Microsomes, Drug Oxidations and Chemical Carcinogenesis V2

Microsomes, Drug Oxidations, and Chemical Carcinogenesis, Volume II, documents the proceedings of the 4th International Symposium on Microsomes and Drug Oxidations held in Ann Arbor, July 1979. The symposium reviewed progress in the understanding of scientific and biomedical problems from a biochemical, biophysical, pharmacological, and toxicological perspective. Volume I contained 117 contributions made by researchers at the symposium, which were organized into three sections (Sections I-III). This second volume contains 122 contributions, divided into four sections (Sections IV-VII). The papers on Section IV examine the metabolic fate of oxygenated compounds. Section V provides studies on microsomal enzymes and lipid metabolism. Section VI includes papers on microsomal enzymes and toxicity of foreign compounds. Section VII covers microsomal enzymes and chemical mutagenesis and carcinogenesis. This book seeks to aid future progress in understanding the complexities of metabolic transformations by these versatile enzyme systems that act on physiologically important lipids as well as on a wide array of foreign substances, including drugs, anesthetics, industrial chemicals, food additives, pesticides, carcinogens, and nonnutrient dietary chemicals.

• Language: English
• Publisher: Academic Press
• Release date: Dec 2, 2012
• ISBN: 9780323143875

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SECTION IV

METABOLIC FATE OF OXYGENATED COMPOUNDS

RATE-LIMITING FACTORS IN XENOBIOTIC METABOLISM BY CYTOCHROME P-450, SULPHOTRANSFERASE, AND GLUCURONYL TRANSFERASE

J.W. Bridges, P. Wiebkin and J.R. Fry,     Institute of Industrial and Environmental Health & Safety and Department of Biochemistry, University of Surrey Guildford Surrey U.K.

The factors determining the formation and subsequent conjugation of phenols are discussed using data obtained from freshly isolated adult rat hepatocytes with biphenyl or simple phenols as substrates. It is shown that tight coupling between the endoplasmic reticulum enzymes P-450 and glucuronyl transferase probably does not occur. Investigations on rate of substrate uptake onto P-450, metabolite excretion, enzyme activity and cofactor levels has led to the conclusion that the rate limiting factor in drug metabolism by normal hepatocytes is either the intrinsic activity of the individual enzymes or their activity after substrate activation.

I. INTRODUCTION

Freshly isolated and cultured intact cell preparations are gaining widespread acceptance as models for investigating various facets of drug metabolism and toxicity (1,2). In the present paper, studies on the metabolism of biphenyl by freshly isolated, viable, adult rat hepatocytes are used in an attempt to answer the following important questions:-

-is it likely that in vivo, substrates gain rapid access to cytochrome P-450?

-is P-450 mediated oxidation coupled to or largely independent of the activities of the conjugating enzymes?

-are cofactor levels or the inherent activities of individual enzymes the primary controlling influence on drug metabolism rates?

II. METHODS

Hepatocytes were isolated from male Wistar rats (80-120g) according to the method of Fry et al (3). Metabolism studies followed the protocol of Wiebkin et al (4). ATP and NADPH were measured according to the methods of Adam (5) and Klingenberg (6) respectively.

III. RESULTS AND DISCUSSIONS

When lipophilic substrates such as biphenyl, barbiturates or aliphatic carbamates are added to suspensions of hepatocytes difference spectra measurements indicate that maximal P-450 type I binding spectra appear within seconds, the spectra then persisting for several minutes (7,8). This suggests that the plasma membrane does not constitute a significant barrier to interaction of P-450 with lipophilic xenobiotics and that reduction of the P-450 substrate complex (or a later stage in the P-450 cycle) is rate limiting for substrate metabolism. The magnitude of the Ks values for the P-450 type I spectra in hepatocytes is similar generally to that obtained in hepatic microsomes, indicating that endogenous lipids probably do not compete significantly with xenobiotics for P-450 binding sites.

The interaction of xenobiotics with P-450 is followed typically by the rapid appearance of metabolites in the suspending medium. For example when biphenyl is used as substrate (70μM, a concentration which has no significant effect on cell viability) and hepatocytes from control, phenobarbitone or 20-methylcholanthrene pretreated adult rats are employed, unconjugated 4-hydroxybiphenyl and 4-hydroxybiphenyl sulphate are found in the medium in significant amounts within one minute (3). 4-Hydroxybiphenyl glucuronide formation shows a lag phase before reaching maximal rates after about 5 min. (table 1). After one hour according to the state of induction of the hepatocytes between 3 and 18.3% of biphenyl is converted to metabolites. There appears to be no significant accumulation of any metabolites in the cell apart from very low levels of apparently covalently bound material. (N.B. For benzpyrene some differential retention of metabolites does occur (9)). The levels of free 4-hydroxybiphenyl begin to decrease after about 10 min. falling to zero within one hour, presumably because the 4-hydroxybiphenyl is readily able to re-enter the cells and become conjugated. This pulse of free 4-hydroxybiphenyl leaving the cell in the early phase of incubation may be ascribable to the failure of glucuronyl transferase at early time points to reach maximal activity. The fact that 4-hydroxybiphenyl can be synthesised and rapidly pass out of the cell in an unconjugated form suggests that, initially at least, there is no tight coupling of P-450 with either glucuronyl transferase or sulphatransferase.

TABLE 1

Metabolism of biphenyl (70µM) by isolated hepatocytes from phenobarbitone pretreated rats

Studies with metabolic inhibitors (table 2) tend to support this view (10). Triethyltin sulphate (1 × 10−5M) which is not a substrate for the conjugation enzymes, increases the amount of unconjugated 4-hydroxybiphenyl being excreted into the medium. In both control hepatocytes and those from phenobarbitone pretreated rats 2,4 Dinitrophenol (2 × 10−4M) also dramatically elevates the excretion of free 4-hydroxybiphenyl. It is noticeable that the overall rate of hydroxylation of biphenyl is not changed in hepatocytes from phenobarbitone pretreated rats by the addition of 2,4-Dinitrophenol while in hepatocytes from control animals 2,4-Dinitrophenol actually boosts the overall rate of metabolism.

TABLE 2a

Effects of inhibitors on phase I and phase II metabolism of biphenyl by hepatocytes from phenobarbitone pretreated rats

TABLE 2b

Effects of inhibitors on phase I and phase II metabolism of biphenyl by hepatocytes from phenobarbitone pretreated rats

*nmoles product / 2×10⁶ cells

When 4-hydroxy- or 2-hydroxy-biphenyl are used as substrates instead of biphenyl the ratio of sulphate to glucuronic acid conjugation of these phenols and the rates of formation of these conjugates is very similar to that observed when biphenyl is employed as substrate. (See table 3). Since sulphotransferase is a cytoplasmic enzyme whereas glucuronyl transferase is thought to be buried in the endoplasmic reticulum it is reasonable to conclude that these phenols once formed by cytochrome P-450 are able to partition freely within the hepatocyte in a very similar manner to the situation which occurs when these phenols are added to the exterior of the cells. The above findings taken overall would strongly suggest that there is no tight coupling of the phase I and phase II reactions for biphenyl metabolism, at least at the early time periods after the addition of substrate. The lag phase before maximal glucuronidation rate is achieved may be due to the activation of latent glucuronyl transferase by the phenols being formed by P-450. Glucuronyl transferase is well established to exist partially in a latent form and to be readily activated by a range of chemicals. Evidence from preincubation studies of substrate (4) with hepatocytes indicates that substrate activation of glucuronyl transferase is likely. If this is the case then although tight coupling of P-450 and glucuronyl transferase may not occur the expression of glucuronyl transferase activity would be at least partially dependent on P-450 activity.

TABLE 3

Relative rates of sulphation and glucuronidation of various phenols (35µM) by isolated adult rat hepatocytes

*nmoles / 2×10⁶ cells / 45 min. in medium

The question arises as to what determines the ratio of glucuronic acid to sulphate conjugates. In view of the fact that cytochrome P-450 and glucuronyl transferase are both located in a lipoid environment whereas sulphotransferase is found in the aqueous cytosol one might speculate that the more lipophilic the phenol formed by P-450 the greater the likelihood that it will form a glucuronic acid rather than a sulphate conjugate. The data presented in table 3 indicates that this is probably not the case, thus the ratio of glucuronide to sulphate for the highly lipophilic 3-hydroxybenzpyrene and for the far more water soluble 7-hydroxycoumarin are very similar. 4-Hydroxybiphenyl which is intermediate in partition coefficient between these two substrates also displays a similar conjugate ratio. A clue to a more important determinant of conjugation is obtained by comparing the conjugation ratios of 2- and 4-hydroxybiphenyls. It would appear that the main cause of the very marked difference in conjugation ratio between these substrates is that 2-hydroxy-biphenyl is a very much poorer substrate for sulphotransferase than is 4-hydroxybiphenyl (Table 4). (N.B. Since these results were obtained in the same hepatocyte preparation, availability of cofactor cannot be responsible for these differences) A similar observation with these two phenols has been made in hepatocytes from other species and also in intestinal cells from rats and guinea pigs.

TABLE 4

Maximal rates of individual reactions involved in biphenyl metabolism (70μM) by isolated adult rat hepatocytes

Interestingly harmalol has been shown to be far less well sulphated than the closely related structure harmol in rat hepatocytes (11). Sulphotransferase(s) would appear to be much more selective in their substrate requirements than glucuronyltransferase(s). Thus steric features of the phenols rather than their lipophilicity would appear to be the primary influence on the glucuronidation sulphate ratio.

Thus the drug metabolism enzymes are not coupled, substrate access appears not to be rate limiting, and lack of product accumulation in the cells suggests that excretion of metabolites is also not rate limiting. Then what is the rate determining factor in metabolism in normal hepatocytes, co-factor availability or intrinsic activity of the enzymes themselves? Addition of dinitrophenol which is a potent uncoupler of oxidative phosphorylation to rat hepatocytes (table 2) causes a pronounced reduction in ATP and NADPH levels. Despite this reduction in cofactor levels the rate of P-450 mediated hydroxylation is actually enhanced, indicating that the cofactor level is probably not rate determining for the expression of P-450 activity unless very severe reduction in cofactor levels occur. It would appear that only in situations where very high levels of P-450 activity occur (eg after maximal induction with phenobarbitone) and unusually low levels of NADPH are present is it likely that NADPH levels will become rate limiting. The situation regarding the effects of depletion of PAPS and UDPGA on sulphotransferase(s) and glucuronyl transferase(s) activity has not been determined directly. The much more marked inhibition by mitochondrial inhibitors of conjugation reactions than P-450 mediated metabolism (table 1) suggests that the conjugations carried out by these enzymes are probably more critically dependant on cofactor levels than are the phase I reactions. However altering the concentrations of glucose or inorganic sulphate in the medium, factors which are known to change fairly rapidly the levels of UDPGA and PAPS in hepatocytes, has no significant effect on the rates of conjugation or on the ratios of conjugates implying that in a normal hepatocyte the level of cofactor is not rate limiting but that it may become so if the supply of cofactor is temporarily interrupted. As 2- or 4-hydroxybiphenyl is added in increasing concentration to hepatocytes the level of sulphate conjugates tends to plateau whereas the levels of glucuronides continue to increase (4). A similar phenomenon has been observed with many substrates. This increasing glucuronide to sulphate ratio with increasing substrate concentration has been commonly ascribed to the PAPS levels becoming rate limiting. However addition of high levels of inorganic sulphate to hepatocytes, which is a well established way of boosting rapidly PAPS levels, does not modify the glucuronide to sulphate ratios at any dose level of phenols. Thus the likely explanation is that as the dose of 4-hydroxybiphenyl is increased the sulphotransferase(s) responsible for its conjugation becomes saturated with substrate whereas the glucuronyl transferase(s) require very much higher levels of substrate in order to become saturated.

It may be concluded that in normal hepatocytes the intrinsic activities of the individual drug metabolising enzymes and/or the ease with which they can be activated/inhibited by the addition of substrate is probably the principal rate limiting factor in drug metabolism.

REFERENCES

1. Fry, J.R., Bridges, J.W.Bridges J.W., Chasseaud L.F., eds. Progress in Drug Metabolism. J. Wiley: Chichester, 1977:71. [2].

2. Fry, J.R., Bridges, J.W.Hodgson E., Philpot R., eds. Reviews in Biochemical Toxicology. Elsevier: N. Holland, 1979:201. [1].

3. Fry, J.R., Bellemann, P., Jones, C.A., Wiebkin, P., Bridges, J.W. Analyt. Biochem. 1976; 71:341.

4. Wiebkin, P., Fry, J.R., Jones, C.A., Lowing, R.K., Bridges, J.W. Biochem. Pharmacol. 1978; 27:1899.

5. Adam, H.Bergmeyer H.U., ed. Methods in Enzymatic Analysis. 1st Edn. Academic Press, 1962:539.

6. Klingenberg, M.Bergmeyer H.U., ed. Methods in Enzymatic Analysis. 2nd Edn. Academic Press, 1971:2045.

7. Moldeus, P., Grandin, R., Von Bahr, Orrenius, S. Biochem. Biophys Res. Commun. 1973; 55:937.

8. Bridges, J. W., Sweatman, B., Sargent, N., and Upshall, D. unpublished data.

9. Jones, C.A., Moore, B.P., Cohen, G.M., Fry, J.R., Bridges, J.W. Biochem. Pharmacol. 1978; 27:693.

10. Wiebkin, P., Parker, G. L., Fry, J. R., and Bridges, J. W., Biochem. Pharmacol., in the press.

11. Mulder, G.J., Hagedoorn, A.H. Biochem. Pharmacol. 1974; 23:2101.

DISCUSSION

Dr. Coscia: Can dinitrophenol inhibit glucuronidation and sulfation by uncoupling electron transport? This could lead to a deficiency in PAPS and UDP-glucuronide.

Dr. Bridges: 2,4-Dinitrophenol lowers PAPS and UDPGA levels in the hepatocytes and this we feel is the major excuse of the large reduction it produces in the conjugation of hydroxybiphenyls. There are two reasons for this, namely an effort on cofactor production, probably due to uncoupling, and an increased utilization due to 2,4-DNP serving as a substrate for the sulfating and glucuronidating enzymes. Interestingly, other compounds which interfere with mitochondrial function but are not substrates for the conjugating enzymes, e.g. triethyltin, also have a greater effect on biphenyl conjugation than its hydroxylation.

Dr. Coscia: Does the glucuronyl transferase which you are studying also conjugate catechols and terpenes?

Dr. Bridges: I do not know. I would expect glucuronyl transferase to be a family of enzymes with overlapping substrate specificities (in common with many other drug-metabolizing enzymes). It is therefore likely that biphenyl will compete at least to some extent with substrates like catechol.

Dr. Felton: What is the role of β-glucuronidase as these conjugated products move through the microsomal lumen?

Dr. Bridges: It is only speculation on my part that the higher molecular weight glucuronides might pass down the lumen of the endoplasmic reticulum into bile whereas the lower molecular weight conjugates might pass via a different route into blood. The role of cellular β-glucuronidase in hydrolysing glucuronides has been a subject of discussion for many years. All I can add is that we can find very little evidence for the cleavage of glucuronides when they are added to normal hepatocytes. Presumably this is because the β-glucuronidase is compartmentalized or endogenous inhibitors are present. In many diseased or damaged cells cleavage of glucuronides does appear to occur to some extent.

Dr. Horwitz: Have you considered the possibility of the phenomenon of stacking between PAPS and the biphenyl substrate to explain the steric effect advanced to explain the differences in sulfurylation of 2- and 4-hydroxybiphenyl?

Dr. Bridges: No, it is a very interesting suggestion.

Dr. Ames: The glutathione transferases make up about 10% of liver proteins. Is it known whether the sulfate and glucuronide transferases are an appreciable percent of liver protein?

Dr. Bridges: The contribution of the conjugation enzymes I was discussing, glucuronyltransferase and sulphatransferase, to the total protein content of the cells is rather small.

EPOXIDE HYDRATASE

¹

F. Oesch,     Institute of Pharmacology, University of Mainz, D-6500 Mainz, West-Germany

Publisher Summary

This chapter discusses the recent aspects of epoxide hydratase—epoxide hydrase, EH. EH catalyzes the transformation of epoxides to dihydrodiols. Epoxides are metabolically formed from many foreign compounds possessing olefinic or aromatic moieties. Epoxides are electrophilically reactive to varying degrees and can, therefore, irreversibly bind to nucleophilic moieties of cellular macromolecules, thereby, disturbing the normal biochemistry of a cell. This can lead to cell death, mutation, or transformation to a cancer cell. The dihydrodiols produced by the action of EH can sometimes be further epoxidized at other parts of the molecule. In some cases, the resulting dihydrodiol epoxides are of special reactivity, and high mutagenic and carcinogenic activity. Thus, by inactivating monofunctional epoxides and providing precursor molecules for dihydrodiol epoxide biosynthesis, EH plays an important role in chemical carcinogenicity and other toxicities.

I. INTRODUCTION

Epoxide hydratase (epoxide hydrase, EH) catalyzes the transformation of epoxides to dihydrodiols (1). Epoxides are metabolically formed from many foreign compounds possessing olefinic or aromatic moieties. Epoxides are electrophilically reactive to varying degrees and can therefore irreversibly bind to nucleophilic moieties of cellular macromolecules thereby disturbing the normal biochemistry of a cell. This can lead to cell death, mutation or transformation to a cancer cell. The dihydrodiols produced by the action of EH can sometimes be further epoxidized at other parts of the molecule. In some cases, the resulting dihydrodiol epoxides are of special reactivity and high mutagenic and carcinogenic activity (2). Thus by inactivating monofunctional epoxides and providing precursor molecules for dihydrodiol epoxide biosynthesis, EH plays an important role in chemical carcinogenicity and other toxicities. Since a recent review (3) on EH is available, only more recent aspects not covered there will be dealt with here.

II. THE EFFECT OF TRANS-STILBENE OXIDE

Recently we reported that trans-stilbene oxide (TSO) selectively induces EH (4). At doses of TSO leading to maximal induction of EH, some monooxygenase parameters such as aryl hydrocarbon hydroxylase (assayed according to Nebert and Gelboin (5)) were never increased and some others such as aminopyrine N-demethylase and cytochrome P-450 content were increased so slightly that statisical significance was never reached (4). Since so many monooxygenases with differing substrate preferences exist, these results, obtained with only five monooxygenase parameters, did not justify the conclusion that EH induction was specific. Thus we termed it selective (4).

In the mean time we discovered that monooxygenase activity toward a further substrate, 7-ethoxycoumarin, was indeed markedly increased, almost as much as EH (6). Also some other laboratories observed increases in some monooxygenase parameters after TSO treatment (7,8). Thus TSO may induce some monooxygenase forms with high preference. In large molecules such as benzo(a)pyrene (BP) this could lead to a marked shift in the sites of the molecule which are preferentially attacked by these monooxygenases.

Therefore [¹⁴C]benzo(a)pyrene was incubated with control and with TSO-induced rat liver microsomes and the metabolites were separated by HPLC. Standard compounds and two different elution systems were used to characterize the metabolites. The results presented in Table 1 are derived from a separation using an aceonitrile-water gradient. Quantitatively similar results were obtained using a methanol-water gradient with the exception that the 4,5-epoxide peak was separated from the 3,6-quinone peak when the acetonitrile-water but not when the methanol-water gradient was used. Furthermore it was confirmed that metabolites which were (from their mobilities) tentatively identified as dihydrodiols disappeared or were markedly reduced in parallel incubations where the epoxide hydratase inhibitor TCPO (9) was present. By these means all of the major metabolite peaks could be characterized. Using [¹⁴C] labelled BP as substrate these peaks were quantitated. The total of the metabolites was not significantly increased after TSO-treatment of the rats. However a most remarkable shift of the metabolism was observed (Table 1). The quantity of metabolites which were oxidized at the benzo ring (7,8-dihydroxy-7,8-dihydro-BP, 9,10-dihydroxy-9,10-dihydro-BP; 9-hydroxy-BP) was drastically decreased, and far more K-region metabolites (4,5-dihydroxy-4,5-dihydro-BP; BP 4,5-oxide) were formed. Compared to controls the ratio between 7,8-dihydroxy-7,8-dihydro-BP and 4,5-dihydroxy-4,5-dihydro-BP was more than 20 times lower with TSO-induced microsomes. As expected from the induction of EH, a much higher percentage of the metabolically produced BP 4,5-oxide was converted to the corresponding dihydrodiol with TSO-induced microsomes in comparison to control microsomes. Only insignificant changes were observed in the peaks containing the quinones and 3-hydroxy-BP.

TABLE I

Effect of TSO Administration on Rat Liver Microsomal Metabolism of BPa

aTrans-stilbene oxide (2 mmoles per kg body weight dissolved in 0.5 ml sunflower oil) was given i.p. to male Sprague-Dawley rats (210-230 g) 72, 48 and 24 h before sacrifice. Controls received sunflower oil only. [¹⁴C]BP, NADPH, NADH and microsomes were incubated at 37°C for 20 min. Ethyl acetate/acetone (2:1 v/v)-extractable metabolites were separated by HPLC with a linear acetonitrile/water gradient. Values represent means + S.D. from 3 (with TSO-treated) or 2 (with control animals) independent experiments and are expressed (1) as the total amount present after the incubation time, corrected for small (range of less than + 10% of the mean) variations in protein content and (2) as percentage of the radioactivity of all but the BP peak. For each experiment organs from two animals were pooled.

bRatio of metabolites after 20 min incubation with microsomes from TSO-treated and control rats.

It was of special interest to investigate how the shift of metabolism which is caused by TSO would affect the mutagenicity of BP, since BP can be activated both at the benzo ring and at the 4,5-K-region to highly mutagenic metabolites; at the benzo ring mainly to dihydrodiol epoxides, at the K-region to the 4,5-oxide (10–12). For this purpose we activated various doses of BP by microsomal or postmitochondrial fractions to mutagens which were detected by the reversion of various his Salmonella typhimurium strains. Induction by TSO decreased the mutagenicity of BP in all instances. Some differences were observed between the two tissue fractions and the different bacterial strains. The greatest reduction of the mutagenic effect (by more than 90%) was obtained when postmitochondrial fraction and the strain TA 100 were used. However, TSO treatment only slightly reduced the mutagenicity with TA 1537. The Salmonella assay is a backward mutation assay which requires specific mutations to reconstruct a functional his gene. Different his strains vary in their susceptibility to reversion by different BP metabolites (11,12). TA 100 and TA 98 are easily reverted by BP 7,8-dihydrodiol 9,10-oxides and by BP 4,5-oxide. The strain TA 1537 is less sensitive towards the 7,8-dihydrodiol 9,10-oxides but is highly sensitive to the 4,5-oxide (12). The greater decrease of the mutagenicity by TSO induction with the strains which are sensitive to 7,8-dihydrodiol 9,10-oxides (TA 100, TA 98) when compared with a strain which is relatively insensitive towards these dihydrodiol epoxides (TA 1537) suggests that the reduction of the mutagenicity is caused to a significant extent by the decreased oxidation of BP at the benzo ring. The decreased benzo ring metabolism is accompanied by increased K-region metabolism, leading to the mutagenic BP 4,5-oxide, but this epoxide is a good substrate of EH (11,13), which is induced by TSO. Thus, the two effects of TSO, shift of the site of metabolic oxidation and induction of EH synergistically provide protection against the mutagenic effects of BP.

These remarkable changes in the BP metabolite pattern and the consequent profound changes in mutagenic effects occur without any significant alteration of the BP monooxygenase activity determined by the widely used assays which measure the fluorescence intensity of the phenolic BP metabolites extracted into alkali (5) (the so-called aryl hydrocarbon hydroxylase activity) or tritium release from [3H]BP (14). Attempts to correlate susceptibility to polycyclic hydrocarbon-mediated toxic effects with aryl hydrocarbon hydroxylase activity are based on the premise that the enzyme activity measured by this assay gives a sufficiently complete indication of the metabolism to toxic BP derivatives. As is evident from the above, this is not always so, and misleading results may be expected when it does not apply.

III. ENDOGENOUS ROLE OF EPOXIDE HYDRATASE

If selective or eventually perhaps specific induction of EH is to be useful as a tool to investigate the exact role of this enzyme in the complicated situation of the whole organism, it should be known what changes in the normal homeostasis of the body would be expected if EH activity is changed. Therefore studies on the endogenous role of EH were initiated. Since microsomal metabolism of the naturally occurring C18-unsaturated estratetraenol and of the corresponding α-epoxide to the naturally occurring trans-diol was known (15) it was investigated whether the same enzyme which converts epoxides derived from carcinogenic environmental compounds is also involved in this endogenous metabolism.

A highly sensitive and rapid radiometric assay for the determination of specific EH activity with the steroid epoxide 16α, 17α-epoxy-1,3,5(10)-estratrien-3-ol (estroxide) has been developed (16).

The EH activity with estroxide and BP as substrates in various organs of male and female rats are shown in Table II.

TABLE II

Estroxide and BP 4,5-Oxide Hydratase Activities in Various Organs of the Pata

aThe organs were taken from Sprague Dawley rats (200-250 g). n = Number of experiments. Duplicate determinations at two different protein concentrations were performed. Values represent means + S.D.

In the male rat the specific activity was second highest (after liver) in testes with both substrates. Likewise in the female rat, specific activity was second highest in the ovary. In both sexes the specific activity in kidney was nearly as high as in the gonads, whilst the specific activities in lung and in the second steroidogenic gland, the adrenal, were lower. The specific activities in the male liver and testis were higher than those of the female liver and ovary, whereas the specific activities in lung and adrenal were similar in both sexes. The ratio of the activities measured with the two substrates was about 2 and was remarkably similar (1.6–2.2) in the ten different organs investigated. These results indicate that either the same enzyme or enzymes which are under common control are responsible for the hydration of both substrates, at least in the organs tested.

Immunological studies were performed to confirm the supposition that steroid epoxides and epoxides derived from polycyclic aromatic hydrocarbons are hydrated by the same enzyme. Antiserum raised against apparently homogeneous EH was used. This antiserum was monospecific as judged from the single precipitation line seen after immuno-diffusion. The antiserum did not inhibit EH activity towards any of the tested substrates when incubated with microsomal suspensions. Thus immunoprecipitation was performed using solubilised microsomes. In contrast to five non-ionic detergents tested, the steroidal surfactants sodium cholate and sodium desoxycholate both solubilised the activity towards estroxide with only small losses of activity. Thus when microsomal solutions containing 1% cholate or desoxycholate were centrifuged at 100,000 × g for 1 hr the EH activity of the supernatant fraction was always more than 85% of that of the untreated microsomal fractions whether measured with estroxide, BP 4,5-oxide or styrene oxide as substrate. Consequently the immunoprecipitation studies were performed with cholate-solubilised microsomal fractions. As shown in Fig. 1, the enzyme activity towards all three substrates is precipitated simultaneously as a function of the amount of antiserum. The precipitation of activity towards all three substrates was complete at high antiserum concentrations. The immunoprecipitation studies, therefore, suggest that the hydration of all 3 substrates is catalysed by the same enzyme. This supports the results obtained by comparison of the activities towards estroxide and BP 4,5-oxide in the various organs reported above.

FIGURE 1 ] and styrene oxide [Π] as substrates. Controls contained preimmune serum. At least two determinations at two different protein concentrations were performed in each case. Values represent means. Deviations from means were always less than 5%.

This and further evidence suggests that the same enzyme which catalyzes the hydration of epoxides derived from carcinogenic polycyclic hydrocarbons also functions in the metabolism of endogenous compounds, at least in the metabolism of the endogenous estrogen which was investigated.

ACKNOWLEDGMENT

The author wishes to thank his collaborators for their work quoted in this paper.

REFERENCES

1. Oesch, F. Xenobiotica. 1973; 3:305.

2. Jerina, D.M., Lehr, R., Schaefer-Ridder, M., Yagi, H., Karle, J.M., Thakker, D.R., Wood, A.W., Lu, A.Y.H., Ryan, D., West, S., Levin, W., Conney, A.H.Hiatt H.H., Watson J.D., Winsten J.A., eds. Origins of Human Cancer. Cold Spring Harbor Laboratory:, 1977; 639.

3. Oesch, F., Epoxide hydrataseBridges, J.W., Chasseaud, L.F., eds. Progress in Drug Metabolism; 3. John Wiley, Chichester, England, 1979:253.

4. Schmassmann, H.U., Oesch, F. Mol. Pharmacol. 1978; 14:834.

5. Nebert, D.W., Gelboin, H.V. J. Biol. Chem. 1968; 243:6242.

6. Bücker, M., Golan, M., Schmassmann, H. U., Glatt, H. R., Stasiecki, P., and Oesch, F., Mol. Pharmacol., in press.

7. Mukhtar, H., Elmamlouk, T.H., Bend, J.R. Chem.-Biol. Interactions. 1978; 22:125.

8. Seidegard, J., Morgenstern, R., DePierre, J. W., and Ernster, L., Biochim. Biophys. Acta, in press.

9. Oesch, F., Kaubisch, N., Jerina, D.M., Daly, J. Biochemistry. 1971; 10:4858.

10. Glatt, H.R., Oesch, F., Frigerio, A., Garattini, S. Int. J. Cancer. 1975; 16:787.

11. Wood, A.W., Levin, W., Lu, A.Y.H., Yagi, H., Hernandez, O., Jerina, D.M., Conney, A.H. J. Biol. Chem. 1976; 251:4882.

12. Bentley, P., Oesch, F., Glatt, H.R. Arch. Toxicol. 1977; 39:65.

13. Bentley, P., Schmassmann, H.U., Sims, P., Oesch, F. Eur. J. Biochem. 1976; 69:97.

14. Hayakawa, T., Udenfriend, S. Anal. Biochem. 1973; 51:501.

15. Breuer, H., Knuppen, R. Biochim. Biophys. Acta. 1961; 49:620.

16. Bindel, U., Sparrow, A., Schmassmann, H.U., Golan, M., Bentley, P., Oesch, F. Eur. J. Biochem. 1979; 97:275.

DISCUSSION:

Dr. Mannervik: I don’t think that we should be left with the impression that the protection against mutagenic epoxides in vivo, which is induced by trans-stilbene oxide, should be ascribed entirely to epoxide hydratase. A major route of detoxification goes via glutathione conjugation (cf. presentation by Plummer and Bend) and the glutathione S-transferases are also induced by trans-stilbene oxide. Using substrate specificity and, more specifically, purification and quantitative immunological techniques, we have shown that each of the transferases A, B, and C¹ in rat liver cytosol are induced 3 to 4 fold after treatment of rats with this inducer. It is also significant that the cytoplasmic enzyme, glutathione reductase, is induced about 2-fold by trans-stilbene oxide.

Dr. Oesch: It is not possible that the protection against trans-stilbene oxide-induced bacterial mutagenicity of benzo(a)pyrene which I described in my talk is mediated by an induction of glutathione S-transferases because the experiment was performed under conditions where glutathione has no influence. The experiment has been performed with washed microsomes and the S9 fraction from which gluththione has been removed. So, in this particular instance glutathione transferase contribution was absent. But I have made clear in my talk that the trans-stilbene oxide influence is by no means specific. It is selective but not specific.

Dr. Kupfer: It is highly interesting that you observed an epoxidation of a Δ¹⁶-steroid and epoxide cleavage by epoxide hydratase. However, is there evidence for such Δ¹⁶-steroid as being an endogenous substrate forming 3,16ß, 17α-trihydroxyestratriene?

Dr. Oesch: Yes, it is a minor pathway but it does also occur in human, it has been proven both in plasma and in urine, and not only with respect to the estrogen series but also with respect to the androgen series. But in both cases it is a minor metabolite.

Dr. Kupfer: So actually the 17α, 16ß metabolite is a product, an endogenous form?

Dr. Oesch: Yes, this is an epi-estriol. It has the configuation at both the 16 and 17 carbon atoms opposite to that of the usual, major estriol.

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¹Supported by the Deutsche Forschungsgemeinschaft.

KINETIC STUDIES ON MECHANISM AND SUBSTRATE SPECIFICITY OF THE MICROSOMAL FLAVIN–CONTAINING MONOOXYGENASE

Daniel M. Ziegler, Lawrence L. Poulsen and Michael W. Duffel,     Clayton Foundation Biochemical Institute and Department of Chemistry, The University of Texas at Austin, Austin, Texas

Intermediate forms of the flavoprotein predicted from kinetic studies are detectable spectrophotometrically. The spectra suggest that the hydroxylating species is a relatively stable peroxyflavin · NADP+ intermediate and some parameters that affect interaction of nucleophiles with this form of the enzyme are described.

I. INTRODUCTION

The flavin-containing monooxygenase is one of the more abundant components of the liver endoplasmic reticulum. It accounts for 3 to 4 percent of the total microsomal protein in liver tissue from adult hogs (1). The concentration (activity) of this monooxygenase is also very high in human liver (2), but somewhat lesser amounts are present in hepatic tissue from rodents and other small mammals (3). In most species the monooxygenase is most concentrated in liver, but substantial amounts are also present in lung and kidney. The amount present in most other tissues is quite low, although a monooxygenase with catalytic and immunological properties similar to the liver enzyme is detectable in all nucleated mammalian cells.

The monooxygenase purified to homogeneity from hog liver catalyzes oxygenation of a variety of nucleophilic organic nitrogen and sulfur compounds (1, 4). The wide tissue distribution of the flavin-containing monooxygenase and its broad substrate specificity suggest that it plays a major role in oxidative metabolism of certain drugs and other foreign