” Today, society not only demands to know whether the agent is carcinogenic; it wants information about the likelihood of developing cancer at each level of exposure. Although it is possible by means of quantitative risk assessments to answer such questions, the issue facing us now concerns the accuracy of the answers provided. Members of the regulatory community have tended to view the predictive models of the toxicologist in much the m e way as the engineers’ black boxwithout understanding what is going on inside it. Regulators accept what the model tells them as directly applicable to human populations. This article offers a look inside the toxicologist’s black box of animal modeling to begin to estimate the extent to which past regulatory actions have been consistent with biological reality. The focus is on three areas of critical importance to the field of predictive toxicology and risk assessment that are used to determine the adequacy of predictive models: The predictive relevance of mouse hepate mas (liver tumors), the role of p h y s b logically based pharmacokinetic (F‘BPK) models in risk assessment, and the capacity of current animal models to deal with the occurrence of human heterogeneity in their predictions of human responses.
Mouse liver tumors Among the major points of controversy in the field of predictive toxicology is the occurrence of chemically induced bepatonm in mice and their extrapolative relevance to humans. livo rodent models normally are used in the standard cancer bioassay performed by the Department of Health and Human
Services’ National Toxicology Program (NTP): the B6C3Fl mouse strain and the Fischer 344 rat strain. Results of several hundred cancer bioassays show that the mouse strain responds much more readily than does the rat strain to the development of chemically induced hepatomas. For example, in a review of 85 chronic exposure assays, the mice developed hepatomas in 45 studies, whereas rats developed tumors in 15 studies (1). The discrepancy between the two animal models has created considerable regulatory concern. The issue emerges as to which model best predicts the risk of human liver cancer. Because of interspecies differences in response to exposure and because of the possibility that the mouse may be uniquely sensitive to developing hepatomas,the Nutrition Foundation organized an ad hoc review panel to assess the predictive relevance of the mouse hepatoma ( I ) . One important conclusion of the panel is that the mouse, in contrast with the rat, develops tumors in the NTF’ bioassay from agents that have been found to be nongenotoxic in mutagenicity assays. Although this area remains under intense investigation, the panel suggested that the mouse strain may possess preinitiated cells; that is, that “nongenotoxic” carcinogens actually may act as cancer promoters rather than as cancer initiators. The panel’s conclusion is that this may explain the negative findings in rats. More recent advances in toxicology appear to be shedding some light on this rather striking difference in suscep tibility to liver cancer. For example, several nonmutagenic chemicals that are liver carcinogens in mice have been found to cause increases in the number of hepatic peroxysomes. It has been proposed that a causal relationship exists beween peroxysome proliferation and the development of hepatocellular carcinoma via the involvement of reactive oxygen species such as hydrogen peroxide (2). Increased peroxysomal @-oxidation of fatty acids leads to the formation of increased steady-state concentrations of hydrogen peroxide, which may damage DNA. Of particular interest is that TCE can stimulate hepatic peroxysomal @oxidation (a hydrogen-peroxide-generating oxidase) in mice but not in rats (2). This increase in enzymatic activity was found to be associated with a similar increase in the number and the volume density of peroxysomes. The reason TCE is a peroxysome proliferator in mice but not in rats is that in mice, TCE displays linear kinetics for the formation of trichloroacetic acid (TCA, the principal TCE m e t a b lite) but saturation kinetics for formation of TCA in rats (seedefinitions). As
Toxicola : a gli Acetylation-conjugation mecha;m in the metabolism of xeno)tics. B6C3F1 mouse strain-strain of ice widely used in cancer studies. Deacetylation-mechanism for ...e removal of acetic acid from a chemical agent. Epigenetic mechanism-a echanism for carcinogenesis in iich there is no evidence of any airect interaction between the carcinogen and the genetic material of the cell. This mechanism involves solid-state carcinogens such a s polymers and metal foils, as well as mnones, immunosuppressants, Earcinogens, and promoters. Fisher 344 rat strain-strain of rats widely used in cancer studies. Genetic polymorphism-the occurrence of multiple forms of the same gene; an example is the A, B. id 0 blood groups. Hepatic peroxysome proliferation-increase of peroxysomes in the liver (see definition of peroxysomes below). Hepatoma-a carcin liver. Linear kinetics-the is proportional to the do Nongenotoxic carcinogen-see 3finition of epigenetic mechanism adove. PB-PK model-physiologically based pharmacokinetic model. Peroxysome-a cytoplasmic ormeile characterized by a single niting membrane and a finely grantar or homogeneous matrix. reroxysomes are associated with various functions such as gluconeonanesis, lipid metabolism, and detxification of hydrogen peroxide. Pharmacokinetics-the quantitare study of the metabolic procjses of absorption, distribution, biotransformation, and elimination. Phenotype-a genetic category group to which an individual may 3 assigned on the basis of one or lore characteristics, observable inically or by laboratory means, iat reflect genetic variations or 3ne-environment interaction. Saturation kinetics-a s u b =iance is not metabolized above a certain dose, and the parent comoound is eliminated unchanaed.
the dose of TCE is increased, a proportionally greater amount of TCA is produced in mice; similar studies with rats show that at exposures of more than 50 mg/kg, no additional TCA is produced and the parent compound is excreted unchanged. Of importance here is that TCA is believed to be the agent responsible for TCE-induced peroxysome Environ. Sci. Technol., Vol. 21, No. 7, 1987 619
proliferation. At TCE doses below 50 mglkg, no peroxysomal enzyme induction occurs. Consequently, the increased risk of mice (but not rats) for developing TCE-induced liver cancer is believed to be controlled by the epigenetic mechanism of peroxysome proliferation, which is related to the linear kinetics of TCA formation.
Physiological models Considerable regulatory attention has been devoted to PB-PK models and their potential use in risk assessment. They are essentially mechanistic models that try to account quantitatively over time for the various pharmacokinetic processes that involve an agent of concern from the time the agent reaches a site of absorption to the time an interaction occurs between the agent, its metabolites, and various body tissues. Once it is determined whether the parent compound or its metabolites are the cause of a carcinogenic response, a PB-PK model can be developed to quantify the magnitude and the duration of exposure to this agent at the critical target site in the animal model. Once the estimates of target tissue dose in the animal model have been made and validated, the information can then be scaled up to enable the estimation of target organ exposure in humans. Such data may then be of assistance in estimating human cancer risk based on the animal response. It should be emphasized that the PB-PK model does not offer an explanation of what the mechanism of cancer initiation is. It cannot predict susceptibility of a target organ or differentiate the susceptibility of one target organ from another or of one species from another (3, 4). Although it has limitations, PB-PK modeling offers an important tool for researchers and regulators alike. Even though these models do not show interspecies differential susceptibility for target organs, they can be used to quantify target organ doses between species. More important, as additional information is obtained about the pharmacokinetics of a chemical it can be incorporated into the model without affecting the basic structure, thus enhancing its predictive capability. The use of PB-PK models provides important advantages over conventional pharmacokinetic analyses (3).In typical pharmacokinetic modeling, time-course curves are determined that reflect the concentration of the administered agent or its metabolites in blood or some other body compartment over time. The resulting curves are then described by curve-fitting biostatistical techniques. Conventional pharmacokinetic models depend more on mathematical 620 Environ. Sci. Technol., Vol. 21, No. 7, 1987
modeling than on the biological systems they purport to represent. PB-PK models, however, are designed to predict kinetic behavior over a wide range of doses and conditions of exposure. This requires enormous amounts of data on matters of anatomy and physiology, the partitioning of test agents into selected tissues, and the biochemical constants for tissue binding and metabolism in various organs. From these data, a series of mass balance differential equations can be written to describe the interactions between the chemical and animal models. Physiologically based pharmacokinetic modeling has been applied to several agents, including methylene chloride and ethylene dichloride (3, 4). The case of methylene chloride illustrates the powerful implications of this approach. One PB-PK model is based on data that indicate two routes of metabolism (oxidation by mixed function oxidase [MFO] and dependence on glutathione s-transferase [GST]) in four species-mice, rats, hamsters, and humans. The model was designed to quantify the contributions of the two metabolic pathways in the lung and liver and to allow for extrapolation from rodents to humans. The kinetic constants were obtained from in vivo experiments and from the literature, and validation involved a comparison of predicted blood concentration timecourse data in rats, mice, and humans. The capacity of methylene chloride to cause tumors in mice was associated with the target tissue dose and was closely related to the amount of methylene chloride metabolized by the GST pathway but not by the MFO pathway. According to the PB-PK model, the target tissue doses in humans exposed to low concentrati'ons of methylene chloride were approximately 50-200 times lower than would have been predicted by linear extrapolation and body surface area factors used in conventional methods of risk assessment. The PBPK analysis therefore suggests that conventional risk analysis greatly overestimates risk to humans exposed to low levels of methylene chloride. The PBPK approach is an attractive development because it increases the biological plausibility of predictive approaches while still incorporating biomathematical approaches for low-dose risk prediction.
Extrapolation to humans Susceptibility to the toxic and carcinogenic effects of chemical, environmental, and medical agents is not equally distributed in the human population. One case in point involves the diphtheria-tetanus-pertussis vaccine. Although there is no reason to doubt the
enormous overall benefit of this vaccine, it must be recognized that one in 300,000 shots will result in the development of permanent neurological illness. Thus, in the United States, several dozen children may be severely affectedby this vaccine each year. The government considers this level of risk acceptable, given the alternative of not using the vaccine. To what extent is this risk a function of chance or a function of biological predisposition? A variety of factors, such as age, sex, diet, genetic makeup, and predisposition to disease, have been reported in the literature as affecting susceptibility to environmentally and medically induced adverse health effects, including cancer (5). That people differ in their responses to environmental agents is not a new idea. It was, in fact, pointed out in 1938 by the geneticist J.B.S. Haldane (6). Several national conferences have concerned themselves with better identification and quantification of those considered to be at higher risk of adverse effects than the general public. Although it is accepted that human susceptibility to toxic substances, including carcinogens, is highly variable, it is believed that the highly inbred rodents used in laboratory studies display a much more homogeneous response than humans to toxic agents, including carcinogens. The question, then, concerns whether specific animal models can be extrapolated to a narrow band of the broad spectrum of human response.
Variations in response Acetylation. Certain aromatic amines known to be animal and human carcinogens tend to be metabolized by acetylation. Typically, aromatic amines require bioactivation to the carcinogenic metabolites via N-hydroxylation, The situation is more complex, because subsequent acetylation of the N-hydroxylated metabolite appears to be a requirement for the occurrence of liver cancer in rodents, whereas the nonacetylated N-hydroxylated metabolite appears to be essential to initiate bladder cancer. Thus, susceptibility to the occurrence of an aromatic amine-induced bladder or liver cancer is contingent on the relative capacity to N-hydroxylate, acetylate, and deacetylate the specific aromatic amine or metabolite (3). Within the human population, there are both fast and slow acetylators in roughly equal proportion, for example, among whites and blacks in the United States. An assessment of the acetylation of about a dozen aromatic amines in humans revealed a 3.7-13.0-fold variation in response, depending on the substance in question (5, s). In a study that used 2-aminofluorene (2-AF) as a
model substrate for hepatic acetylation, it was shown that considerable variation in acetylation exists among commonly used laboratory animals. Compared with rats, which are arbitrarily given a relative acetylation value of 1, mice, guinea pigs, and hamsters are given acetylation values of 8, 12, and 18, respectively. In constrast, no Nacetylation of any of the three carcinogenic arylamines studied was detected in dogs. Among the four commonly used animal species-hamsters, guinea pigs, mice, and rats-the capacity of the hamster to acetylate 2-AF is about 10 times greater than that of a fast human acetylator and about 120 times greater than that of a slow human acetylator. Guinea pigs and mice are about 6 and 5 times more efficient acetylators of 2-AF, respectively, than is the fast human acetylator. In marked contrast, rats acetylate 2-AF with an efficiency that lies between that of fast and slow human acetylators. Rats display activity that is about 7 times greater than that of the slow human acetylator and only about 50% that of the fast acetylator. In contrast with rats, hamsters, and guinea pigs, rabbis display a genetic polymorphism (the occurrence of multiple forms of the same gene) with re-
spect to N-acetylation that is similar to that found in humans. The fast rabbit acetylator displays a rate 10-50 times greater than that of the human fast acetylator; the slow rabbit acetylator a p proximates both human fast and slow acetylators depending on substrate. Figure 1 shows how the acetylation capacity of these four animal models compares with the capacity of human fast or slow acetylators. Deacetylation. The capacity to deacetylate carcinogenic arylacetamides has been shown to be a significant risk factor in the occurrence of bladder cancer in dogs. Observations indicate that an N-hydroxy nonacetylated metabolite is the carcinogenic agent involved. As a result of dogs’ ability to N-hydroxylate aromatic amines and because of their poor ability to acetylate such compounds, dogs are at increased risk of bladder cancer. Similarly, when dogs are exposed to carcinogenic arylacetamides, they exhibit an enhanced risk of developing bladder cancer as a result of their capacity for N-hydroxylation and then deacetylation. It is important to recognize that although nonacetylated arylamines that have been N-hydroxylated are pctential bladder carcinogens, N-hydrox-
ylated acetylated arylamines have been found to be probable causes of liver cancer. The enzymes that effect N-acetylation of arylamines and deacetylation of arylacetamides, respectively, seem to determine susceptibility to liver and bladder cancer (5, 6). The dog’s relative capacity to acetylate and deacetylate arylamines is an important factor for arylamine-induced cancer. In rats and mice, the ratios of N-acetyltransferase activity to deacetylase activity are relatively higher than in dogs. This quality may provide rodents with some protection against bladder cancer that they do not have against liver cancer (6). Little is known about the range of human variation with respect to deacetylation. However, in vivo studies indicate that humans and rabbits, unlike dogs, are poor deacetylators of acetylated aromatic amines. Because humans have greater ability to acetylate and lesser ability to deacetylate aromatic amines than do dogs, it is likely that humans, especially fast acetylators, are at considerably lower risk than dogs of developing arylamine-induced bladder cancers (Figure 2). Liver epoxide hydrase activity. In humans, the procarcinogen TCE is
Human Slow
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Envimn. Sci. Technal., MI. 21, No. 7,1987 621
bioactivated to its epoxide, which has teen hypothesized to be the proximate carcinogenic form of the compound. Detoxification of the epoxide is believed to occur by means of the activity of epoxide hydrase (EH), an enzyme that converts the epoxide to the less toxic and presumably noncarcinogenic diol form. In a survey of 163 individuals, most of whom had liver diseases, a 63-fold range of variation in hepatic EH activity was found, with benzo[u]pyrene 4,5-oxide as substrate for EH. Between 80% and 90% of the human subjects showed activity within a IO-fold range. Liver cytosol EH activity, determined with rrans-stilbene oxide as substrate for EH, showed a 539fold variation among 135 subjects! The activities of 90% of the samples deviated by a factor of three from the median activity (6). Hepatic microsomal levels of EH in humans have been compared with those in several commonly used animal spec i s , including mice (NIH strain, C3H strain), rats (Sprague-Dawley), guinea pigs (Hartley), rabbits (New Zealand), and Rhesus monkeys. In general, mice display the lowest activity level, regardless of strain (Table 1) (6). If one arbitrarily assumes a distribution with a 250-fold range of EH activity variation in humans, the mouse would have a value at the low end of the human range. Consequently, the significantly lower activity of EH in mouse liver may be an important factor in helping to explain the high risk the mouse faces for developing TCE-induced liver cancer. Similarly, the higher enzymatic capacity of the rat for hepatic EH is consistent with its lower risk of TCEinduced liver cancer (7).
Human population variation The previous information has indicated that human susceptibility to biochemical factors affecting carcinogenesis, including the ability to bioactivate and deactivate compounds, varies widely in the population (@. The extent of this variation differs according to the I susceptibility Io arylamine-
specific predisposing condition and is likely to be related to the sample size and to the characteristics of the population from which samples are derived. In most studies, the human sample pop ulation has been relatively small, often consisting of fewer than several hundred individuals. At times, fewer than 20 persons have been studied. Furthermore, the selection of the sample has never been statistidy representative of a given larger group, because methods used to select the study group have been nonrandom in nature. In general, the samples from most studies of human population variation were obtained by investigators who were fortunate to obtain any samples at all. Consequently, even though the data indicate that human variation in the capacity to bioactivate certain mutagens or carcinogens exists-and the potential for this variation is considerable4 is not possible to make a general statement about the degree of heterogeneity in the human population or about the distribution of any such range. Even in the face of the general inability to describe the extent of human heterogeneous responses to carcinogenic agents, it is clear from the data that human variation in response is generally much greater than that observed in commonly used animal species. Yet there is almost never a reference in toxicological reports to which segment of the human population the animal model used in toxicological studies is thought to be related. In the absence of specific comments it is g e n e d y assumed that the model represents the human population mean. The purpose of this article is to provide information about the groups of humans to which animal models are l i l y to be extrapolated. With respect to acetylation, the most appropriate animal model may depend on the human phenotype. To estimate arylamine-induced cancer risk accurately, information a b u t human phenotype must be viewed in concert with data concerning variations :- -Wty to N-hydroxylate ed bladder cancer
I
622 Environ. Sci. Technol., Vh. 21, NO.7. 1987
I
elative values of liver
ouse (C3Hstrain)
and deacetylate arylamines. Such assessments offer a population-based a p proach for evaluating the human significance of bioassay data. The heterogeneity of the human pop ulation with respect to the risk of developing chemically induced cancer has a parallel in the rather large interspecies differences in animals. For example, one animal may reasonably simulate the response of one segment of the p o p ulation (such as fast acetylators), and another may best simulate another segment of the population (such as slow acetylators). Another approach along these same lines may be to permit testing agencies to select animal models, before studies begin, to coincide with the segment of the population that the agency desires the model to simulate. Thus, it may be possible to select an animal model that more closely simulates a potential highrisk group of humans. This knowledge may be of considerable value in interpreting estimates of cancer risk to humans that are made on the basis of biomathematical models. This approach also may assist the further development of prospective assessments and of new protocols for testing, and it may help in the reinterpretation of past studies to assess their extrapolative relevance. For example, the Occupational Safety and Health Administration has used data from studies of dogs in making quantitative assessments of bladder cancer risk from exposure to methylene bis-ochloroaniline (MOCA). Because it has been observed that MOCA must be N-hydroxylated (and not acetylated) to be a bladder carcinogen, it is important to note that dogs have an extremely low capacity for acetylating aromatic amines. Because dogs show a greater deacetylaseacetylase ratio than humans do with respect to aromatic amines, using dogs as models will likely result in overestimation of human risk, especially with respect to the human fast acetylator phenotype (Figure 2).
Basis of risk assessments Despite significant uncertainty in the process of predicting human cancer risk
from animal studies, recent advances in various aspects of toxicological research shed light on the relevance of animal model data to human responses. Three critical areas are the relevance of mouse hepatoma for humans, the use of PB-PK models in risk assessments, and the problems engendered by human heterogeneity when data from highly h e mogeneous animal models are extrapolated. It is hoped that toxicologists and regulators will continue to direct major efforts toward trying to understand the causes of interspecies and intraspecies variations so that risk assessments can be based on enhanced understanding of the biology of the predictive models and on the distribution of human responses to carcinogenic exposure.
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Acknowledgment Before publication, this article was reviewed for suitability as an ES&T feature by lulian B. Andelman, University of Pittsburgh, Pittsburgh. Pa. 15261; and Robert L. Harris, Jr., University of North Carolina, Chapel Hill, N.C. 27514. References (I) "The Relevance of Mouse Liver Hepatoma toHuman CarcinogenicRisk.'' Report of the International Expert Advisory Committee; Nutrition Foundation: Washington, D.C..
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1983; p. 34. ( 2 ) Elcombe. C.R. Arch. Toxicol. 1985, 8. 6I,
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(3) Anderson, M.E. et i Phormacol. 1987 87(2). t 0 3 - m ~ . (4) DSoula. R.W. et al. Presented at the National Academy of Sciences Conference on Physiologically Based Pharmacokinetic Models.Washington, D.C.. October 1986. ( 5 ) Calabrese, E.J. Ecogenerics: Wiley: New York. 1984. (6) Haldane. J.B.S. Heredity ond Politics: George Allen and Unwin: London. 1938:
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(7:I Calabrese. E.]. 1. Phorm. Sei. 1986. 75(1). IWI-46. (8) Calabrese. E.J. Principles of Animal Errropolorion: Wiley: New York. 1983.
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Edwad 1. Cahbrese is professor of roxicology at rhe Universiry (fMassachuserrs School of Public Healrh, Amherst. He has published numerous orricles on toxicology and has wrirten books on animal extrapolarion and rbe causes of human differenrial susceptibility to roxic subsrances. Calabrese has been a consulranr ro rhe governmenr and 10 rheprivate secro1: Currenrly he is on rhe National Academy of Sciences Safe Drinking Water Committee and is coordinating a study for €PA on the health risks of drinking-water rrrarmenr technolo gies.
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Environ. ki.Techna1.. Val. 21. No. 7. 1987 623
