Document V3jMVYXnRyRBb99m9nZVG7224

Arch. ToxicoL 39, 97-108 (1977) yZu-f'ZgetfiA>+ ,>yr>? **?&f3*& <tflWw> of TOXICOLOGY f by Springer-Vcrlag 1977 Species Differences in Activating and Inactivating Enzymes Related to the Control of Mutagenic Metabolites* F. Oesch, D. Raphael, H. Schwind, and H. R. Glatt ** olofische* lnstitut dcr Univerritit Mainz, hlbacher Strafle 67, D-6500 Mainz, Federal Republic of Germany 7ct. Microsomal monooxygenases catalyze the biosynthesis of epoxides 'efinic and aromatic compounds whilst microsomal epoxide bydratase iplasmic glutathione S-transferases are responsible for their further bionation. Although catalytically very efficient the cytoplasmic glutathione erases play, due to their subcettular localization, a minor role in the ation of epoxides derived from large lipophilic compounds and were, therefore, not included in this study. It was shown with such a lipophilic com pound, benzo(a)pyrene, as a mode! substance and with liver enzyme mediated bacterial mutagenesis as biological endpoint that species and strain differences in epoxide hydratase and monooxygenases are reflected in very dramatic differ ences in mutagenicity of benzo(a)pyrene which varied from extremely potent to a degree which could easily be overlooked. In order to investigate whether the differences in enzyme activities were causally linked to the observed differences in mutagenicity, the enzyme activities were modulated by inhibition and induc tion. These manipulations were always accompanied by the corresponding changes in mutagenicity. It is concluded that species such as mice which possess high monooxygenase activity but very low epoxide hydratase activity are much more susceptible than man to those toxic effects which are mediated by metabolically formed epoxides which are substrates of epoxide hydratase. In this regard, it is especially note worthy that mice possess a much lower hepatic epoxide hydratase activity than man. Key words: Epoxide hydratase -- Benzo(a)pyrene -- Inactivation -- Mutageni city. Zusammenfaasung. Mikrosomale Monooxygenasen oxidieren olefinische und aromatische Stoffe zu Epoxiden, mikrosomale Epoxidhydratase und zytoplas- Smd offprint requests to F. Oesch at the above address * Presented at the Symposium "Influence of Metabolic Activations and Inactivations on Toxic Effects" held at the 18th Spring Meeting of the Deutsche Pharmalcologiscbe Gesellschaft, Section toxicology, D-6500 Mainz, March 15, 1977 SL 066172 V a matische Glutathion-S-Transferasen setzen diese Epoxide weiter um. Obwoh1 katalytisch sehr aktiv, spielen zytoplasmatische Glutathion-S-Transferasen au grund ihrer subzellularen Lokalisicrung nur eine untergeordnete Rolle bei dt. Inaktivierung von Epoxiden, die von groBen lipophilen Substanzen gebildet werden, und wurden daher in dieser Studie nicht untersuebt. Mit Benzo(a)pyrr als Modclisubstanz und mit Leberenzym-vermittelter bakterieller Mutagenese a biologischem Endpunkt wurde gezeigt, daB sich Spezies- und Stammesunterschiede von Epoxidhydratase und Monooxygenasen sehr massiv in der Mutage nitat widerspiegeln: Je nach Herkunft des aktivierenden Leberpraparates wir fur Benzo<a)pyren eine auBerst Starke oder eine versebwindend geringe, durch aus ubersehbare Mutagenitfit beobacr*:.. Um festzusteUen, ob die Unterschiede in den Enzymaktivitaten mit den be ichteten Unterschieden der Mutagenita' kausal zusammenhangen, wurden die Enzymaktivitaten durch Inhibition um Induktion manipuliert Diese Manipulationen hatten in jedem Faile entsprechende Veranderungen der Mutagenitiit zur Folge. Es wird geschlossen, daB Herarten wie die Maus, die eine hohe Monoox;, genase- und eine sehr niedrige Epoxidhydratase-Aktivitat aufweisen, viel anfa tiger sind als der Mensch fBr solche toxischen Wirkungen, welche durch metabolisch gebildete Epoxide verursacht werden, die durch Epoxidhydratase inaktiviert werden. In dieser Hinsicht ist erwahnenswert, daB Mause eine sehr vie geringere Epoxidhydratase-AktivitSt aufweisen als der Mensch. Introduction Evidence is accumulating that a vast array of substances which after application to an animal produce mutagenic and carcinogenic effects need metabolism to electrophilicaliy reactive species before they can exert such effects (for a review see Millet and Miller, 1974). Aromatic and olefinic compounds are metabolized by microsomal monooxygenases to electrophilically reactive epoxides. On the one hand, such epox ides can spontaneously bind with nucleophilic moieties in tissue such as DNA, RNA and proteins. On the other hand there exist enzymes which compete for these epox ides (for reviews see Daly et aL, 1972; Oesch, 1973; Sims and Grover, 1974; Heidelberger, 1975). Suedes differences in activatine and inactivating enzymes are likely to plav an imortant role in the determination of the tissue levels of reactive metabolites;. For large and lipophilic species such as epoxides derived from polycyclic hydrocar bons, the cytoplasmic glutathione S-transferases play a minor role due to their local ization (Glatt and Oesch, 1977). Therefore, in this study, the relationship between species and strain differences in monooxygenases (mixed function oxidases) and epoxide hydratase (epoxide hydrase, E.C. 4.2.1.63), and the relative accumulation of benzo(a)pyrene metabolites mutagenic for Salmonella typhimurium strains was investigated. Materials and Methods C3H/HJ mice (18--23 g) wer from the Jackson Laboratory. Bar Harbor, Maine. USA; CF-1 mice (15-20 g) from Issa Credo, Les Oncins, Saint Germain sur L'Arbresle. France; Sprague-Dawley rats SL 066173 Species Differences in Drug Metabolizing Enzymes Related to Mutagenicity 99 (200-250 g) from the Versuchstier-Zuchtanstalt Wiga, Sulzfeld, West Germany; Fischer rats (220-250 g) from the Zentralinstitut fur Versuchstierzucht, Hannover-Linden, West Germany. All mimals used in this study were adult males with the exception of the C3H mice, which were adult females', immediately after killing the animals the livers were removed and all subsequent steps in the preparation of subcellular fractions were performed at 0--4 C. The livers were homogenized in 3 volumes of 150 mM KCI containing 10 mM potassium phosphate buffer, pH 7.4, using a PotterElvehjem all glass homogenizer. The homogenates were centrifuged at 9000; for 15 min. The resulting post-mitochondrial supernatant fractions were divided into two portions. One portion was used as such and is called "post-mitochondrial supernatant" in this study. This fraction is analogous to the fraction referred to as "S-9" by Ames et al (1973). The other portion was centrifuged at 100.000 g for 1 h. The resulting pellets were rinsed with 150 mM KCI containing 10 mM potassium phosphate buffer and then resuspended in the same medium. This preparation is called "microsomes". The mutagenicity was tested as described by Ames et al. (1973) with some minor modifications (Glatt et aL, 1975) but without albumin. The test compounds were added in dimethylsulfoxide (20--60 pi) and were, together with the enzyme systems and histidine-dependent Salmonella typhimurium TA 1537 or TA 98, poured in a histidine-poor top agar onto a minimal agar plate. After incubation at 37s C for 2 days, the his" revertant colonies were counted. Monooxygenase activity with benzo(a)pyrene as substrate was determined, measuring fluorescent phenolic metabolites and using 3-hydroxybenzo(a)pyrtne as standard (Nebert and Gelboin, 1968). Monooxygenase activity with 7-ethoxycoumarin as substrate was determined according to the method of Ullrich and Weber (1972) using 3 diagnostic inhibitors as a crude measure for different monooxy genase forms (Ullrich et aL, 1975). Epoxide hydratase activity with styrene oxide (Oesch et aL, 1971a) and benzolii)pyrene 4,5-oxide (Schmassmann et al,, 1976) was determined as described. Protein con centration* were determined by the method of Lowry et aL (1951). ['HI-* eat oxide was prepared at described (Oesch et aL, 1971a), [,H)-benzo(a)pyiene 44-oxide following tne synthetic procedure for the unlabelled compound (Dansette and Jenna, 1974) using generally labelled t*H1-benzo(a)pyrene (Radiochemical Centre, Amersham. England) as starting mate rial Synthesis, isolation and handling of benzo(a)pyrene and its derivatives were performed under dim light. Results end Discussion Spedes Differences in Enzyme Patterns and Mutagenic Effects. The parent hydro carbon, benzo(a)pyrene did not revert any of the Salmonella typhimurium strains tested {TA 1537, TA 98) to histidine prototrophy in absence of a metabolically activating liver preparation (data not shown). However, in the presence of liver homogenate or liver microsomes and NADPH, the cofactor necessary for mono oxygenase activity, benzo(a)pyrene was transformed to metabolites which reverted the Salmonella strains investigated (Figs. 1--5). This was true for the liver preparations from all mammalian species and strains investigated, but the extent of increase in the number of revertant colonies differed dramatically for the various species and strains. As an example, it can seen in Figure l, that the maximal increase in the number of revertant colonies is 1.7-fo)d with respect to spontaneous mutations when benzo(a)pyrene is activated with liver micro somes from untreated Sprague-Dawley rats, but more than 20-fold with liver micro somes from untreated C3H mice. The 1.7-fold increase using Sprague-Dawley rat microsomes was statistically highly significant (P < 0.01), but would, according to widespread practice, not be considered "biologically" significant -- a positive label of1 1 From earlier experiments (unpublished) we know that female C3H mice are not significantly (< 10%) different from males with respect to the parameters of this study St 06617* 100 PvEF?TANT COLONIES ITA 1537) F. Oesch et aL Fig. 1. Activation or benzo(a)pyrese to mutagenic metabolites by rat and mouse liver microtomes. Benzola)pyrene in 40 ul dimethyisulfoxide and an enzyme system Giver microtomes corresponding to about 1 mg protein, 1.7 mg NADP*, 1 mg glucose-6-phosphate, I unit glucose-6-phosphate dehydro genase in 0.5 ml 100 mM phosphate buffer pH 7.8,8 mM MgClj, 33 mM KC1) and 1--2 x 10* bacteria (Salmonella typhimurfum TA 1537) were mixed with 2 ml of a histidine-poor top agar (0.55% NaCl, 0.55% agar, 50 uM biotin, 50 uM histidine) and poured onto a Petri dish with minimal agar. After 48 h at 37s C, the hie* revertant colonics were counted. Hatched bars represent activation by SpragueDawtey rat microtomes, open bars by C3H mouse microsomes. Values are means of 2--4 incuba tions "mutagenic" most commonly only being attributed to substances increasing the numbeT of revenant colonies at least twofold. Increasing the amount of benzo(a)pyrene above the maximum shown in Figure 1 did not lead to a further increase in mutagenicity (data not shown). Thus, one of the most potent carcinogens known might have been dismissed as non-mutagenic if submitted to mutagenicity testing as an unknown in a limited screening procedure using only a single metabolically acti vating system if this system had an enzyme pattern unfavorable to the accumulation of mutagenically active metabolites. When using liver microsomes from Sprague-Dawley rats which had been pre treated with Arochlor 1254 according to the schedule recommend by Ames et aL (1975)1 the increase in mutation rate was very high (similar to that after methylcholanthrene pretreatment, data not shown), but the important fact remains obvious (see Fig. 1 and Table 1) that metabolically activating systems possessing different pat terns of activating and inactivating enzymes may lead to very drastically different accumulations of mutagenically active metabolites. This may occur to the extent that what is obviously an extremely powerful mutagen when activated by a liver preparation from a particular species or strain may be missed as a mutagen when : Arochlor 1254 induces 4 wider spectrum of monooxygenase forms (Alvares al.. 1973) than the more classical types of monooxygenase inducers whose prototypes are phenobarbital, 3-methylcholanthrene and pregnenolone 16o-carbonitrUe (Conney et al,, 1973). Although Arochlor 1254-induction may be expected to lead to a liver preparation which increases the sensitivity of the liver enzyme mediated bacterial mutagenicity test toward many compounds, the opposite may occur in cases where the control monooxygenase forms mediate metabolic pathways leading to more potent mutagens than the induced monooxygenases. Moreover, it is important to realize that Arochlor 1254 also inducts epoxide hydratase (Oesch et aL, 1977) SL 066^-7 Species Differences in Drag Metabolizing Enzymes Related to Mutagenicity 101 Table 1. Enzyme activities in liver microsomes or the untreated Sprague-Dawley rats and C3H mice which were used for the mutagenicity experiment shown in Figure 1 Species and strain Enzyme activity* Epoxide hydratase (nmoles styrene glycol/min/mg protein) Monooxygenase (pmoles 3-OH-benzo(a)pyrene fluorescence equivalents/ min/mg protein) Sprague-Dawley rat C3H mouse 7.2 0.2 1.0 0.1 410 + 32 850 + 54 Values represent means S.E.M. of 4 incubations (duplicate determinations at 2 protein concentra tions) using a different species or strain- This clearly underlines the crucial importance of differences in enzyme patterns. Demonstration ofCausal Link between Enzyme Patterns and Mutagenic Effect. As indicated in Table 1, epoxide hydratase activity (with styrene oxide as substrate) is lower in liver microsomes from untreated C3H mice compared to Sprague-Dawley rats by a factor of about 7, monooxygenase activity [with benzo(a)pyrene as sub strate] is higher by a factor of about 2. Both factors would be expected to lead to a higher accumulation of intermediate epoxides in the former species. Thus, the much higher number of revertant colonies during metabolic activation of benzo(a)pyrene by liver microsomes from C3H mice as compared to Sprague-Dawley rats appears quite logical. However, very many other parameters will also be different between microsomal preparations from two different species. If the difference in mutagenic potential were in fact causally linked to these differences in enzyme patterns, it should be possible to compensate for them by enzyme manipulations. Figure 2 and Table 2 show that inhibition of epoxide hydratase in Sprague-Dawley rat liver mierosomes indeed led to a dramatic potentiation of the mutagenic effect This was true for two quite different types of epoxide hydratase inhibitors: Cyclohexene oxide, a moderately potent inhibitor of the non-competitive type, and 1,1,1-trichloropro- , pene 2,3-oxide, an extremely potent inhibitor of the uncompetitive type (Oesch et al., 1971b). Both inhibitors were used at concentrations where no influence on mono oxygenase activity [with benzo(a)pyrene as substrate] was observed. Similarly, it can be appreciated from the data in Figure 3 and Table 3, that an increase in monooxygenase activity in the liver microsomes from Sprague-Dawley rats due to pretreatment of the animals with the inducer 3-methylcholanthrene also leads to a dramatic potentiation of the mutagenic effect However, the situation with respect to monooxygenases is complex. While inhibitor and antibody studies had shown that there exists only a single epoxide hydratase catalyzing the hydration of both styrene oxide and benzo(a)pyrene 4,5-oxide (Oesch and Bentley, 1976) (and quite likely of many other epoxides), several different monooxygenase forms exist They possess overlapping but quantitatively quite different specificities for attack of l*rge molecules at different positions (Lu et al., 1972; Nebert et al., 1973; Holder et SL 066176 102 F. Oesch al. BENZOlAlPYRENE [jug/ptat| Fig. 2. Activation of benzo(a)pyrene to mutagenic metabolites: Effect of epoxide hydratase inhibitors. Benzo(a)pyrene was activated, as described in Figure 1 to mutagenic metabolites by rat liver micro somes. The open bars show the effect ofthe epoxide hydratase inhibitors cyclohexene oxide (2.4 mM in the top agar, CHO) and 1.1,1-trichloropropene 24-oxide (0.36 mM, TCPO). The hatched bars repre sent the number of revertant colonies in the absence of the inhibitors. Values are means of 2--4 incubations Table 2. Effect of cyclohexene oxide and 1,1,1-trichloropropene oxide on microsomal epoxide hy dratase and benza(a)pyrcne monooxygenase activities in the untreatet Sprague-Dawley rats which were used in the mutagenicity experiment shown in Figure 2 Inhibitor Enzyme activity* Epoxide hydratase (nmoles styrene glycol/min/mg protein) None (control) 2.4 mM Cyclohexene oxide 0.36 mM 1.1,1-Trichloropropene oxide 9.8 0.7 1.2 + 0.6 0.8 0.5 Monooxygenase (pmoles 3-OH-benzo(a)pyrene fluorescence equivalents/ min/mg protein) 364 41 390 66 421 24 * Values represent means + S.E.M. of 6 incubations (duplicate determinations at 3 protein concentra tions) al., 1974; Rasmussen and Wang, 1974; Haugen et al., 1975; Wiebel et al., 1975). Thus modulation of monooxygenase is not only an alteration in the rate of produc tion of oxidative metabolites. Much rather induction or inhibition by different agents will afTect differently various monooxygenase forms (Conney et al., 1973; Ullrich et al., 1975) and thereby lead to an alteration of the pattern of metabolites. Thus metabolic activation of benzo(a)pyrene by liver microsomes from untreated (control) or phenobarbital-induced C3H mice ("cytochrome P-450-dependent monooxygenases") leads to mutagenic benzo(a)pyrene metabolites which are very readily inacti vated by homogeneous epoxide hydratase while, in contrast, liver microsomes from SL 066177 Specie Differences in Drug Metabolizing Enzymes Related to Mutagenicity 103 Fig. 3. Activation of benzo(a)pyrene to mutagenic metabolites: Effect of enzyme induction by 3-methylcholanthretie, Benzo(a)pyrene was activated to mutagenic metabolites, as described in Figure 1, by liver microtomes from untreated male SpragueDawley rats (hatched bars) or from rats that had been pretreated intraperitoneally with 10 mg/fcg of 3-methylcholanthrene in sunflower oil and killed 3 days later (open bars). Values represent means of 2--4 incubations BENZ01A) PYRENE [jug/plote ] Table 3. Effect of 3-methylcholanthrene pretreatment on enzyme activities in microtomes of the Sprague-Dawley rats which were used in the mutagenicity experiment shown in Figure 3 Pretrestment* Enzyme activities* Epoxide hydratase (nmoles styrene glycoi/imn/mg protein) Monooxygensse (pmoles 3-OH-benzo(a)pyrene fluorescence equivalents/ min/mg protein) None (control) 3-MethylchoUmhrenc 7.3 + 0.4 7.6 0J 429 36 1780 + 60 1 Treated rats received a single intraperitoneaJ injection of 3-methylcholanthrene (10 mg/kg) in sun flower oil 3 days before sacrifice * Values represent means + S.E.M. of 4 incubations (duplicate determinations at 2 protein concentra tions) 3-methylcholanthrene pretreated C3H mice ("cytochrome P-448-dependent monooxygenases") lead to a fundamentally different pattern of mutagenically active benzo(a)pyrene metabolites on which epoxide hydratase has a weaker and more complex (muldphasic) effect (Bendey et al., 1977). Thus, an increase in monooxy genase activity does not necessarily mean that the mutagenic effect will be poten tiated. While Figure 3 and Table 3 show that metabolic activation of benzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats leads, indeed, to a dramatic potentiation of the mutagenic effect as compared to control microsomes. Figure 4 and Table 4 show that pretreatment with a dose of phenobarbital leading to a similar increase in "overall" monooxygenase activity (in this ex periment determined with 7-ethoxycoumarin as substrate) did not lead to an increase, rather to a slight decrease in mutagenicity. This underlines the importance of keep ing the overall complexity of such systems in mind. Phenobarbital induces a pattern of monooxygenase forms which differ greatly from the forms induced by 3-methyl cholanthrene. Moreover, the dose of 3-methylcholanthrene used in this study did not lead to any change in epoxide hydratase activity, but phenobarbital led to a consid- SL 066178 104 F. Oesch iu. benzchaipyrene f>jg/pioti Fig. 4. Activation of bcnzo(*)pyrcne to mutagenic metabolites by rat liver postmitochondrial superna tant fraction: Effect of enzyme induction by phenobarbital. Benzo(a)pyTene in 20 ul dimetbylsulfoxidc and an enzyme system (liver postmitochondrial supernatant fraction corresponding to about 4 mg protein. 1.7 mg NADP+, 1 mg glucose-6-phosphate in 0.5 ml 100 mM phosphate buffer pH 7.4, 8 mM MgClj, 33 mM K.C1) and 1--2 x 10* bacteria (Salmonella iyphimurium TA 98) were mixed with 2 ml of top agar (0.55% NaCl, 0.55% agar. 50 uM biotin, 50 uM histidine) and poured onto a Petri dish with minimal agar. After 48 h at 37 C, the his* revertant colonies were counted. Hatched bars represent the mutagenicity in the presence of the postmitochondrial supernatant fraction from un treated male rats, open bars from rats that had been pretreated intraperitoneally with 80 mg/kg pheno barbital in 0.9% NaCl on the 4th, 3rd, and 2nd day before sacrifice. Values represent means of 2 incubations erable increase in epoxide hydratase. Both factors would be expected to lead to a diminished mutagenicity of benzo(a)pyrene activated by microsomes from the phe nobarbital treated (Fig. 4) as compared to 3-methylcholanthrene treated SpragueDawley rats (Fig. 3) which is borne out by the data shown in the figures. Generalization of the Link between Enzyme Patterns and Mutagenic Effects. -The data described so far show that species differences in activating and inactivating enzymes will drastically influence the mutagenicity mediated by liver preparations from these species. All data, except those depicted in Figure 4, were obtained using S. typhimurium TA 1537 as detector strain, microsomes as metabolically activating system, Sprague-Dawley rats and C3H mice as source of the liver microsomes, styrene oxide as substrate for epoxide hydratase and benzo(a)pyrcne as substrate for monooxygenase. To investigate the generality of the observed phenomena, the fol lowing experiments (Fig. 4 and 5) were performed with S. typhimurium TA 98 as detector strain, liver post-mitochondrial supernatant as metabolically activating sys tem, Fischer rats and CF-1 mice as source of the liver post-mitochondrial superna tant, benzo(a)pyrene 4,5-oxide as substrate for epoxide hydratase and 7-ethoxycoumarin as substrate for monooxygenase. Moreover, three diagnostic inhibitors (metyrapone, 7,8-benzoflavone and tetrahydrofuran) (Ullrich et al., 1975) were used as a crude measure of the different monooxygenase forms present. Figure 4 shows that S. typhimurium TA 98 was reverted by benzo(a)pyrene metabolically activated by liver post-mitochondrial supernatant from untreated Sprague-Dawley rats more than TA 1537 (Fig. 1) by liver microsomes from the SL 066179 Species Differences in Drug Metabolizing Enzymes Related to Mutagenicity Txbl* 4. Effect of pbenobarbilal (raiment on microsomal epoxide hydratase (nmoles 4<5-dihydrtnty-4>5-dihydrobenzo(a)pyrene/mln/mg protein) and monooxy genase activities (pmoles 7-OH-coumarin/min/mg protein) in the adult male Spragoe-Dawiey rata, Fischer rati and CF-1 mice which were used in the mutageni city experiment shown in Figures 4 and 5 Animal Epoxide Monooxygenase* in presence of modifier Species and strain Pretreatment* None (control) Metyrapone 10-' M 7,0-Benzoflavone 2 10-' M Tetrahydrofuran lO-'M SpragueDawley rat Fischer rat CF-I mouse None Phenobarbital None Phenobarbital None Phenobarbital 7.4 + 0.3 13.3 0.7 3.2 0.2 10.7 0.5 17 i 0.3 2.3 0.1 372 1 40 (100%) 1210 66 (100%) 414 + 12(100%) | 16401 200 (100%) 900 40(100%) 24101 110 (100%) 344141 (93%) 593 1 52 (49%) 369 1 50 (90%) 623141 (38%) 693 1 44 (77%) 988 1 58 (4)%) 451 1 62 (122%) 13101 HO (108%) 504 + 65 (123%) 18701 210 (114%) 855 62 ( 95%) 2220 160 ( 92%) 104 11 (2B%) 448 36 (37%) 123 + 16 (30%) 1017 + 82 (62%) 423 1 21 (47%) 1010+ 88 (42%) * Phenobarbetal treated animals received intraperitoneal injections of phenobarbetal (80 mg/kg body weight) in 0.9% NaCI on the 4th, 3rd. and 2nd days before sacrifice 11 Values represent means + S.E.M. of 4 incubations (duplicate determinations at 2 different protein concentrations) ofot> 106 F. Oesch tt al. i REVERTANT j COLONIES ! ITA 98) 300 200 FISCHER RAT 100 n ! 11 f] , | __ .fl B 1 0 2 4 8 16 32 BENZ0IA1PYRENE l>jg/plot*l Fig. 5- Activation of benio(a)pyrene by Ever postmhochondrial fraction of phenobarbital treated rats and mice. Benzo(a)pyrene was activated to mutagenic metabolites by Ever postmitochondria] superna tant fraction of phenobarbital treated male Fischer rats and CF-1 mice as described in Figure 4. The open bars represent the mutagenicity when the epoxide hydratase inhibitor 1,1,1-trichloropopene 2.3oxide was added to the top agar at a concentration of 1 mM. The hatched ban show the mutagenicity in the absence of the inhibitor. Values represent means of 2 incubations same strain (maximally 3-fold as compared to 1.7-fold with respect to the sponta neous rate). When the same metabolic activation was performed with liver postmitochondrial supernatant from Fischer rats, the mutation rate was considerably higher compared to Sprague-Dawley rats (9-fold as compared to 3-fold with respect to the spontaneous rate). Table 4 shows that the "overall"' monooxygenase activity (with 7-ethoxycoumarin as substrate) as well as the contributions of the various monooxygenase forms are similar for the two strains. Only the epoxide hydratase is markedly lower in Fischer rats as compared to Sprague-Dawley rats. The higher accumulation of mutagenically active metabolites from benzo(a)pyrene by the liver preparation from Fischer rats is in line with their lower epoxide hydratase activity. Phenobarbital leads to a similar induction of "overall" monooxygenasc activity in the two strains and the contribution of the various monooxygenase forms is not drastically different between the two strains after phenobarbital-induction. Interest ingly. epoxide hydratase is increased more in the strain with the lower constitutive activity than in the other strain, leading to similar epoxide hydratase activities in the two strains after phenobarbital treatment. Accordingly, the differences in mutageni city also virtually disappear. Table 4 shows that the differences in enzyme activities are much less between Fischer rats and CF-1 mice after phenobarbital induction than was the case between untreated Sprague-Dawley rats and C3H mice (Table 1). Accordingly, the differ ences in mutagenicity are also much less (compare Fig. 5 with Fig. 1). If, in addition, epoxide hydratase activity is inhibited by 1,1,1-trichloropropene oxide, the rather small difference in monooxygenase activity which persits is now accompanied by an accordingly smaller difference in mutagenicity. In conlusion, differences between species and strains in enzymes activating com pounds to electrophilically reactive metabolites and in enzymes inactivating such metabolites, invariably lead to differences in potential of tissue preparations from such species or strains to mediate mutagenic effects of compounds which are sub- SL 066181 Species Differences in Drug Metabolizing Enzymes Related to Mutagenicity 107 strates of these enzymes. For lipophilic compounds, whose active forms are metabolically produced epoxides which are substrates for epoxide hydratase, liver prepa rations of several mouse strains (e.g. C3H, CF-1) will lead to an accumulation of such epoxides to an extent much higher than many other mammalian species due to high monooxygenase and to very low epoxide hydratase activities (Oesch, 1973). In some special cases where dihydrodiol-epoxides of an especially high reactivity due to location of the epoxide ring at a bay region can be formed (Lehr et al., 1977) epoxide hydratase will have both activating and inactivating properties (Bentley et al., 1977). However, with the majority of aromatic and olefinic compounds of industrial intrest it is to be expected that epoxide hydratase plays a simple inactivating role. Since man has a much higher epoxide hydratase activity (Oesch et aL, 1974) than any of the many investigated mice strains (Oesch et al., 1973) it is to be expected that mice ^pre much more susceptible to toxic effects which are mediated by metabolically produced epoxides which are substrates for epoxide hydratase, at least for com pounds large and lipophilic enough to preclude efficient inactivation by glutathione (Giatl and Oesch, 1977). Whether a metabolically produced epoxide will be a subgfttte for epoxide hydratase can also be predicted for several structural features, nee-structure-activity relationships have been studied for rodent and human hepatie-iflpode hydratase (Oesch, 1974). & "Adknmledgemenis. This wort was supported by the Stiftung Volkswagenwerk. We thank Miss i&fiooc Schweizer for excellent technical assistance and Mr. A. J. Sparrow for synthesis of [JH|benzo(a)pyrene 4,5-oxide. References Alvares, A. P~ Bickers, D. R, Kappas, A.: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc. oat Acad. Sci. (Wash.) 70, 1321--132S (1973) Ames, B. N., Durston, W. E., Yamasaki, E, Lee. F. D.: Carcinogens are mutagens: A simple test system combining fiver homogenates for activation and bacteria for detection. Proc. nat Acad. Sci. (Wash.) 70, 2281-2285 (1973) - Anna, B. 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