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rfgulatory toxicology and pharmacology 5, 182-189 (1985)
The Role of the Chimpanzee in the Evaluation of the Risk of Foreign Chemicals to Man1
W. F. Mueller,2 F. Coulston,* and F. Korte
(JesellsehaH liter Strahlen* und Umweltforschuny mbH Muenchen. Institutfuer Oekologische Chemie. P-8042 Nettherberg. West Germanv; and White Sands Research Center and *Coulston International Corporation. Albany. New York
Received February I, 1985
Various species of laboratory animals are used to evaluate the efficacy and safety of drags in man. However, the extrapolation of data from animals to man is often complicated by species differences in the disposition of foreign chemicals. The findings of comparative metabolism studies are used to illustrate species differences in metabolic pathways, rates of biotransformation, kinetics, and excretion routes. Biochemical and structural consequences of the significant differences in enzyme induction between rodents and primate species are discussed. Among the primates, the chimpanzee has been shown to be the most closely related to man not only in the disposition of xenobiotics, but also in the aspects of endocrinology, serology, and immunology. It would, therefore, be the best possible model to predict the fate and effects of foreign chemicals in man. Due to the limited availability of chimpanzees, however, they can only be used for the evaluation of the most critical chemicals and drags. Comparative metabolism and disposition studies, e.g., of compounds representative of classes of chemicals in chimpanzees and other animals can be used to select suitable species for toxicological testing with larger numbers of animals. Only when the pertinent differences between the selected test species and man are known can correct extrapolations to man be made. ivss Academic Press, Inc.
Laboratory animals are used as models to evaluate the safety of drugs and other chemicals for man. Unfortunately the extrapolation of experimental results from animals to man is often complicated by species differences in the handling of foreign materials. Differences in xenobiotic disposition may be qualitative; i.e., the animal species forms other metabolites from a given chemical than man, or they may be quantitative: the same metabolites are formed, but at a different rate or in different proportions. If an animal species metabolizes a chemical along pathways which are not available to man, it should not be used as a model for safety evaluation. Quantitative differences, which will be unavoidable in most cases, can be taken into
consideration when extrapolating, but only if they are clearly recognized and well defined.
1 Presented at the International Symposium "Bioavailability of Environmental Chemicals," 12-14 Sept. 1984, Schmallenbcrg. West Germany.
1 To whom correspondence should be addressed: 1300 LaVclIc Rd., Alamogordo, N. Mcx. 88310.
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RISK OF FOREIGN CHEMICALS TO MAN
183
METABOLISM
Biotransformation reactions are generally categorized as Phase I reactions, which include oxidation, reduction, and hydrolysis, or Phase II, or conjugation reactions. A classical example of species variations in Phase I reactions is the biotransformation of amphetamines (Williams. 1971). The two main routes of biotransformation are /7-hydroxylation and side chain deamination to phenylacetone, followed by oxidation to benzoic acid (Fig. 1). Reduction or enolization of phenylacetone can also occur, but is of minor importance, except in the rabbit. The newly formed hydroxyl and carboxyl groups are then conjugated with glucuronic acid, sulfate, or glycine. Part of the amphetamine is also excreted unchanged. Table 1 shows that man. monkey, and dog metabolize amphetamine similarly, excreting 25-30% unchanged and 20-30% as total benzoic acid within 24 hr after dosing. /7-Hydroxylation is of minor importance in these species, but it is the main reaction in the rat. which excretes 60% as (conjugated) 4-hydroxyamphetamine and only 13% unchanged and 3% as benzoic acid. The rabbit is the only species listed here that excretes a larger amount of the enolized and conjugated phenylacetone. Metabolism of this drug in the mouse is not very different from that in man with the exception of a higher rate of p-hydroxylation. Looking at the overall rate of excretion in 24 hr, we find that man eliminates 56%, monkey 60%, and rat and mouse 76% each.
Among the Phase II reactions, we found conjugation with glucuronic acid to be the most common in the primates, as is also reported for rodents and most other mammals except the cat, which is deficient in glucuronyltransferase (Dutton, 1966). Summer and co-workers have compared glutathione conjugation in rats and chimpanzees, measuring urinary excretion of mercapturic acids after administration of diethyl maleate or naphthalene (Summer et al., 1979). In untreated chimpanzees the excretion of mercapturic acids was found to be about one-fifth that in untreated rats, which corresponds well to the values the authors found for humans. Treatment with naphthalene failed to increase urinary mercapturic acids in the chimpanzee, which is in agreement with data published for man (Boyland and Sims, 1958). In the rat, extensive formation of mercapturic acids was found. Diethyl maleate
CHjC0CH3
reduction
enol Witl(m
CHjCHCH,
CH-CCHj
NH2 OR OR
II IV
V VI
IK;. I. Metabolism ol amphetamine (Williams, 1971). In II and V, R = glucuronide; in VI, R = sulfate; in IV, R =- glycine.
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184 MUELLER, COULSTON, AND KORTE
TABLE 1 Metabolites of Amphetamines in Various Species
% of dose of ,4C-labeled drug excreted in the urine in 24 hr as
Species
Amphetamine
4-Hydroxyamphetamine"
Benzoic acid"
Phenyiacetone
Benzylmethylcarbinol"
Man Rhesus monkey Dog Rabbit Guinea pig Mouse Rat
30 25 30
3 18 30 13
3 20
3
6 29 0
6 28
1
6 23 22 4
1 65 0
15 31
0
60 3 0
0 0 1 8 0 0 0
Source. Williams (1971). " Total including conjugates, 4 Suspected to be present as the enol sulfate.
increased the mercapturic acid excretion in rats twice as much as in chimpanzees. The finding that primates are much poorer glutathione conjugators than rats is supported by the observation that the specific activity of glutathione-.S'-aryltransferase in liver homogenates or in the soluble supernatant fraction from humans and rhesus monkeys is only a fraction of that in rats (Grover and Sims. 1964).
The synthetic estrogen diethystilbestrol (DES) is conjugated with glucuronic acid by most species. Its metabolic pattern shows some significant differences between primates and rats (Fig. 2), Rhesus monkeys, chimpanzees, and humans form the glucuronides of DES, dienestrol, w-hydroxydienestrol, and, to a small extent, the oi-hydroxy-DES, but none of the ring-hydroxyl or methoxy derivatives which have been reported as DES metabolites in the rat (Metzler, 1975; Metzler et al., 1977).
HCO
O)
Pr tiiij tl
Rt
CHCHvQH
(0>-
o OH
HtCCH
Rit (minor)
Primittl
Fir,. 2. Metabolites of dicthylstilbcstrol in rats and primates (Metzler et al., 1977).
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12 OH * Oisldrin
185
4,5 Ald'in tr - diol
Fig. 3. Major metabolites of dieldrin in mammals.
Earlier in our work with environmental chemicals, we compared the metabolism of the chlorinated cyclodiene insecticide dieldrin in mice, rats, rabbits, rhesus monkeys, and chimpanzees (Mueller et al., 1975). Figure 3 shows the two major metabolic Phase I reactions of dieldrin: hydroxylation at the unchlorinated methylene bridge, resulting in 12-hydroxydieldrin (Baldwin et al., 1970), and opening of the oxirane ring to yield 4,5-franj-dihydroaldrindiol (Korte and Arent, 1965). When we administered 0.5 mg/kg body wt of dieldrin orally to the five species, we found the two metabolites in the proportions shown in Table 2. Unchanged dieldrin was excreted only in the feces during the first 48 hr after administration and represents unabsorbed material that passed through the gastrointestinal tract. Regarding the ratio of the two metabolites, the rat seems comparable to primate species; direct oxidation to the monohydroxy derivative is the main common metabolic route. In the mouse and the rabbit, on the other hand, opening of the epoxide to the diol is the predominant reaction. The total metabolization rate was the highest in the mouse.
ENZYME INDUCTION
Although the rat handles dieldrin much as the primate does after a single-dose or short-term exposure, chronic feeding studies with the pesticide revealed a very significant difference; while the rat reaches a peak level of storage in 50-60 days
TABLE 2
Relative Amounts (% of Dose) of Dieldrin and Metabolites Excreted in 10 Days after Single Oral Dose of 5 mg/kg
Mouse Male Female
Rat Male Female
Rabbit Male Female
Male rhesus monkey
Female chimpanzee
Dieldrin
5.5 3.2 0.8 2.8 0.3 0.5
12-OII-dicldrin
13.0 7.5 8.8 4.6 -- 0.2
iran.v-Dihydroaldnndiol 20,0 26.0 2.3 2.4 1.5 2.0
Total
38.5 36.9 11.9 9.8 1.8 2.7
9.0 9.4 2.0
20.7
3.2 2.0 1.1
6.3
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186 MUELLER, COULSTON. AND K.ORTE
(Coulson and McCarthy, 1963), after which time the intake of dieldrin and the excretion of its metabolites are in equilibrium, it took rhesus monkeys 300 days to approach a storage plateau with much higher tissue and organ concentrations than in rats at comparable exposure levels (Mueller et al., 1979). This indicates that the induction of the enzyme systems responsible for the biotransformation of dieldrin is much less pronounced in the primate species than in the rat, resulting in very different long-term kinetics. These toxicokinetic findings in monkeys correspond well to data of dieldrin accumulation published for man (Hunter et al.. 1969). We must conclude, therefore, that, concerning long-term exposure to organic chemicals which are accumulated, rodents are a poor model for man, whereas accumulation in the primate species largely parallels that in man. The toxicokinetics of such chemicals are, however, only one aspect of the evaluation of the risk they constitute for humans. The high degree of enzyme induction which permits rodents to metabolize and eliminate chemicals like dieldrin much more efficiently than primate species, is reflected histologically by pronounced proliferation of the smooth endo plasmic reticulum of liver cells, leading to enlargement of hepatocytes, increased liver weight, and the well-described symptoms of hepatotoxicity, which may eventually result in hepatocellular carcinoma (Wright et al. 1972). With comparable exposure to dieldrin, rhesus monkeys, with their much lower degree of enzyme induction, did not show any significant structural changes in the liver (Wright et al., 1978). This indicates that, at least for chemicals which have a tendency to accumulate and which can induce metabolizing enzyme systems, rodents not only are poor models for man in terms of toxicokinetics under chronic exposure conditions, but that extrapolation of biological- effects like hepatotoxicity from rodents to humans is likely to lead to exaggerated risk expectations.
EXCRETION ROUTES
The extent of excretion through the bile depends largely on molecular weight and polarity. As a rule of thumb it can be said that polar compounds with a molecular weight of less than 300 are generally excreted through the kidney by all species. In the molecular weight range 300 to 500, excretion through the bile becomes the major route in some species, but not in others (Williams, 1971).
In our metabolism studies with chlorinated cyclodiene insecticides, like endrin, dieldrin, chlordane, etc., we have consistently found that the rat excreted most of the material in the feces and the rabbit almost exclusively in urine. In the rhesus monkey and the chimpanzee, dieldrin metabolites were excreted about equally in urine and the feces (Mueller et al., 1975), and monkeys eliminated pentachloronitrobenzene (Koegel et al., 1979) and pentachlorophenol, and their metabolites, in similar proportions in the feces and urine. However, using cholestyramine, an ionexchange resin that binds conjugated biliary metabolites in the intestines and thereby prevents hydrolysis by bacterial glucuronidases and reabsorption of the free metabolites into the blood stream, we found that excretion of dieldrin metabolites and pentachlorophenol was shifted almost completely to the feces (Mueller, 1980), This shift shows that these compounds are excreted mainly as biliary conjugates which are subsequently hydrolyzed to some extent and eliminated through the kidneys.
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RISK OF FOREIGN CHEMICALS TO MAN
187
When we compared the metabolism of trichloroethylene in rhesus monkeys, baboons, and chimpanzees to published data on the fate of this solvent in humans (Kimmerle and Eben. 1973), we found a somewhat surprising difference between the primate species (Table 3). While all three nonhuman primates formed the same metabolites, trichloroacetic acid and trichloroethanol, as humans, in proportions very similar to man, the ratios of urinary excretion and fecal excretion differed considerably between man and the chimpanzee on one side, and the baboon and the rhesus monkey on the other. Man and the chimpanzee excrete only a few percent in the feces, whereas fecal excretion is considerable in the two lower primate species (Mueller et al,, 1982). It appears therefore that rhesus monkeys and baboons are satisfactory models for man as far as metabolite patterns are concerned, but in cases where excretion routes are critical, only the chimpanzee parallels man very closely.
REPRODUCTIVE PHYSIOLOGY
In the area of reproductive physiology, significant differences in 'he endocrinology of rodents and humans have resulted in increasing the use of nonhuman primates as human endocrine and toxicological models. While the use of rhesus monkeys has led to greater understanding of human endocrinology and has been valuable in studies of reproductive toxicology, more recent findings have pointed out important differences in the reproductive physiology of the rhesus monkey and man, whereas the chimpanzee has been found to be much more closely related to man (Hobson el al.. 1978). Some of the reproductive statistics are reviewed in Table 4, In most aspects, the chimpanzee is intermediate between the rhesus and man. Chimpanzees, unlike rhesus monkeys but similar to humans, continue to ovulate and undergo menstrual cycles throughout the year.
While radioimmunoassays for human hormones have not proved suitable for use in measuring rhesus monkey gonadotrophins, human assay systems have been found to be very reliable for the chimpanzee. The gonadotrophins of the chimpanzee appear therefore to be immunologically more closely related to their human counterparts than the gonadotrophins of other primate species. The patterns of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, and pro-
TABLE 3
Excretion of Radioactivity by Primate Species after Administration of ['`CJTrichloroethylene
Chimpanzee
Baboon
Rhesus monkey
Male
Female
Male
Female
Male
Female
Recovery (% of dose)
Urine (% of total excretion)
Feces <% of total excretion)
59.1 44.2 23.5 13.2 36.6 29.8 95 98 75 66 77 82
5 2 25 34 23 18
Human
40-60 >95
<5
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188 MUELLER. COULSTON, AND KORTE
TABLE 4 Comparative Primate Reproductive Statistics
Man
Rhesus monkey
Life span (years) Weight (kg) Gestation (days) Menarche (years) Menopause (years) Menstrual cycle (days) Seasonal infertility
72 55 280 12 44 28 No
25 6
165 2.5
25? 27 Yes
Source. Hobson et al., (1978).
Chimpanzee
50? 45 225
9 ? 32 No
gesterone, which control the sequence of the menstrual cycle, are similar in the human, the chimpanzee, and the rhesus monkey. This indicates that both the rhesus monkey and the chimpanzee can be used as models to study effects of drugs on reproductive hormones outside pregnancy. One reservation in this respect may be that rhesus monkeys do not respond uniformly to externally administered gonado trophin-releasing hormones, while chimpanzees do. For studies of drug influences on the level of hypothalamic-pituitary interaction, the chimpanzee would therefore be a more appropriate model than the rhesus monkey.
Very significant differences have been found between the hormone levels in chimpanzees and rhesus monkeys during pregnancy. (Hobson et al., 1976). Chorionic gonadotrophin in the rhesus reaches a peak at Day 25 of pregnancy and disappears completely by the 40th day. In humans and chimpanzees, chorionic gonadotrophin peaks at a level 100 times higher and is measurable throughout pregnancy. Estradiol and estrone levels in chimpanzees are near or within the human range, whereas levels of both hormones are 20- to 100-fold lower in rhesus monkeys. Serum progesterone concentrations in chimpanzees and humans rise throughout pregnancy and reach values of up to 100 ng/ml in both species. In contrast, progesterone peaks near the 35th day of pregnancy in rhesus monkeys and remains at levels of 2 to 4 ng/ml until parturition, Estriol is not measurable at all in the rhesus monkey during pregnancy but rises dramatically in the human and the chimpanzee. The 16hydroxylated precursors of estriol are produced by the fetal liver and adrenals and converted to estriol by the placenta; therefore the measurement of estriol serves as a means of assessing the health of both the fetus and the placenta during pregnancy. Obviously the availability of this indicator to detect trouble during pregnancy, which seems to be unique in the chimpanzee and the human, as well as the similarity of the hormone pattern during pregnancy, makes the chimpanzee a particularly wellsuited model for reproductive physiology.
CONCLUSION
It appears that the chimpanzee is the best available model for man, paralleling the human very closely in xcnobiotic disposition and in reproductive endocrinology. Due to the limited availability of chimpanzees, however, they can only be used for the evaluation of the most critical drugs and chemicals. The large numbers of animals which protocols of toxicology testing usually require make it difficult to use the chimpanzee for this purpose. A way out of this dilemma appears to be to
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RISK OF FOREIGN CHEMICALS TO MAN
189
compare the metabolism and disposition of the compound to be tested in the chimpanzee and other animals in order to select the most suitable species for testing with the necessary animal numbers. When the pertinent differences between the selected test species and man are well defined, they can be taken into consideration when the results are extrapolated to man.
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Boyland, E., and Sims, P. (1958). Metabolism of polycyclic compounds. Biochem. J. 68, 440-447. Coulson, D. M., and McCarthy, E. M. (1963). Effects of pesticides on animals and human beings.
Stanford Res. Inst. Rep. 13.
Dutton, G. j, (1966). The biosynthesis of glucuronides. In Glucuronic Acid, Free and Combined. Chemistry, Biochemistry. Pharmacology and Medicine (G. J. Dutton, ed.), pp. 185-299, Academic Press, N. Y.
Grover, P. L.. and Sims, P. (1964). Distribution of glutathione-S-aryltransferase in vertebrate species.
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Hobson, W. C,, Coulston, F.. Faiman, C., Winter, J. S. D., and Reyes, F. (1976), Reproductive endocrinology of female chimpanzees: A suitable model for humans. J. Toxicol, Environ. Health 1, 657-668.
Hobson, w. C., Fuller, G. B.. Mueller. W. F., Korte, F., and Coulston. F. (1978). The reproductive endocrine system of nonhuman primates--A model for prediction of toxicity. Ecotoxicol. Environ. Saf. 2, 257-266.
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Kimmerle, G-, and Eben. a. (1973). Metabolism, excretion, and toxicology of trichloroethylene after inhalation. 2. Experimental human exposure. Arch. Toxicol. 30, 127-138.
Koegel. W,, Mueller, W. F,, Coulston. F., and Korte. F. (1979). Fate and effects of pentachloronitrobenzene in rhesus monkeys. J Agnc. Food Chem, 27, 1181-1185.
Korte, F., and ARENT, H. (1965). Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrin-1 JC. Life Sci. 4, 2017-2026.
Metzler, M. (1975). Metabolic activation of diethylstilbestrol. Biochem. Pharmacol. 24, 1449-1453.
Metzler, M., Mueller, W. F., and Hobson, W. C. (1977). Biotransformation of diethylstilbestrol in the rhesus monkey and the chimpanzee. J Toxicol. Environ. Health 3, 439-450.
Mueller, W. F. (1980). Metaboiismus and kinetische Unlersuchungen ausgewaehher Chemikalien in Rhesusaffen und Schimpansen. Gesellschaft fuer Strahien- und Umweltforschung, Muenchen, GSFReport Oe 599, pp. 120-128.
Mueller, W. F,, Coulston, F,, and Korte, F. (1982). Comparative metabolism of l4C-trichloroethylene in chimpanzees, baboons, and rhesus monkeys. Chemosphere II, 215-218.
Mueller, W. F,, Nohynek, G., Woods, G., Korte, F,, and Coulston, F. (1975). Comparative metabolism of dieldrin-"C in mouse, rat, rabbit, rhesus monkey, and chimpanzee. Chemosphere 2, 89-92.
Mueller, W. F,, Nohynek, G., Coulston, F., and Korte, F. (1979). Aufnahme. Verteilung, Umwandlung und Ausscheidung von Dieldrin in nicht-menschlichen Primaten und anderen Laborticren. Z Naturlorsch. C 34, 340-345.
Summer, K. H.. Rozman, K,, Coulston. F, and Greim, H. (1979). Urinary excretion of mercapturic acids in chimpanzees and rats. 'Toxicol Appl. Pharmacol. 50, 207-212.
Williams, R. T, (1971). Species variations in drug biotransformalions. In Fundamentals of Drug Metabolism and Drug Disposition (B. N. LaDu, H. G. Mandel, and E. L. Way, eds.). pp. 187-205. Williams & Wilkins, Baltimore.
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Wrigii r, A. S.. Pottir, D., Wr rider. M. I'., AND Donninger, C. (1972). I lie ctt'ects of dieldrin on the subeellular structure and function in mammalian liver cells. Food Cosmet. Toxicol. 10, 311-332.
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