Dual role of epoxide hydratase in both activation and inactivation of benzo(a)pyrene

The effect of epoxide hydratase upon the mutagenicity of benzo(a)pyrene was investigated using two Salmonella typhimurium strains (TA 1537 and TA 98). These two bacterial strains were found to differ characteristically in their susceptibility to different mutagens biologically produced from benzo(a)pyrene providing a diagnostic tool to investigate which types of mutagenic metabolites were produced in various metabolic situations. The results showed that the pattern of mutagenic metabolites produced by microsomes from methylcholanthrene-treated mice was very different from that produced by microsomes from phenobarbital-treated or untreated mice. However in all cases at least two mutagenic metabolites were produced. Epoxide hydratase was very efficient at reducing the mutagenic effect when benzo(a)pyrene was activated by microsomes from untreated or phenobarbital-treated mice. However, when microsomes from methylcholanthrene-treated mice were used the effect of hydratase depended upon the benzo(a)pyrene concentration. At low concentrations the mutagenicity was increased by addition of epoxide hydratase and decreased by inhibition of the hydratase. At high concentrations the reverse was true. These findings indicate that when microsomes from untreated and phenobarbital-treated mice were used the main contributors to the mutagenicity were simple epoxides (or compounds arising non-enzymically from them). The activation of dihydrodiols must, however, contribute to a significant extent when microsomes from methylcholanthrene-treated mice were used. Thus the role of epoxide hydratase was determined by the monooxygenase form present in the microsomes in the activating system.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Epoxide hydratase and benzo(a)pyrene monooxygenase activities in liver, kidney and lung after treatment of rats with epoxides of widely varying structures.

The effect of treatment with various epoxides on epoxide hydratase and benzo(a)pyrene monooxygenase activities in rat liver, kidney and lung was tested with the aim possibly to find a selective inducer of epoxide hydratase. In a first series of epoxides, good substrates of epoxide hydratase and relatively small molecules did not lead to an increase in epoxide hydratase activity in any organ tested. Treatment with the poor substrates dieldrin and trans-stilbene oxide(TSO), however, increased epoxide hydratase activity in the liver and with TSO also in the kidney (3-fold). In contrast to all other epoxide hydratase inducers so far discovered, TSO did not affect the benzo(a)pyrene monooxygenase activities, even after doses leading to maximal induction of epoxide hydratase ( ~ 350 per cent of controls). In an attempt to find an even more potent selective inducer of epoxide hydratase several trans-stilbene oxide derivatives were synthesized. All modifications of the TSO molecule led to compounds which were less effective inducers of liver epoxide hydratase than the parent compound and also either increased or drastically decreased the benzo(a)pyrene monooxygenase activities. With TSO, 4-methoxy-TSO, 4-chloro-TSO and 4-nitro-TSO the tests were extended to three other parameters of the monooxygenase system, the cytochrome P450 content, the NADPH-cytochrome c reductase and the aminopyrine N-demethylase activities. All these derivatives but not TSO itself affected part of the monooxygenase system. Thus, with respect to five measured monooxygenase parameters TSO was found to be a selective inducer of epoxide hydratase in rat liver. An influence of TSO on the pattern of the various cytochrome P450 forms not leading to observable changes in monooxygenase activity towards two substrates known to be preferential substrates of different cytochrome P450 forms (aminopyrine and benzo(a)pyrene) is unlikely but cannot be excluded.     

Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Specificity of human, rat and mouse skin epoxide hydratase towards K-region epoxides of polycyclic hydrocarbons

Epoxide hydratase activity has been measured in microsomal fractions of skin from mouse, rat and humans. The skin enzyme was able to hydrate all epoxides tested. The specific enzyme activities decreased in the order human > mouse > rat. The relative activity towards K-region epoxides of various polycyclic hydrocarbons in skin microsomal fractions from all three species decreased in the order phenanthrene 9,10-oxide > benz(a)anthracene 5,6-oxide ? benzo(a)pyrene 4,5-oxide ? 7-methylbenz(a)anthracene 5,6-oxide > 3-methylcholanthrene 11,12-oxide > dibenz(a,h)anthracene 5,6-oxide. The activity of epoxide hydratase in human skin microsomal fractions showed little pH dependence and was inhibited by small molecular weight inhibitors in a manner similar to that of the liver microsomal enzyme. Interindividual variation of epoxide hydratase activity in skin microsomal fractions from six human subjects was considerable, namely from 175 to 447 pmoles benzo(a)pyrene 4,5-dihydrodiol/min per mg protein. This variation was not due to skin disease or treatment and had no apparent correlation with age or sex. A possible correlation with the part of the body from which the skin sample was taken could not be excluded since the activity in skin samples from the abdomen seemed lower than that in samples from leg or breast.     

Species and organ specificity of the trans-stilbene oxide induced effects on epoxide hydratase and benzo(a)pyrene monooxygenase activity in rodents.

It was investigated, whether the selective induction of epoxide hydratase by trans-stilbene oxide (TSO) represents a general phenomenon or is confined to the liver of male rats where it was discovered. Therefore the effect of treatment with TSO on epoxide hydratase and benzo(a)pyrene mono-oxygenase activities were investigated in other organs (kidney, lung, skin, testis), other species (mice, hamsters) and also in female rats. In female rat livers the effect of TSO on the measured enzyme activities was very similar to that found in the male rat liver, i.e. a large induction of epoxide hydratase activity to 300–400 per cent of controls without affecting the benzo(a)pyrene monooxygenase activity. The potency of TSO to induce liver epoxide hydratase activity expressed as per cent of controls was 350:180:140 in rat, mouse and hamster, respectively. Selective induction of epoxide hydratase was found in rat and hamster liver, but not in the mouse liver, where benzo(a)pyrene monooxygenase activity was induced to about the same extent as the epoxide hydratase activity. The only extrahepatie organ in which an increased epoxide hydratase activity was found after TSO treatment was the rat kidney. Subcutaneous and topical treatment with TSO for 12 and 10 days respectively did not induce rat skin epoxide hydratase activity, instead a decrease of the enzyme activity to about 70 per cent ofthat found in control animals was found. Thus, TSO which was demonstrated to be a selective inducer of epoxide hydratase in rat liver can be utilized so far only in a limited number of carcinogenicity test systems, since it failed to induce the skin epoxide hydratase activity, which would have been an excellent tool to study directly the role of epoxide hydratase in the mechanism of skin tumor formation caused by polycyclic hydrocarbons. Interestingly, the epoxide hydratase of the hamster, investigated for the first time in this study, proved quite different from that of rat and mouse in that it hydrated styrene oxide remarkably faster than benzo(a)pyrene 4,5-oxide. This was true for all organs investigated. Also, the organ distribution of epoxide hydratase proved to be very different from that in rat and mouse. In the mouse the activity (with benzo(a)pyrene 4,5-oxide as substrate) was amongst all organs investigated highest in the testis (2.5 fold as compared to liver) but in the hamster the activity was more than 100 fold lower in testis as compared to liver. On the other hand, the activity in kidney was about 50 fold higher in hamster as compared to mouse.     

Styrene induced modifications of some rat liver enzymes involved in the activation and inactivation of xenobiotics.

Rats were injected intraperitoneally with single doses of styrene. Its effects on the kinetic parameters of liver microsomal monooxygenases and epoxide hydratase were investigated. The results were compared with those produced either by ethylbenzene, the vinyl-saturated analog of styrene or by phenobarbital and 3-methylcholanthrene, the classical inducers of those enzymes. The biochemical modifications were correlated with the altered ability of homogenates obtained from similarly pretreated rats to activate benzo(a)pyrene into intermediates mutagenic towards Salmonella typhimurium. Administration of styrene or 3-methylcholanthrene decreased the K_m, of benzo(a)pyrene hydroxylase and aldrin epoxidase; styrene, but not 3-methylcholanthrene, decreased the K_m of styrene oxide hydratase; none of the two compounds modified the K_m of styrene epoxidase.Pretreatment of the rats by styrene or 3-methylcholanthrene enhanced the S₉ mediated mutagenicity of benzo(a)pyrene several-fold, when compared to the mutagenic response mediated by liver preparations from control rats. Phenobarbital and ethylbenzene did not modify either the K_m of the investigated enzymes or the liver-mediated mutagenicity of benzo(a)pyrene.     

Effects of Panax Ginseng Extract on the Benzo(a)Pyrene Metabolizing Enzyime System

ABSTRACT

Ethanol extract of Panax ginseng C. A. Meyer, which has been used for centuries as a tonic in Asian countries, exhibited a selective induction of epoxide hydratase and cytosolic glutathione transferase activity without the concurrent induction of aryl hydrocarbon hydroxylase activity. Thus, Panax ginseng appears to have the potential to alter the metabolic patterns of benzo(a)pyrene and its reactive metabolites.     

Metabolism of benzo[a]pyrene in isolated human scalp hair follicles.

Human scalp hair follicles contain an enzyme system that metabolizes the carcinogen benzo[a]pyrene. The major ethyl acetate soluble metabolites are 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene,9,10-dihydro-9,10-dihydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene. Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO), an inhibitor of epoxide hydratase, prevents the formation of the dihydrodiols. The overall metabolism can be inhibited by the addition of alpha-naphthoflavone. The metabolism of benzo[a]pyrene in a cell culture of human scalp hair follicles has also been investigated. The results show that the activity of arylhydrocarbon hydroxylase (AHH) and epoxide hydratase (EH) is maintained in culture.     

Chapter 2: METABOLISM OF CARCINOGENS,POSSIBILITIES FOR MODULATION

Abstract: One of the structural elements which are widely occurring in very many chemical mutagens and carcinogens are aromatic and olefinic moieties. These can be transformed into epoxides by microsomal monooxygenases. Such epoxides may spontaneously react with nucleophilic centers in the cell and thereby covalently bind to DNA, RNA and protein. Such a reaction may lead to cytotoxicity, allergy, mutagenicity and/or carcinogenicity, depending on the properties of the epoxide in question. An important contributing factor is the presence of enzymes controlling the concentration of such epoxides. There are several microsomal monooxygenases which differ in activity and substrate specificity. With large substrates, some monooxygenases preferentially attack at one specific site different from that attacked by others. Some of these pathways lead to reactive products, others are detoxification pathways. Also important are the enzymes which metabolize epoxides, such as epoxide hydrolases and glutathione transferases. Such enzymes can act as inactivating and in some specific cases also as co-activating enzymes. Moreover, precursor-sequestering enzymes such as dihydrodiol dehydrogenase, glucuronosyl transferases and sulphotransferases are important for the control of reactive epoxides. These enzymes themselves are subject to control by many endogenous and exogenous factors. By virtue of their contribution to the control of carcinogenic metabolites such modulators can act as modifiers of tumorigenesis and can be used experimentally to study the role of the various individual enzymes.     

Initial characterization of drug-metabolizing systems in the liver of the Northern pike, Esox lucius

NADPH-cytochrome c reductase, cytochrome -450, benzo[a]pyrene mono-oxygenase, epoxide hydratase, and glutathione S-transferase activities in the liver of the Northern pike (Esox lucius) have been measured and partially characterized. The level of these systems in pike liver is between 13.2 and 133% of the corresponding levels in rat liver, with the exception of glutathione S-transferase, whose specific activity in the high-speed supernatant fraction of pike liver is 305% of that in rat liver. In addition, pike liver contains about 23% of the mammalian level of reduced glutathione. Drug-metabolizing systems in pike liver are distributed in essentially the same manner in subfractions as the corresponding systems in the liver of mammals. Benzo[a]pyrene mono-oxygenase and epoxide hydratase activities display the expected pH maxima of 7.5 and 9.5, respectively, and have temperature maxima of 37 degrees and 47 degrees C, respectively. NADPH-cytochrome c reductase and glutathione S-transferase activities are relatively independent of temperature. Intraperitoneal treatment of Northern pike with methylcholanthrene induces the benzo[a]pyrene mono-oxygenase activity of liver microsomes 33-fold.     

Metabolic activations of polycyclic hydrocarbons

Considerable evidence now points to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrenes as ultimate mutagenic and carcinogenic forms of benzo(a)-pyrene. Quantum mechanical calculations have been performed to assess the possible general role of diol epoxides in polycyclic aromatic hydrocarbon (PAH) mutagenesis and carcinogenesis. The calculations enable a prediction of relative reactivity (ease of carbonium ion formation) for diol epoxides derived from a single PAH and also for diol epoxides from different PAHs. The calculated reactivity has so far been found to provide a good estimate of diol epoxide mutagenicity. Results of the metabolic activation of benzo(a)anthracene dihydrodiol derivatives and of the mutagenicity of benzo(a)anthracene diol epoxides are reported. Limitations inherent in predictions of polycyclic aromatic hydrocarbon carcinogenicity using a model based upon the calculated reactivity of a potential metabolite are discussed.Presented at the Symposium Influences of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, March 15, 1977, Mainz     

Effects of Panax ginseng extract on the benzo(a)pyrene metabolizing enzyme system

Ethanol extract of Panax ginseng C. A. Meyer, which has been used for centuries as a tonic in Asian countries, exhibited a selective induction of epoxide hydratase and cytosolic glutathione transferase activity without the concurrent induction of aryl hydrocarbon hydroxylase activity. Thus, Panax ginseng appears to have the potential to alter the metabolic patterns of benzo(a)pyrene and its reactive metabolites.     

Dose-related induction of rat hepatic drug-metabolizing enzymes by diuron and chlorotoluron, two substituted phenylurea herbicides

Dose-related induction of various hepatic drug-metabolizing enzymes has been investigated after short-term treatment of rats by diuron and chlorotoluron, a dichlorinated and a monochlorinated phenylurea herbicide, respectively. Results suggest that 'saturation' of the induction system of benzo(a)pyrene monooxygenase, 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities may occur in the same range of the molar doses of both compounds, and with the dichlorinated herbicide at much higher activities. Induction of epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases also shows saturation curves in the function of molar doses. However, the structural difference is not reflected in the enhancement of enzyme activities.     

K-region epoxides of polycyclic hydrocarbons: formation and further metabolism by rat-lung preparations

Epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene that have been identified as the K-region epoxides, 7-methylbenz[a]anthracene 5,6-oxide and benzo[a]pyrene 4,5-oxide, have been detected as microsomal metabolites using preparations from the lungs of rats that had been pretreated with the microsomal mixed function oxidase inducer, 3-methylcholanthrene. It was also possible, using lung microsomal preparations from uninduced animals, to demonstrate the formation of an epoxide identified as the K-region derivative, benz[a]anthracene 5,6-oxide, as a microsomal metabolite of benz[a]anthracene. The K-region epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene could not always be detected as metabolites when lung microsomal preparations from uninduced rats were used. The activities of two other enzymes present in pulmonary tissue fractions that are involved in the further metabolism of polycyclic hydrocarbon epoxides have also been measured and the values compared with those obtained with rat-liver. When benz[a]anthracene 5,6-oxide was used as substrate, much lower levels of microsomal epoxide hydrase activity were found in lung than in liver, but soluble-supernatant fractions of rat-lung appeared to possess higher levels of glutathione S-epoxide transferase activity than were present in rat-liver.The significance of these results in relation to the metabolic activation of polycyclic hydrocarbons by epoxide formation and to the induction of tumours of the respiratory tract by members of this class of chemical carcinogens is discussed.     

The apparent ubiquity of epoxide hydratase in rat organs

Using the recently developed sensitive assay with [³H] benzo [a] pyrene 4,5-oxide as substrate, epoxide hydratase was shown to be present in 26 rat (Sprague-Dawley) organs and tissues investigated. Only blood showed no detectable activity, which indicates that the low enzyme activity found in some organs is not due to the presence of blood components in the tissues. In earlier studies with a less sensitive assay, epoxide hydratase activity was detected only in rat liver and kidney but not in organs such as muscle, spleen, heart and brain. Epoxide hydratase was also measured in 6 organs of the mouse (NMRI). The distribution pattern was quantitatively quite different in the two species. The sp. act. in the rat were in the order liver > testis > kidney > lung > intestine ～- skin. In the mouse, very surprisingly, testis had the highest specific epoxide hydratase activity. Moreover, the order of sp. act. in the mouse organs was remarkably different from that in the rat, namely testis > liver > lung > skin > kidney > intestine. The fact that the sp. act. in kidney was much lower than in lung or skin is most striking. Pretreatment of rats with Aroclor 1254 (a mixture of polychlorinated biphenyls) increased the epoxide hydratase activity in the liver to 175 per cent of the control level. However, the enzyme activity in the 13 extrahepatic tissues investigated was not significantly changed. In organs possessing sufficiently high enzyme levels, epoxide hydratase activity was also measured with styrene oxide as substrate. The ratio of the sp. act. of the two substrates was very similar in rat liver, kidney, lung and lestis. This supports the assumption that in these organs a single enzyme is responsible for the hydration of both substrates—as was earlier shown by several methods for the rat liver.     

Relationships of quantum mechanical calculations, relative mutagenicity of benzo[a]anthracene diol epoxides, and "bay region" concept of aromatic hydrocarbon carcinogenicity.

Evidence supporting the conclusion that 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes are ultimate mutagenic and carcinogenic forms of benzo[a]pyrene (BP) is summarized. Qauntum mechanical calculations that predict reactivity of diol epoxides derived from BP and other polycyclic aromatic hydrocarbons are described. The calculations predict that diol epoxides in which the oxirane ring forms part of a "bay region" of a tetrahydrobenzo ring should be the most reactive for a given aromatic hydrocarbon. Experiments with dihydrodiols and diol epoxides from benzo[a]anthracene (BA) are described. The ability to metabolically activate BA 3,4-dihydrodiol to species much more mutagenic that those obtained from other BA dihydrodiols ant the much greater mutagenicity of the diastereoisomeric 3,4-diol 1,2-epoxides of 1,2,3,4-tetrahydro BA relative to other diol epoxides of BA are in accord with predictions of the quantum mechanical calculations.     

Mechanisms in inactivating reactive metabolic products of drugs (author's transl)

An in vitro test system was used to study the relative contribution of epoxides metabolically produced from aromatic or olefinic drugs to the total of mutagenically reactive metabolites. As an epoxide-specific tool the enzyme epoxide hydratase was purified to homogeneity. Using several aromatic and olefinic hydrocarbons as model substrates the following was observed, exceptions being discussed in the main text: 1. Epoxides represent the most important and generally the almost exclusive metabolites responsible for the observed toxic endpoint. 2. These epoxides generally are efficiently inactivated by epoxide hydratase. The rather narrow limitations of exceptions are outlined in detail. 3. Amongst the enzymes metabolizing epoxides, for the substrates investigated epoxide hydratase represents the system which is most critical for the control of tissue levels of epoxides. 4. Several practical consequences following from these facts are outlined in the last paragraph of this paper. 5. A generalization from these model experiments will be justified only after further experimental validation.     

Increased cytochrome P-450 independent drug metabolism and mutagen activation in rat liver by octachlorostyrene

Single intraperitoneal injections of 200 mg/kg octachlorostyrene (OCS) increased the activities of flavin-containing monooxygenase, epoxide hydrolase and glutathione S-transferase in the livers of male Wistar rats. UDP-glucuronyl transferase activities measured with aglycones increased by methylcholanthrene or phenobarbital treatment, were both slightly increased by OCS treatment. A liver 9,000 X g supernatant fraction from OCS pretreated rats increased the bacterial mutagenicity of 2-acetylaminofluorene and 2-aminofluorene compared to controls, while insignificant or only minor effects were seen on N-hydroxy 2-acetylaminofluorene and benzo(a)pyrene mutagenicity. The effect of OCS on mutagen activation was similar to that seen after phenobarbital treatment. The use of monolayers of hepatocytes instead of 9,000 X g subfractions did not reveal any qualitative differences in mutagen activation.     

Organ specific induction of drug metabolizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied on the activities of arylhydrocarbon hydroxylase, ethoxycoumarin deethylase, cytochrome c reductase, epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase in the liver, kidney, lung, small intestinal mucosa, and testis of male Wistar rats. There was a severalfold increase in the activity of monooxygenase in the liver, kidney, and lung, whereas virtually no effect could be detected in the intestine or testes. The proportion of 3- and 9-hydroxylation of the total hydroxylation of benzo(a)pyrene decreased in the liver, but increased in the kidney. TCDD had no significant effect on epoxide hydratase or glutathione S-transferase activities in any tissues. UDP glucuronosyltransferase exhibited a sevenfold increase in the liver, less than twofold in the kidney, and none in other tissues. Treatment of the microsomes with digitonin, trypsin, or phospholipase A did not reveal additional induction UDP glucurnosyltransferase, although all were able to increase measurable enzymatic activity in control and TCDD-treated animals. TCDD seems to be different from phenobarbital and polycyclic hydrocarbons as an effector of not only monooxygenase but also epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase.