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xenobiotica, 1972, vol. 3, no. 5, 303-340 Review Article Mammalian Epoxide Hydrases: Inducible Enzymes Catalysing the Inactivation of Carcinogenic and Cytotoxic Metabolites Derived from Aromatic and Olefinic Compounds FRANZ OESCH Biozentrum of the University of Basel, CH-4056 Basel, Switzerland {Rtcavtd 6 April 1973) 1. Several aromatic and olefinic compounds are converted to intermediate srene and alkene oxides by rnammalian mono-oxygensees. Intermediata arena oxides tearranga non-enzymically to phenols. Arene and alkene ozidai an convened by epoxide hydrases to vicinal diols and by glutathione poxida conjugates to glutathione conjugates. Due to their high electrophilic reactivity, such oxirtnes also bind to proteins, RNA and DNA. Mutagenic, cardnogenie and cytotoxic effects of several aromatic and olefinic compounds appear to be due to the formation of intermediate epoxides and their reaction with tissue cotwti- tuents. Whether a given aromatic or olefinic compound produces such an effect would thus depend on a variety of factors, such as the relative rate of formation and degradation of the intermediate oxirane, on its stability with respect to spontaneous isomerization to tha corresponding phenol and on tea L-.I electrophilic reactivity. 2. Epoxide hydrases, which convert such intermediate oxirsnes to much lees reactive vicinal diols, have been studied in greater detail. Epoxide hydrate activity is found in mouse, rat, guinea-pig, rabbit, pig. Rhesus monkey and human liver. Activity is high in liver, low in kidney, very low in intestine and 1ung and not detectable in muscle, spleen, heart and brain. Tha enzyme is located exclusively in microsomal membrane*. Epoxide hydruc activity is markedly increased after pretreatment of rats with phenobsrbital or 3-mcthyi- cholanthrene and during maturation of rata. These increases are reminiscent of similar increases in microsomal roono-orygenases. However, the extents of induction of total levels of these two enzymes or enzyme families are not com parable and are under separate genetic control. 3. Several stereochemical properties of tha reaction catalysed by epoxide hydrases have been studied with microsomal preparations. With styrene oxide and naphthalene oxide as substrates, attack by H,'*0 occurs virtually exclusively at the 2-position. Product glycols which are stereochemicaily fixed by a ring structure invariably have the tram-configuration. Hydration of some acyclic _ _ alkene oxides has also been found to proceed vis a tram-opening of the oxirane* ' ring. Cyclohexene oxide, benzene oxide and naphthalene 1,2-oxide are con verted predominantly to l&lR-tranr-diols, while in the esse of phensnthrene 9,10-oxide the liS,25,-trdMf*diol predominates. 4. Epoxide hydraae from guinea-pig liver mkroeomea was solubilized and purified, based on an assay with styrene oxide as substrate. The specific activity after thelastpurification step isabout+O times higher than in the crude homogenate. This increase is not due to the removal of an inhibitor. About 30% of the activity of the purified preparation it lost within 1-2 days. However, the remaining activity is remarkably stable. Gel electrophoresis of the final (stable) preparation SL 66136 2 306 F. Oesch thaw* one major band corresponding to a mol. wt. of approx. 50 000. However, several minor bands are also present. 5- Several properties of epoxide hydrase were investigated with this purified" preparation. While no desrcut pH optimum could be observed with microsomal preparations (broad' optimum' between 7 and 9) a sharp pH profile was obtained with the purified preparation with its optimum at pH 9. Non-enzymic hydration was significant (5%) only below pH 6-5. The Kst with respect to styrene oxide ss substrate is 2-8 x 10~*m and the apparent Pmaa. 2-4 pmol product/mg N per 5 min. Mo metal ions or other low mol. wt. co-factors ere necessary foe maximal activity. High concentrations of substrate inhibit the enzyme, whereas product diols have no effect. Several inhibitors of drug-metabolizing enzymes . (SKF 525-A), piperonyl butoadde, e-nsphthofUvone) do not influence epoxide hydrase activity, while sulphydryi reagents slightly, but tignificandy, inhibit the enzyme. Several alcohols, ketones snd imidazoles stimulate the enzyme. Kinetic analysis of the activation by the potent stimulator, metyrapone, indicates negative co-operativity with the substrate. 6. Hie active site of the enzyme readily accommodates, as substrates or competitive inhibitors, mooosubstituted axiranes with a lipophilic substituent larger than an ethyl group, suggesting hydrophobic binding sites near the active site. With axiranes having such s lipophilic substituent, the enzyme interacts with mono-, 1,1-di- snd cii-t ,2-disubstituted oxiranes, but not with treat-1,2* diaubttituted oxiranes or tri- or tetra-substitutsd oxiranes. suggesting increasing bulk around tha oxirane ring prevents the approech of the oxirane to the active site. Several oxiranes fused to tltcydic rings (cydohexene oxide, 144,4tetrahydronaphthalene 1 4-epoxide) are potent inhibitors but very poor substrata*. Kinetic analysis revealed ism-competitive inhibition with respect to the sub strate, styrene oxide. Styrene sulphide (an analogue of the competitive inhibitor, styrene oxide) but not cydohexene sulphide (sn analogue of the noe-competidve inhibitor, cydohexene oxide) has inhibitory activity, suggesting differing structural requirements for the sites involved in competitive end now-competitive inhibition. The most potent inhibitor discovered so far is 1,1,1-trichloropropena; 24-oxide, which is on the other hand a poor substrate. Inhibition by this compound is of the ua-competitive type. Structure-activity relationship for substrates and inhibitors of purified human epoxide hydrase are qualitatively identical to the ones discussed above for the purified guinea-pig enzyme. 7. Evidence suggesting the presence of more than one liver enzyme capable of hydrating epoxides indude differential stabilities, different ratios of hydrsae activity towards various epoxides in preparations from different species, a different purification factor (activity of the purified preparation compared to liver homo genates) towards benzene oxide as compared to several other epoxides, inability to inhibit hydration of styrene oxide (in purified or particulate preparations) with much higher concentrations of benzene oxide. 8. Evidence indicating the presence of a coupled mono-oxygsnase-epoxide hydrate multienzyme complex include the following observations. Substantial amounts of dihydrodiola in the urine of animals treated with aromatic hydro carbons, despite the high instability of intermediate arena oxides, lack of equilibria" don between pools of naphthalene oxide formed in situ and of exogenous naph thalene oxide, differential inhibition of * free epoxide hydrates * at concentrations of 1,1,1 -trichloropropent 2,3-oxide which do not effect the * coupled monooxygenase-epoxide hydrase system *, selective induction of. the coupled system by 3-methylcholanthrene, snd high epoxide hydrase activities in solubilized and purified cytochrome P-450 snd P-448 fractions, where other microsomal enzymes (glucose-6-phosphatase, cytochrome c reductase) were absent. Such a coupled mono-oxygenase epoxide hydrase system may be of great relevance to problems such as carcinogenic properties of arena oxides derived from several polycyclic hydrocarbons, and hepatotoxicity of intermediate Irene oxides derived from halobenzenes, by circumventing these adverse effects by their rapid conversion si O6613; 4 I 00. However, h thi* purifi 1 ith microaomal le was obtain d ymic hydndon pect to styrene 10I product/mg e necessary for tzyme, whereas tlizing enzymes luence epoxide ttly, inhibit die the enzyme, pone, indicates > substrates or tlic substituent near the active izyme interacts with trans-\uggesdng that e oxirane to the oxide, 1,2,3,4toot substrates, ct to the sub* ntive inhibitor, ton-competitive idng differing ton-competitive ichloroptopene; ibition by this eladonahip for ce qualitatively zyme. nxyme capable doe of hydrate tie*, a different to liver homotides, inability oaradons) with ,'enaae-epoxide *. Substantial 'omadc hydroclc of equilibr*/ ogenoua naphconcentrations coupled monotoupled system solubilized and somsl enzymes such a coupled ce to problems erst polycyclic derived from pid conversion Epoxide Hydrases 307 to dihydrodiols. Indeed, pretreatment of rats with 3-methylcholanthrene, which selectively induces this coupled system, provides protection from chlorobenzeneevoked hepttotoxicity, whereas pre-treatment with phcnobarbital increases the toxic effect, although it induces the total level of epoxide hydraae to a much greater extent than 3-methylcholanthrene. Introduction For many years the mechanism(s) whereby chemically unreactive sub stances, e.g. aromatic hydrocarbons, exerted their carcinogenic or cytotoxic action has puzzled researchers. However, in recent years evidence has ac cumulated which shows that many of these compounds are converted enzymically to chemically highly reactive derivatives which act as the ultimate carcinogens or cytotoxins (for references see Shuster, 1964 ; Miller & Miller, 1966 ; Uehleke, 1971). Their high reactivity makes such ultimate agents often so unstable that proof of their formation is a difficult task. Boyland proposed the formation of arene oxides* from aromatic hydrocarbons and suggested their importance in carcinogenicity as early as 1950, but the first proof of the biological formation of an arene oxide was not realized until many years later (Jerina, et al., 1968 b; 1970 b). Strong evidence has recently been put forward that such arene oxides are the causative agents for the carcinogenicity of (at least some) polycyclic aromatic hydrocarbons (Marquardt, et al., 1972), and fon the liver necrosis evoked by several halobenzenes (Brodie, et aL, 1971). These intermediate oxides rearrange non-enrymically- to phenols, are metabolized to glutathione conjugates by glutathione 5-epoxide conjugascs and to vicinal diois by epoxide hydrases. The glutathione 5-epoxide transferases are localized in the cyto plasm (Boyland Sc Chasseaud, 1969), are not inducible (E. Boyland, personal communication) and the glutathione 5-epoxide conjugation reaction also occurs non-enzymically to a high extent (Boyland & Chasseaud, 1969; Jerina, et al., 1970 b). The epoxide hydrases are localized in the endoplaamic reticulum, i.e. possibly near the site of formation of the arene oxides (Oesch, Jerina 2c Daly, 1971 a), they are inducible (Oesch, et al., 1971 a) and non-enzymic hydration of arene oxides has not been observed (Vogel Sc Klamer, 1968; Jerina, et al., 1968 b, 1970 a ; Sims, 1971). These epoxide hydrases appear therefore to be especially worthy of detailed investigation. Biological formation of oxiranes from aromatic and olefinic substrates Arene oxides would be expected to be chemically very unstable because of the great gain of resonance energy obtained upon isomerization to the corresponding phenol. It is therefore not surprising that the first arene oxides became syntnetically available only recently, when Newman Sc Blum (1964) prepared Kregion oxides of phenanthrene and benz(a)anthracene, closing the oxirane ring from Air-aldehyde precursors with the aid of Mark's reagent, frir-dimethvlaminophosphine. Soon afterwards arene oxides which were not of the Kregion oxide type became synthetically available via dehydrohalogenation of cyclic haloepoxides (Vogel, Bdll & Gunther, 1965 ; Vogel Sc Gunther, 1967). Jerina, * Compound* which formaliatically result From the epoxidation of on* of the double bond* of an aromatic nuclcua are called arene oxide*. For thoae reaulting from the epoxidation of an olefinic double bond, the term alkcne oxide i* suggeated. SL 066138 - vtfr*;\s 308 F. Oesch Boyd & Daly (1970 a) showed arene oxide formation by direct transfer of oxygen from photo-excited pyridine-fV-oxide to naphthalene. This constituted the first example of chemical epoxidation of an aromatic double bond. With the aid of synthetically available arene oxides it became possible to demonstrate their formation upon incubation of aromatic hydrocarbons with liver microsomes fortified with NADPH. Jerina, et al., (1968 b, 1970 b) demonstrated the formation of 14C-naphthalene 1,2-oxide from 14C-naphthalene by trapping the radioactive oxide in a large pool of unlabelled synthetic naph thalene 1,2-oxide. Using short incubation times and low temperatures, they also succeeded, without using trapping techniques, in isolating by countercurrent distribution a sufficiently large quantity of enzymically formed naph thalene 1,2-oxide to allow identification of this metabolite by chemical and chromatographic characteristics. Subsequently arene oxides were also reported as microsomal metabolites of several polycyclic hydrocarbons, namely dibenz(a,h)anthracene (Selkirk, Huberman 8e Heidelberger, 1971), phenanthrene and benz(a)anthracene (Grover, Hewer & Sims, 1971 b), pyrene and benzo(a)pyrene (Grover, Hewer 8c Sims, 1972), and 7,12-dimethylbenz(a)anthracene (Keysell, et al., 1972). Finally, formation of naphthalene oxide was demonstrated in re constituted enzyme systems, consisting of solubilized and purified cytochrome P-450 or P-448, NADPH, cytochrome c reductase and lipid fraction (Oesch, et aL, 1972). Although no other examples are available to date, it may be concluded that in all likelihood the formation of arene oxides from aromatic hydrocarbons by the action of mono-oxygenases is a general phenomenon. However, attempts to trap radioactive benzene oxide formed from radioactive benzene with rabbit liver microsomes, in the presence of exogenous carrier benzene oxide have not been successful, possibly due to relatively great in stability of benzene oxide caused by the gain of resonance energy through isomerization to a monocyclic aromatic compound. Thus, the recovery of added benzene oxide was very poor. Moreover, benzene is a poor substrate for the microsomal mono-oxygenases (Jerina, et al., 1970 b). Isolation of alkylarene oxides after incubation of the corresponding aromatic hydrocarbons is also un feasible, since arene oxides with alkyl substituents at the oxirane ring are not formed by hepatic microsomal mono-oxygenases (with the possible exception of mesitylene oxide) as demonstrated by comparing isomerization products of synthetic alkylarene oxides to microsomal metabolites of the parent hydro carbons, whereas arene oxides with alkyl substituents at other ring positions are very unstable, rearranging spontaneously even at 0s (Kaubisch, Daly 8c Jerina, 1972). Experiments using an epoxide substrate as a competitive inhibitor proved the naphthalene oxide to be the obligatory intermediate in the formation of dihydrodiol and glutathione conjugates (Jerina, et al., 1970 b). The absence of deuterium in the 4-chlorocatechol and its O-methylated products produced by in vivo metabolism of chloro[4-*H]bcnzene demonstrated that catechols arise by dehydrogenation of dihydrodiols rather than by further hydroxylation of a phenol (Jerina, Daly & Witkop, 1967). Observation of dihydrodiols, catechols or glutathione conjugates (the latter with the exception of those formed by a direct replacement of a labile halo- or nitro-group : Booth, Boyland 8c Sims, 1961 AlKassab, Boyland 8c Williams, 1963 ; Clark, Darby 8c Smith, 1967) as metabolites of foreign aromatic compounds is therefore a good indication for the formation SL 066139 :ransfer of oxygen ; constituted the nd. came possible to ,-drocarbons with (1968 b, 1970 b) 114C-naphthalene i synthetic naphmperatures, they ting by counter* lly formed naphby chemical and .ere also reported namely dibenzthenanthrene and d benzo(a)pyrene hracene (Keysell, monstrated in rerified cytochrome fraction (Oesch, date, it may be es from aromatic ral phenomenon, from radioactive ,`xogenoua carrier latively great in: energy through the recovery of poor substrate for ition of alkylarene irbons is also unfane ring are not 'ossible exception ation products of ie parent hydroring positions are h, Daly Sc Jerina, libitor proved the formation of di- The absence of ucts produced by catechols arise by lation of a phenol ols, catechols or >rmed by a direct Sims, 1961 ; Al57) as metabolites for the formation Epoxide Hydrates 309 of an intermediate arene oxide. Such metabolites have been shown to be formed from several compounds of common concern, namely from carcinogenic aromatic hydrocarbons (e.g. dibenz(a)anthracene, Boyland Sc Sims, 1965 a ; benzolpyrene, Sims, 1967 c; 3-methylcholanthrene and 7,12-dimethylbenz(a)anthracene, Sims, 1970), from halobenzenes which are widely used as solvents and which can produce liver necrosis (e.g. chlorobenzene, Smith, Spencer Sc Williams, 1950 ; fluoro-, bromo- and iodobenzene, Azouz, Parke Sc Williams, 1953 ; Jollow, et al,, 1972; dichlorobenzenes, Parke Sc Williams, 1953), from pesticides (e.g. 2,6-dichiorobenzonitrile, Wit Sc van Genderen, 1966 ; Carbaryl, Sullivan, et al., 1972), and from clinically used pharmaceuticals (e.g. diphenylhydantoin. Horning, et al., 1971 ; Lorazepam, Schillings, Schrader Sc Ruelius, 1971 ; phenobarbital and mephobarbital, Harvey, et al., 1972 a). Hydroxylation of deuterated zoxazolamine is the only reported case (Tanabe, et al., 1970) which is not compatible with a hydroxylation mechanism proceeding via an intermediate arene oxide, since the rate of phenol formation appeared to be different whether hydrogen or deuterium was present at the position of hydroxylation.. This suggests insertion of oxygen rather than addition. A preliminary report of isotope effects during hydroxylation of nitrobenzene has appeared (Daly, Jerina Sc Witkop, 1972). However, in all other investigated cases no such primary isotope effects could be observed during hydroxylation of substrates which were deuterated or tritiated at the appropriate position (Guroff Sc Daly, 1967 ; Tanabe, et al., 1967 ; Perel, et aL, 1967 ; Daly St Jerina, 1969). Several olefinic compounds are metabolized to alkene oxides* which are resistant enough to spontaneous and enzymic degradation so that they can be isolated as such without recourse to special techniques such as enzyme inhibition and/or radiotracer trapping. Examples are heptachlor oxide (Davidow Sc Radomski, 1953), dieldrin (Winteringham Sc Barnes, 1955 ; Bann, et al., 1956), 16a, 17-epoxy-estratricne-3-ol (Breuer Sc Knuppen, 1961), 1,4: 2,3-diepoxy1,2,3,4-tetrahydronaphthalene (Sims, 1965), endrin (Wong Sc Terrierre, 1965) and chlordane epoxide (Brooks, 1966). 1 -A*-Tetrahydrocannabinol, the com pound believed responsible for most of the psychopharmacoiogical effects of marihuana (Gaoni Sc Mechoulam, 1964), allyl-substituted barbiturates, such as secobarbital, allobarbital and alphenai, sedatives of short, intermediate and long duration respectively, and carbamazepine, widely used as an antiepileptic agent or in the treatment of trigeminal neuralgia, are interesting examples of drugs which are converted to such alkene oxides, as demonstrated very recently (Gumy, etaL, 1972; Harvey, et al, 1972 b ; Frigerio, et al., 1972). However, frequently glycols (and no epoxides) are observed as products arising from olefinic com pounds by oxidative metabolism (Brooks & Young, 1956; El Masri, Smith & Williams, 1958 ; Boyland & Sims, 1960 ; Booth, et al., 1960 ; Douglas, 1965 ; Waddell, 1965 ; Fukami, Yamamoto & Casida, 1967 ; Leibman Sc Ortiz, 1968, 1969, 1971 ; Fukami, et al., 1969; Sisenwine, et al., 1970). Until the late sixties the role of epoxides in the biological formation of glycols was not estab lished. Glycols could conceivably arise by enzymic or spontaneous hydrolysis of intermediate epoxides or by direct dihydroxylation of an ethylenic moiety. Maynert, Foreman Sc Watabe (1970) showed that addition of a potential competitive inhibitor (4,5-epoxy-a-octane) of an enzymic hydration of an * See footnote p. 3, I 'gXJ 7TM"^' SL 066140 310 F. Oesch intermediate epoxide to a microsomal incubation mixture containing n-1 -octene as substrate allowed isolation of a considerable quantity of 1,2-epoxy--octane. In the absence of the potential inhibitor only the corresponding glycol was formed. -4-Octene was also converted to the corresponding glycol with no trace of epoxide in the absence of an excess of another epoxide, 1,2-epoxy--octane, but only to epoxide in its presence. This indicates that the epoxide is an obligatory intermediate in the conversion of n-4-octene to the glycol and that in many cases where glycols are observed as metabolites from olefinic compounds epoxides may be intermediates. Leibman St Ortiz (1970) also reported observation of inter mediate alkene oxides from olefinic compounds (styrene, cyclohexene and indene) which hitherto had been known only to produce glycols. Hence it appears that oxiranes, although in many instances not stable enough to survive isolation, are very widespread primary oxidation products in the meta bolism of aromatic and olefinic foreign compounds. Firm establishment of the generality of the formation of (intermediate) oxiranes must await further in vestigation. Fate of oxiranes The fate of arene oxides is summarized in Fig. 1. Reaction 1 shows the valence bond tautomerism to oxepins. Both extremes of the oxide-oxepin equilibrium have been observed : Naphthalene 1,2-oxide exists exclusively as the oxide (Vogel St Klirner, 1968 ; Boyd, Jerina St Daly, 1970) presumably because tautomerization to the corresponding oxepin would cause loss of aromaticity in the benzene ring. Attempts to prepare naphthalene 2,3-oxide, on the other hand. R (2) r nuy R (1) Non-Mitynie R (3) Na*-an*yntie ftutarftioA* 5-WOmM tramfmw 13) Non-anty"** Coaotantly bound .0 to protain, RNA, DNA \ (<) CwMl \ hydma RR Fig. 1. Biological formation and degradation of arene oxides. SL 0661^1 -1-octeneas -octane. In was formed, no trace of octane, but n obligatory i many cases poxides may on of interhexene and able enough in the metament of the further in- 1 shows the xide-oxepin sively as the bly because iticity in the other hand, vtrientty bound protein, NA, ONA siMt SrM Epoxide Hydrates 311 have produced only the oxepin (Jeffrey & Jerina, 1972). Oxepins which are not in equilibrium with the corresponding arene oxide are stable and do not isomerize to phenol. As yet, only arene oxides which exist at least partly in the oxide form have been established as metabolic intermediates. Reaction 2 (Fig. 1) shows the isomerization of arene oxides to phenols. This reaction occurs non-enzymically as a spontaneous rearrangement in a neutral or basic environment and as a acid-catalysed isomerization at pH < 6 as indicated by recent kinetic studies (Kasperek Sc Bruice, 1972 ; Kasperek, et ai., 1972; Yagi, et ai, 1972). The rate determining step in the spontaneous rearrangement is the heterolytie opening of the carbon-oxygen bond to the zwitter ion (A). In thjT acid-catalysed isomerization the protonated form (B) predominates (Kasperek, et aL, 1972). The isomerization of arene oxides to phenols is accompanied by an (A) (B) intramolecular displacement of a substituent X (e.g. isotopic hydrogen, halogen* . ^ - y ? methyl) from the place of the final Ideation of the phenolic group to a neighbour-, . - ;X ing carbon atom. This phenomenon, the * NIH Shift *, has now been observed' r< ` ^ concomitant with the rearrangement of many arene oxide* to phenols Daly Sc Witkop, 1968 a; Jenna, Kaubisch & Daly, 1971; Boyd, Daly Sc Jenna, ^ 1972 *, Kaubisch, et ai., 1972). A complete mechanism for the isomerization ^ f ( of arene oxides to phenols must therefore be compatible with the observation of the NIH shift. Formation of a keto tautomer (C) prior to enolizatton to the a phenol (D, E) satisfies the requirement. The mechanism is shown for the spontaneous reaction (A) which predominates at physiological pH. A more detailed description of this mechanism is given by Boyd, et at. (1972). (C) (D> Reaction 3 (Fig. 1) is a conjugation of the arene oxide with glutathione (Boyland Sc Sims, 1965 a, b ; Jerina, et aL, 1968 c, 1970 b). Such glutathione conjugates lead after loss of glutamate and glycine and ;V-acetylation of the remaining cysteine residue, to the premercapturic acids which in acidic environ ment easily dehydrate to the mercapturic adds (Boyland, Ramsay Sc Sims, 1961). This conjugation of arene oxides with glutathione proceeds spontaneously but there is also a cytoplasmic liver enzyme, glutathione 5-epoxide transferase, which catalyses this reaction (Boyland Sc Sims, 1965 a, b ; Jerina, et al., 1970 b). Reaction 4 (Fig. 1) is the hydration of arene oxides to the corresponding trmu-dihydrodiols. This reaction is catalysed by epoxide hydrases which are ,,,,SL 066142 312 F. Oesch localized in the hepatic endoplasmic reticulum (Oesch, et al., 1971 a). These enzymes will be dealt with in greater detail in the Bio-inactivation section of this review. The dihydrodiols are then conjugated with glucuronic acid or are de hydrogenated to catechols by a soluble hepatic dehydrogenase (Ayengar, it al., 1959 ; Jerina, Ziffer 5c Daly, 1970 c). Catechols may then be meta or para 0methylated by a non-particulate (Creveling, et al., 1970, 1972; Meyer & Scheline, 1972) or microsomal (Inscoe, Daly 5c Axelrod, 1965; McCormick, Flanagan Sc Lloyd, 1972) hepatic catechol O-methyltransferase. Reaction 5 of Fig. 1 shows covalent binding of arene oxides to tissue macro molecules. Arene oxides form covalent bonds with many nucleophiles such as methanol, azide and sulphydryl compounds (Jerina, et al., 1968 c, 1970 b ; De Marines & Berchtold, 1969; Yagi, et al., 1972). They would therefore be expected to react with nucleophilic moieties in proteins, DNA and RNA In fact, aromatic hydrocarbons bind to DNA, RNA and proteins m vitro if a metabolically active oxidative system (liver microsomes) is present but not if such a system is absent or relatively inactive (no NADPH added) (Grover Sc Sims, 1968 ; Gelboin, 1969). Moreover, the amount of the hydrocarbon which is bound to DNA is considerably increased if microsomes are used from "! pre-treated with 3-methylcholanthrene (Gelboin, 1969). Such treatment is known to induce the microsomal enzyme system which oxidatively transforms aromatic hydrocarbons (Conney, 1967). Thus, the oxidative enzyme system in the endoplasmic reticulum appears to transform aromatic hydrocarbons to a reactive intermediate which then binds to tissue constituents. In the light of the studies which are discussed in this section and which show that arene oxides are formed by liver microsomes it appears'likely that the active metabolites which bind to DNA RNA and proteins are arene oxides. Gelboin (1969) showed further more that the binding was probably not an adsorption but rather of a co valent nature since the ratio of aromatic hydrocarbon : DNA remained con stant during repeated precipitation, extractions with organic solvents and fractionation in a CsCl gradient. Grover Sc Sims (1970) showed that in the absence of a microsomal enzyme system arene oxides did firmly bind in vitro to DNA RNA and histone whereas the corresponding aromatic hydrocarbons and dihydrodiols did not. In cell cultures possessing some oxidative metabolizing potential, aromatic hydrocarbons, arene oxides, dihydrodiols and phenols did firmly bind to DNA, RNA and proteins, with arene oxides dearly binding to a much greater extent than the other compounds (Grover, Forrester Sc Sims, 1971 a; Kuroki, et al., 1972). Kuroki & Heidelberger (1972) report similar results in mouse embryo cells with respect to binding to the 1 h ' protein, which they believe plays a role in chemical oncogenesis. Arene oxides react in vitro readily with poly(G), slower with poIy(A), but not with poly(U) or poly(C) (Grover 5c Sims, 1972 a). Arene oxides also behave as active alkylating agents towards p-nitrobenzylpyridine (Sims, 1972 b), a widely used model acceptor for alkylating agents. Physical affinity of arene oxides to nucleic adds (i.e. ' solu bility ' in nucleic add solution as compared to buffer solution) may be an essential prerequisite for the formation of covalent bonds since for aromatic hydrocarbons positive correlations appear to exist between this physical affinity and the chemical reactivity with nucleic acids in vivo (Brookes 5c Lawley, 1964; Goshman 5c Heidelberger, 1967) and in vitro (Grover 5e Sims, 1968). The lifetime of arene oxides, which under physiological conditions would be expected to be SL 066143 ). These ion of this or are de. gar, et al., ( >r para O; Meyer & ' cCormick, j ' ue macroj es such as | <70 b; De /, erefore be iRNA. In * if a meta- t if such a j ims, 1968; 11 bound to jre-treated known to aromatic in the a reactive the studies Iare formed .Inch bind < ,`d further- r of a co* Iained con* 1 vents and I j that in the j in vitro to 11 irbons and etabolizing henols did Jinding to a r Sc Sims, ort similar j :ein, which * ict in vitro or poly(C) | ting agents i.) cceptor for (i.e. ` soiu> inessential [ Jrocarbons ` ie chemical Goshman 1 lifetime of cted to be Epoxide Hydrates 313 in the order of minutes, should allow for diffusion from the site of formation, the endoplasmic reticulum, to nucleic adds, where close physical contact (e.g. inter* calation) may precede the establishment of covalent bonds. Alkene oxides are also substrates of glutathione 5*epoxide*transferases (Boyland & Williams, 1965) and epoxide hydrases (Oesch, et al., 1971 c and references therein) and bind covalently with DNA or related compounds (see below). Thus, they undergo metabolic reactions which are analogous to re* actions 3, 4 and 5 of the arene oxides (Fig. 1). Lawley Sc Wallick (1957) showed that ethylene oxide and propylene oxide react with gusnosine at the N~7 position. Windmueller Sc Kaplan (1961) noted that ethylene oxide reacts with adenine nucleotides predominantly at the jV-1 position. Recently, propylene oxide was * reported to bind covalently with DNA (Lawley & Jarman, 1972). At neutral pH values at 37" 7*(2-hydroxypropyl)guanine and 3-(2-hydroxypropyl)adenine hydrolysed out of the alkylated DNA. Quite a number of alkene oxides have been assessed to have strong alkylating properties using p-nitrobenzyipyridine as a model acceptor (Preussmann, Schneider & Epple, 1969). In studies on transformation and disposition of aromatic or olefinic drugs measurable amount is often unaccounted for after adding together what B' excreted from the body by various means. This missing amount may represent that which haa bound covalently as a consequence of formation of electro* V philically reactive intermediate oxiranes. This suspicion becomes even stronger, if measurable levels can be observed in tissues and if these levels are persistent, although drugs, especially those with a high lipid solubility, can also be retained in tissue fat without being covalently bound. Covalent binding of low molecular sv material to substances which play a key role in the normal functioning of a cell such as proteins, RNA and DNA, would be expected to lead to disturbances. Some adverse effects of arene oxides and alkene oxides will be discussed in X following chapter. C -deity and cytotoxicity of oxiranes !,, <e oxides have been synthetically available for a much longer time th.. ides, due to the considerably greater stability of the former, such ** alkene oxides have for a longer time been known to cause malignant tumours. Several di-epoxides having the two oxirane rings separated from each other by a ' simple aliphatic hydrocarbon chain (Hendry, et aL, 1951 ; Van Duuren, et al,, 1963, 1967 a) as well as some mono-epoxides with additional functional groups, fdr example glycidaldehyde, (Van Duuren, et al., 1967 a, b) have been reported-to be carcinogenic. Propylene oxide and ethylene oxide are mutagenic for Drosophila (Loveless, 1966). Recent indirect evidence suggests that the destruc tion of cytochrome P-450 which is observed in vivo and in vitro after administra- ( tion of several allyl-containing compounds (secobarbital, aprobarbital, alio- 1 barbital, 2-ally1-2-isopropylacetamide) is due to the formation of an alkene oxide in the ally! moiety of these compounds (De Mattcis, 1971; Levin, et al,, 1972 b; Lfcvin, Jacobson St Kunxman, 1972 a). Early investigations on the carcinogenicity of arene oxides in whole animals were inconclusive (Boyland St Sims, 1967 ; Miller St Miller, 1967 ; Sims, 1967 a ; Van Duuren, et al., 1967 a) which is not surprising considering their great instability. In order to produce adverse effects they may have to be generated 'v SL 066144 F. Ottch Table 1. Malignant transformation of cell* derived from moult protlatc by epoxide* and other derivatives of aromatic hydrocarbon* The G 23 clone of cell* derived from C3H mouie ventral prostate wa* used. . 10* cell* were plated in 60 mm dithe*. 'Teat compound* were dissolved in dimethyl aulphoxide or acetone and the cell* were treated with them for a period of 24 h beginning 24 h after plating. The compounds were then removed by media change* which were twice weekly. After 56 day* the dithe* were fixed, stained and scored for piled-up foci. Data from Maripjardt, el of., (1972). Derivative* of: Benx(a)*nlhracene Di bcnz(a ,h )anthraccnc 3-Methylcholanthrene Phcnanlhrene Number of transformed foci/number of dishes* produced by Control Parent hydrocarbon /(region epoxide 0/30 (DMSO, 0-5%) 0/30 (DMSO, 0-5%) 0/13 (1-0) 0/18 (3-0) 0/1S (10) 0/18 flO-0) 0/30 (DMSO, 0-5%) 1/13 (1-5) 10/33 (10-0) 9/19 (0-S) 23/25 (1-0) 4/20 (0-5) 9/17 (1-0) 12/13 (10-0) 38/27 (0-75) 58/26 (I S) K-region -cir- dihydrodiol 0/10(1-0) 0/10 (104)) 0/12 (141) 0/1S (1041) 0/10 (341) 0/18 0041) K-rcgion -tramt- dihydrodiol 0/10 (5-0) 0/8 (1041) 0/10 (5-0) 0/12 (10-0) 0/10 (5-0) 0/12 (100) 0/20 (Acetone, 0-3%) 0/12 (1-0) 0/12 (5-0) 0/12 (1-0) 0/15 (S-0) -- * Number* in parenthesis represent concn. of teat compound* in pg/ml. -- JC-region phenol 0/12 (1-0) 0/18 (3-0) 0/6 (141) 0/10 (S-0) 0/8 (1-0) 8/16 (2-5) 0/10 (S-0) -- SL 066145 Epoxide Hydrous 315 in situ. Recent studies in cell cultures showed more clearcut effects. Hamster embryo cells (Grover, tt al,, 1971 c) and cells derived from mouse prostate (Marquardt, et al,, 1972) underwent more malignant transformation in the presence of oxides of benz(a)anthracene, dibenz(aji)anthracene, and 3-methylcholanthrene than in the presence of their parent hydrocarbons, dihydrodiola or -ht-.ola. The 9,10-oxide of the non-cardnogenic aromatic hydrocarbon nanthrene was inactive (Table 1). The latter study also showed /C-region oxides to be more active at producing malignant transformation than non-ATregion oxides of the same hydrocarbon. The piled-up fod of these cell cultures were injected into mice and found to produce malignant tumours. Cells isolated from non-piled-up areas of carcinogen-treated dishes and cells from control dishes did not give rise to tumours. This ceil culture system therefore appears to be a valid model for carcinogenesis. Although free radicals (Nagata, Kodama & Tagashira, 1967) and carbonium ions (Dipple, Lawley & Brookes, 1968) have been invoked as reactive intermediates involved in the induction of cancer by polycyclic hydrocarbons, it now appears from the facts discussed in this and in the preceding chapter that arene oxides are the causative agents. This seems to be true at least for polycyclic hydrocarbons without substituents. Since cancer is inherited from mother to daughter cells it may be caused by mutation of the genome. Experiments with Chinese hamster cells (Hubertfism, et a/., 1971) showed that aromatic`hydrocarbons require metabolic activation in order to become mutagenic. In the benz(a)anthncene and 3-methyicfaolanp threne series the /.-region oxides were clearly more mutagenic than pererit hydrocarbons, dihydrodiola or phenols. In the dlben2(aji)anthracene series, however, the phenol appeared more mutagenic than the epoxide. JC-rcgioo oxides of dibenz(a,h)anthracenc, benz(a)anthrscene and 7-methylben2(a)anthritcene produce frame shift mutations in SahnauUm typMmssrmm whereas fMken^, hydrocarbons, AT-region dihydrodiola and phenols are inactive (Ames, Simsflt Grover, 1972). AT-region oxides are also effective mutagens in bacteriophage (Cookson, Sims & Grover, 1971). There is good, although indirect evidence that the causative agents for necrosis after treatment of animals with halobenzcnes are also arene Bromobenzene is converted by microsomes in vitro in an NADPH and Qj dependent reaction to an active intermediate which forms a covalent complex with glutathione. Pre-treatment of rats with phenobarbital, which stimulates the synthesis of microsomal enzymes involved in the oxidative metabolism of many foreign compounds (Remmer, 1959), potentiates both covalent binding of ' bromobenzene and necrosis elicited by this halobenzene, while blockage ofthis metabolism by B-diethylaminoethyl diphenyipropylacetate (SKF 525-A) : (Axelrod, Reichenthai & Brodie, 1954) prevents both binding and necrosis. Moreover, radioautography of paraffin liver sections after administration of (14C]bromobcnzene showed that the label was firmly bound at the fit** of necrosis (centrolobular zones), indicating a dose relationship between covalent binding and necrosis (Brodie, et ai., 1971 ; Mitchell, et e/., 1971). Duncan 9tf Brookes (1971) also report a positive correlation between the extent of binding of dibenz(a,c)anthracene and dibenz(a,h)anthracene and their carcinogenic potency. With mouse embryo cells in culture the binding index (i.e. the extent of binding of the hydrocarbon to the macromolecule in question resulting from metabolism of 1 nmol of hydrocarbon per ml of medium) to DNA and RNA was at least "V1 " y ve f ?A- ' SL 066146 316 F. Oesch 10 times higher for the carcinogenic dibenz(a,h)anthracene than for the noncardnogenic dibenz(a,c)anthracene. Bio-inactivation of oziranes by epoxide hydrases Several intermediate oxiranes bind firmly to proteins, RNA and DNA, cell constituents which are of prime importance for the normal functioning of the cell (see section on fate of oxiranes) and several, as would be expected, appear to be ultimate mutagens, carcinogens or cytotoxins (see section on carcinogenicity and cytotoxicity). Whether a given olefinic or aromatic com pound does produce such adverse effects may depend on the chemical reactivity of the intermediate alkene oxide or arene oxide and on the relative rate of its formation and degradation. The level and activity of epoxide hydrases, which transform such reactive oxiranes to much less reactive vicinal diols (see section on carcinogenicity and cytotoxicity) may therefore be of critical significance. The importance of epoxide hydrases for the degradation of arene oxides is illustrated by the recent observation of Grover, et al. (1972) that arene oxides can be isolated after incubation of pyrene and benzo(a)pyrene with liver microsomes in the presence but not in the absence of a potent non-competitive epoxide hydrase inhibitor, 1,2,3,4-tetrahydronaphthalene 1,2-epoxide (Oesch, et aL, 1971 c). Moreover, Sims (1970) reported that rat liver preparations transform benz(a)anthracene and dibenz(a,c)anthracene predominant! o dihydrodiols but convert dibenz(a,h)anthracene to about equal amounts c; dihydrodiols and phenols. Thus, the arene oxide(s) formed from the former two hydrocarbons appear to be much better substrates for epoxide hydrase(s) compared with those formed from dibenz(a,h)anthracene. This may be causally related to the much weaker carcinogenic activity of the former hydrocarbons compared with the latter. Although correlating these two facts largely oversimplifies the very complex events which eventually lead to cancer after exposure to a chemical carcinogen, it nevertheless shows the potential importance of this enzyme. Moreover, a great variety of arene and alkene oxides, widely differing from each other with respect to the chemical environment of the oxirane ring, are converted to the cor responding diols by liver preparations (Table 2). It therefore seems that the enzyme($) which catalyse this reaction deserve detailed investigation. Assay Arene oxides are impractical as substrates for a routine assay of epoxide hydrase activity because of their generally great instability. A sensitive and rapid radiometric assay has therefore been developed using the relatively stable [sH]styrene oxide as a substrate (Oesch, et al., 1971 a). Only after a whole complex of questions had been solved with this convenient assay was it then checked whether the information also held true for arene oxides and other alkene oxides (Oesch, Jcrina & Daly, 1971 b ; Oesch & Daly, 1972). The substrate [7-3H] styrene oxide, was synthesized from commercially available (7-1H]styrene. After incubation the unreacted substrate is extracted quantitatively into petroleum ether, while the enzymically formed styrene glycol is polar enough to be almost completely unaffected by this extraction. The low freezing point of the petroleum ether allows freezing of the aqueous phase in acetone-dry ice followed by simple decantation of the petroleum ether. After SL 066147 Epoxide Hydrates 317 thawing, the styrene glycol is extracted into ethyl acetate and an aliquot assayed by scintillation spectrometry. Radiochromatography proved the radioactivity in this extract to be solely associated with carrier styrene glycol (Oesch, it al.t 1971 a). This assay is very quick. However, for organs where epoxide hydrase is very low, this assay is not sensitive enough, the blank being the limiting factor. (With liver preparations the blank can easily be kept below 3%). The same problem was encountered when it was attempted to determine epoxide hydrase activity in needle biopsy specimens of human liver. For such problems an assay was developed with increased sensitivity at the expense of simplicity and rapidity (Oesch, Thoenen Sc Fahrlinder, 1973 c). (For epoxide hydrase assays with other substrates see Jerina, it at., 1968 c ; Leibman & Ortiz, 1968 ; Brooks, Harrison & Lewis, 1970 ; Maynert, it al., 1970 ; Oesch, it al,, 1971 b; Oesch, et at., 1971 c; Watabe Sc Akamatsu, 1972.) Distribution of epoxide hydrase Diol formation from arene or aikene oxides (Table 2) has been demonstrated almost invariably with liver homogenates or microsomes from rats, rabbits or pigs. Using the above assay with [*H]styrene oxide as substrate, quantitative data on the levels of hepatic epoxide hydrase in various species were obtained (Oesch, et at., 1971 a ; Oesch, it at., 1973 c). Figure 2 shows that levels at the human enzyme are more comparable to rodents than to Rhesus monkeys. Nb significant (0-3 > P> 0-21 differences in eooxide hydrase activity were neted Human Monkey Rabbit Guinea Pig Rot Mouse Fig. 2. Epoxide hydrate activities in liver homogenates from various species. Data from Oesch, et al. (1973 b). Aaaey with [*H]styrene oxide as substrate. F. Oesch 6^ p8 5? 0 Boylaml and Simt, 1965 b o5^ Breutr and Knupptn, 1961 Wataba,af at, 1971 b Sim*, 1972 o &> R - CHICH5)- (CH2)3-CH(CHj)j Grover, el ot, 1972 ? 00 Wataba, et at 1971 b Brooks, Ltwl* amt Horrltoa 1968 3t>Grow,to/, 1972 Brooks, 1969 Cl Sfc? Cl * Stereochemistry not Indlcotsd Brook*,at at, 1970 Brooks, et at, 1968 Brooks, at at, 1970 SL 066150 Cony, of at., 1966 <yt* cm. Moynsrt, e/at, 1970 Rj**CHs,R2-H fetch, et at, 1971 b Clayton, tt a/., 1968 R, - H, R2 CHj Clayton, et at, 1968 R,*>CzH9>R2-HO Corey, et of., 1968 Epoxide Hydrates 320 F. Oesch intestine and lung and not detectable in brain, heart, spleen and muscle. Adult mouse skin also exhibited very low epoxide hydrase activity, whereas in foetal mouse skin, the enzyme could not be detected (Oesch, et al., 1971 a ; 1973 a). After differential centrifugation of rat and rabbit liver homogenates prepared with isotonic KCI, epoxide hydrase activity was associated with mitochondrial and microsomal fractions to a similar extent on a per protein basis. However, the same was also true for a microsomal marker enzyme, glucose-6-phosphatase, suggesting heavy microsomal contamination of the mitochondrial fraction. In contrast, when isotonic sucrose was used, both epoxide hydrase and glucose-6phosphatase activities were found virtually exclusively In the microsomal fraction (Oesch, et al., 1971 a). This was also true for human epoxide hydrase (Oesch, et al., 1973). Because of the simplicity of the above assay, epoxide hydrase might be used as a marker enzyme for microsomal membranes. Properties of epoxide hydrase determined in microsomal preparations Epoxide hydrase activity in liver microsomes as determined with styrene oxide as substrate amounted to 150% of controls after pre-treatment of rats with 3methylcholanthrene and to 300% of controls after pre-treatment with phenobarbital (Oesch, et at, 1971 a). The relative increases after these pretreat ments were similar with respect tostyrene oxide, naphthalene 1,2-oxide or benzene oxide as substrates, while benzene had no effect on activity towards any of these substrates (Oesch, et al., 1973 a). During maturation of male rats an increase of hepatic epoxide hydrase by a factor of 4 took place between 28 and 40 days of age (Oesch, et al., 1971 a). These increases in specific activity of epoxide hydrases are reminiscent of similar increases in other microsomal drug metabolizing enzymes, i.e-, mono-oxygenase(s) (Conney, 1967; Nebert & Gelboin, 1969). However, the extents of induction of total levels of these two enzymes or enzyme families are not comparable (cf. Conney & Burns, 1963, and Oesch, et al., 1971 a) and are under separate genetic control (Nebert, et al., 1972 ; Oesch, et al., 1973 c). These latter observations strictly refer to total levels of epoxide hydrases, whereas certain epoxide hydrase(s) and monooxygenase(s) appear to exist as fairly stable multienzyme complexes as will be discussed below. The stereochemistry of the enzymic hydration of several oxiranes has also been studied in microsomal preparations. With naphthalene 1,2-oxide (Jerina, et al., 1970 b), and styrene oxide (Jerina, et al., 1970 c) the stereochemical course of the entering oxygen was investigated. With both substrates, attack by HtuO occurred almost exclusively at the 2-position. Formation of dihydrodiol from naphthalene by microsomes involves incorporation of oxygen from air into the 1-position and hence from water into the 2-position (Holtzman, Gillette & Milne, 1967). Thus, dihydrodiol formation from naphthalene and from naphthalene 1,2-oxide is identical with regard to the source of oxygen at the 2position proriding an additional argument for the intermediacy of oxiranes in the formation of diols from aromatic and olefinic compounds. Product glycols which arc stereochemically fixed by a ring structure invariably have the frcmr-configuration (Breuer & Knuppen, 1961 ; Leibman Sc Ortiz, 1968 ; Jerina, et al,, 1968 c; 1970 b ; Daly, et al., 1970 ; Pandov & Sims, 1970 ; Brooks, et al., 1970 ; Watsbe, et al., 1971 b ; Sims, 1971; 1972 a). Hydration k Epoxide Hydrates 321 of some acyclic alkene oxides has also been shown to proceed via a tranr-opening of the oxirane ring. Thus, threo-9,10-dihydroxy-stearic acid was produced from eir-9,10-epoxy-stearic acid, tAreo-1,2-diphenyl-1,2-ethanediol from cisstilbene oxide but meso-1,2-diphenyl-1,2-ethanediol from the traw-isomer (Watabe fit Akamatsu, 1972). It is noteworthy, that urinary diol metabolites of arenes and cycioalkenes also possess trans-configuration. The only exceptions known to-date are the metabolites of indene (Brooks -fit Young, 1956) and acenaphthylene (Hopkins, Brooks 2c Young, 1962) representing a mixture of cis~ and traw-dihydrodiols. However, even there, in all likelihood, the primary metabolites are the trans-isomers since cis- and trans-mixtures were found in rat urine after administration of the carefully purified trons-dihydrodiols (Hopkins, Lewis fie Young, 1964). With trons-indene-l,2-dihydrodiol the interconversion appears to occur through an intermediate ketol, 2-hydroxy-indan-1 -one (Lewis, 1970). The absolute stereochemistry of several trans-diols produced by the action of microsomal epoxide hydrase(s) has also been investigated. Cyclohexene oxide, benzene oxide and naphthalene 1,2-oxide are converted predominantly to If?, 22?-trons-diols whereas in the case of phenanthrene 9,10-oxide the 15,25trons-diol predominates. Optical purity varied between 30-70% with these product diols. Incubation of either naphthalene or naphthalene 1,2-oxide with liver microtomes from several species lead to diols with the same absolute stereochemistry and similar ( -- 30-50%) optical purity. Benzene dihydrodiol produced as an in vivo metabolite from benzene also had the same absolute stereochemistry as the diol produced from benzene oxide by incubation with liver microsomes (Jerina, et al., 1970 c). This implies that the enantiomers of the intermediate arene oxide are either in equilibrium with the oxepin (Vogel, et al., 1965) leading to a racemic mixture, or that the arene oxide is enzymically formed with very low stereoselectivity. The latter appears to be true for naphthalene 1,2-oxide which is not in equilibrium with the corresponding oxepin. No spontaneous racemiration of optically active naphthalene oxide takes place at neutral pH. However, the optical activity of naphthalene di hydrodiol is --10% lower when racemic naphthalene oxide is employed as substrate for microsomal epoxide hydrase in place of naphthalene oxide enzymic ally generated in situ from naphthalene. This suggests an asymetric synthesis of naphthalene oxide by microsomal mono-oxygenases of a low stereoselectivity (Boyd, et al., 1970). Due to molecular symmetry, the ease of opening of the oxirane ring of ea-stilbene oxide would be expected to be equal for either C-0 bond. However, enzymic hydration stereo-selectivity produces Ut,2R-l,2diphenyl-l,2-ethanedioI, probably due to the structure of the enzyme-substrate complex. Similarly, racemic cu-9,10-epoxystearic acid is enzymically hydrated in an optically stereoselective manner to yield the t/ireo-glycol with dextro rotatory properties (Watabe fit Akamatsu, 1972). Enzymic hydration of 2s, 3aand 2/l,3/?-epoxy-5a-choIestanc occurs with a high stereospecificity. Only one of the four possible stereoisomeric diol products was observed, namely the 2,3-trans-diaxiaI dihydroxysteroid 20,3-dihydroxy-5a-cholestane. The same product was formed from either of the two isomeric substrates although at very different rates (Watabe et al., 1971 b). Acid-catalysed hydration of these two isomeric epoxystcroids also leads to the same stereoisomer (Schoppee, Jones fit Summers, 1957). This may be an indication that hepatic epoxide SL 066152 322 F, Oesch hydraae(s) exert their catalytic effect by means of protonation of the oxirane ubatratea as a conaequence of the presence of an easily dissociable proton in the n active site of the enzyme. On the other hand, in an extensive study on structure- activity relationship with a purified epoxide hydrase preparation, the relative yr< activity of various oxiranes as substrates or inhibitors of epoxide hydrase did V , + not correlate with that expected from a mechanism invOfejM an initial acid- catalysed opening of the oxirane ring (Oesch, et aL, flmS. The enzymic hydration of indan-8,9-oxide is unique in that it occurs ^m^Ujdically by 4,9- and not by 8,9-addition of water (Daly, if aL, 1970). factors possibly prevent * normal * hydration and lead to ttereospedfic entry of water at the homo- ailylic position. ,' y5: V* '?'< v - ' '-if , ',tiV ' ir," ,X` i The arene dihydrodiol products are in turn substrates for a dihydrodiol dehydrogenase which converts them to catechols. Surprisingly, the more readily dehydrogenated isomer is the minor enantiomer formed by the action of the epoxide hydrase. This inverse correlation between the two enzymes holds true with all the substrates studied, Le. even with pbenanthrene 9,10-dihydrodiol .. in which the absolute stereochemistry of the major isomer formed by the hepatic - epoxide hydrase(s) is S,S and not R,R as in all other dihydrodiois which were investigated (Jenna, f L, 1970 c). Sohdn&tatum and purification of epoxide hydrate Hepatic epoxide hydrase is localized exclusively in the microsomal fraction (Oesch, et aL, 1971 a). Solubilization of epoxide hydrase activity from guineapig liver tnicrosomea met with difficulties. Use of hypotonic buffer or high salt concentrations (e.g. 6m guanidine hydrochloride) preserved the activity, but failed to release the enzyme from the microtomes. Attempts to prepare acetone powders were unsuccessful and use of several detergents destroyed the enzymic activity. Finally, solubilization without loss of activity was effected under optimal conditions with a neutral detergent, Cutscum. The solubilized preparation contained more than 200% of the epoxide hydrase activity originally present in the liver homogenate (Oesch 8c Daly, 1971). Epoxide hydrase activity in the homogenate was linear with respect to protein concentrations. Repeated dialysis of the liver homogenate or Sephadex G-25 chromatography of the solubilized preparation did not alter the enzymic activity and combinations of the homogenate and the solubilized preparation in various proportions always resulted in an additive activity, Le. an activity corresponding to the sura of the two components (Oesch, et aL, 1973 c). Therefore, this increase in activity . after solubilization cannot be due to the removal of an inhibitor but rather may reflect exposure of active sites by the action of the detergent. No activity was - observed in the sediment after centrifugation at 100 000 x/ for 1 h. This i* the criterion by which the preparation was called * solubilised **. Jl electron micro scopic investigations have been performed with the preparing SL 066153 n92w -<SaI < ,, e p 5 a E, ii Q 8 *.-5: Epoxide Hydrates Table 3. Purification of an epoildt hydrate from guinea pig liver Data from Ocach & Daly (1971). Fraction Protein recovery (%) Specific activity* Activity recovery (%) Liver homogenate Microsomes Supernatant after Cutscum treatment and centrifugation at 100 000 x g for 1 h Supernatant after removal of top layer and filtration (Nil,),SO, ppt. after Sephadex G-25 chromatography Calcium phosphate gel adsorption and desorption too 204 149 104) 2-5 OS 102 334 1242 1918 2837 4210 too 44-7 204-2 188-0 494 20-6 * Specific activity expressed as nmol styrene glycol/mg N/5 min. Purification factor f 3-3 12-2 18-8 27-8 41-3 SL 066154 u fci 324 F. Oefch Table 3 summarizes the purification procedure. An overall purification of 40-fold with a recovery of 20% of the epoxide hydrase activity originally present in liver homogenates was achieved (Oesch Sc Daly, 1971). The final preparation lost about 30% of its activity within 1-2 days at - IS or at 0-5. However, the remaining epoxide hydrase activity was very stable and lost virtually no (< 10%) activity during storage at -- 15s for at least 6 months. Polyacryl amide disc gel electrophoresis showed under several condft$^ns a single protein band which moved into the separating gel, in addition top^ttein which did not migrate from the stacking gel. No hydrase activity could be detected in slices of unstained gel. Sodium dodecyl sulphate gel electrophoresis allowed all protein to move into the separating gel. One major band corresponding to a molecular weight of approximately 50 000 dominated the picture. However, several minor bands were also present (unpublished results). Fig. 3. Epoxide hydrate activity in purified epoxide hydrate preparation from guinea pig liver at a function of pH. Assay with (*H]*tyrene oxide as substrate (Oesch, Jerins It Daly, 1971 a) ; phosphate ; , Tria ; A. glycine. Data from Oesch St Daly, 1971. Subsequent to our preliminary report (Oesch, et al,, 1970), a procedure was described for solubilizing and purifying epoxide hydrase from rabbit liver microsomes (Watabe St Kanehira, 1970). The method appears less satisfactory, SL 066155 irification originally The final r at 0-5*. ;t virtually Polyacryl;le protein ;h did not in slices of all protein molecular :t, several Epoxide Hydrates 325 resulting in a final purification factor from liver microsomes of 3*8 with only 5% recovery of enzyme activity. Properties of the purified epoxide hydrase Full enzymic activity is not dependent on the presence of metal ions or other low molecular weight co-factors, since chelating agents such as EDTA or *,dipyridyl, or repeated dialysis or Sephadex G-25 or G-200 chromatography had no effect on epoxide hydrase activity. Moreover, if the purified preparation was combined with the original homogenate in various proportions, the resulting activity was always additive. Carbonyl reagents such as hydroxylamine, did not influence the enzymic activity. On the other hand, sulphydryl reagents, such as mersalyl or p-chloromercuribenzoate, slightly but significantly inhibited epoxide hydrase activity, possibly indicating that sulphydryl group(s) are important for the most favourable conformation of the enzyme protein but are probably not involved in the catalytic mechanism at the active site. Product diols, such as styrene glycol and several dihydrodiois derived from arene oxides did not inhibit the enzyme even at twice (4 m.\i) substrate concentration whereas high substrate concentrations (8-16 mM styrene oxide) inhibited enzyme activity, by 20-50%. 'itinta pig liver 1971 a); , rocedure was rabbit liver s satisfactory. Fig; 4. Effect of concentration and chain length of 1 -alkanoU upon epoxide hydrate activity tcith PffJ*tyrtne oxide as substrate. Alcohols were added in 20 fd acetonitrile at aero time with no pre-incubation. Purified epoxide hydrase preparation from guinea pig liver (Oeach St Daly, 1971) was used. Assay with ['HJstyrene oxide (2 mM) aa substrate (Oeach, et ai., 1971 a). Data from Oeach, et ai. (1971 e). 4I SL 066156 1 326 F. Ouch /J-Diethylaminoethyl diphenylpropylacetate (SKF 525-A), piperonyl bytoxide and a-naphthoflavone, potent inhibitor* of several reactions catalysed by microsomal drug-metabolizing enzymes (Axelrod, tt al., 1954; Anders, 1968 ; Wiebel, tt al., 1971), had no effect on epoxide hydrase (Ocsch, tt al., 1971 a, c, 1973a,c). >^ In contrast to crude homogenates or microsomal preMgjUions, where a very broad pH profile with its optimum for enzymic hydnUwo of styrene oxide between 7 and 9 eras observed, the purified preparation displayed a sharp pH profile with its optimum at pH 9 (Fig, 3). Non-enzymic hydration of styrene oxide was significant (>5%) only below pH 6*5 (Oeach 8c Daly, 1971; Oesch, tt aL, 1973 c). 5J000 10,000 1/sH-*tyr*ns oxide] Fig. 5. Activation of tpoxidt hydras* by mttyrspear. Double-reciprocal plot of velocity (pmot styrene glycol/mg of NI5 min) versus concn. of substrate styrene oxide (m) (Oeach, st at., 1971 c). Assay as described (Oesch, tt at,, 1971 a). Metyrapone (A) was added in 20 pi acetonitrile at zero time. Purified epoxide hydrase preparation from guinea-pig liver was used (Oeach it Daly, 1971). Compounds which stimulate epoxide hydraae activity could serve as tools for the study of the rote of this enzyme in drug metabolism and they might even prove useful in alleviating or preventing mutagenic, carcinogenic or cytotoxic : effect! of aromatic or olefinic compounds which are caused by their epoxide derivatives. A number of simple alcohols such as cydohexanol, 2-cydohexen-l- 'ol and giycidol were found to activate epoxide hydrase n activation by 1-alkanola was maximal at very high (1-2 M) concentra a function of chain length (Fig. 4). Certain imidazoles, such as ylphenyl)- imidazole and certain ketones, such as -tetralone, also ie enzyme. SL 066157 .-I bytoxide talyscd by Jers, 1968; /., 1971 a, c, here a very /rene oxide a sharp pH i of styrene 71 ; Oesch, versus concn. :ribed (Oeach, at zero time. Deach tt Daly, e as tools for might even or cytotoxic heir epoxide ydohexen-1he activation as a function opylphenyl)the enzyme. Epoxide Hydrates 327 A marked activatiod was obtained with the ketonic itr-pyridine, metyrapone, whereas pyridine or nicotinamide were inactive. The stimulating effect was also completely lost on reduction of the ketonic group of metyrapone to the alcohol. Kinetic analysis of the activation of epoxide hydrase by metyrapone (Fig. 5) indicated negative co-operativity with the substrate (Oesch, et al., 1971 c, 1973 a). Attempts to stimulate epoxide hydrase in vivo so far have been un successful. Table 4. Monotubetituted oxiranes as inhibitors towards hydration of pH] styrene oxide Data from Oeach (1973 b). Substrate ([*H]styTene oxide) and inhibitor concn. 2 mM. The inhibitor was added in 20 pi acetonitrile at aero time with no preincubation. Thit amount of solvent had no significant effect on enzyme activity. Incubation mixture, conditions and assay as described (Oeach, et al., 1971 a). Inhibitor % Inhibition* of epoaide hydrate purified from liver of: Mon_________ Guineo piq Rot na ns ns na ns ns ns ns ns 12 1-2 9 2-3 17+2-1 37 t 5 2 39 + 21 43 60 59 4-4 30 + 3 1 82 3 2 cr^ 56 2-3 28 1-3 40 21 37 5-4 38 +6-2 28 2-7 51 2-1 49+ IS 52 2 2 68 43 74 9-2 79 2-3 'Significant effect with P < 0-001 (nawnot significant). SL 066158 328 F. Ouch The rapidity of the radioa$ay (Oesch, et al., 1971 a) allowed indirect de lineation of structural requirements necessary for effective interaction with epoxide hydrase by measuring the inhibitory effect of more than 100 compounds towards hydration of [*H]styrene oxide. Assays for determination of product formation were then developed for several key-compounds and their relative ability to inhibit hydration of styrene oxide compared to their relative ability to serve as substrates of epoxide hydrase. Moreover, kinetic analysis of the type 5,000 10,000 5,000 1/[H-*tyrene oimje] 10,000 Fig. 6. Inhibition of epoxide hydrate tcirh pH] ttyrtnt oxide at tubttrate. Inhibitor: (A) 4-chlorophenyt 2,3-epoxypropyl ether, (B) 1,1,1 -trichloropropene 2,3-oxide, (C) cyclohexene oxide, and (D) l,2-epoxy-l,2,3,4-tetrahydronaphthalene. Double-reciprocal plots of velocity* (pmol styrene glycol formed 'mg N'5 min) versus concn. of substrate (m). Assay as described (Oesch, et al., 1971 a). Inhibitor was added in 20 pi of acetonitrile at xero time with no pre-incubation (Oesch, et al., 1971 c). Cither 10 pi (A) or 20 pi (B-D) of purified epoxide hydrase preparation from guinea-pig liver (Oesch & D>lv, 1971) was used per inruhnton. SL 066159 ndt.*ect dei with impounds of product leir relative adve ability i of the type -el., preparation Epoxide Hydrates 329 of. inhibition was performed with several representative compounds (Oesch, et aL, 1971 b, c). Table 4 shows that mono-substituted oxiranes interact with epoxide hydrase only if they have a lipophilic substituent larger than an ethyl group such as an isopropyl, f-butyl, n-hexyl or phenyl group. This may indicate lipophilic binding sites of the enzyme near its active site. Several monosubstituted oxiranes with a large lipophilic substituent, such as n-octene 1,2oxide, styrene oxide, phenylpropene 2,3-oxide and several phenyl 2,3-epoxy propyl ethers were also demonstrated to be excellent substrates for epoxide hydrase and kinetic analysis of the inhibition exerted by one of them, 4-chIorophenyl 2,3-epoxypropyl ether, indicated that the inhibition was competitive with respect to substrate, [*H]styrene oxide, as expected (Fig. 6). The most potent Inhibitor of epoxide hydrase discovered so far is a monosubstituted oxirane 1,1,1trichloropropene 2,3-oxide. This oxirane completely inhibited hydration of [*H]styrene oxide at concentrations of one fifth of substrate. The bulk and strong electron-withdrawing effect of the trichloromethyl group as well as the oxirane ring appeared to be essential, since analogous epoxides with either the bulky f-butyl group or the smaller but strongly electron-withdrawing trifluoromethyl group were much less potent and a variety of analogous compounds containing a trichloromethyl group but no oxirane ring had no effect on the hydration of [*H]styrene oxide. This potent inhibitor was on the other hand a very poor substrate for epoxide hydrase, and kinetic analysis proved the inhibition to be uncompetitive with respect to the substrate, styrene oxide (Fig. 6) (Oesch, et al.t 1971 c, 1973 a). In a series of styrene oxides (Table S) the enzyme readily interacted with mono- and 1,1-disubstituted oxiranes, although less effectively as the bulk of the two substituents was increased. Strikingly, the enzyme interacted with 1,2disubsrituted oxiranes only if they had rir-configuration. All the above styrene oxides not only inhibited hydration of [*H] styrene oxide, but also served as substrates for epoxide hydrase. However, quantitatively their relative activity as substrates or inhibitors did not always correlate well. For more complete data and discussion see Oesch, et ai., (1971 c). With tr<mr-l,2-disubstituted, tri- and tetra-substituted oxiranes of the styrene oxide series (Table 5) no inter action was observed either with respect to inhibition of hydration of [*H]styrene oxide or with respect to product formation from these compounds. This dependency on the substitution pattern of the oxirane ring was also found with several other classes of compounds (Oesch, et aL, 1971 c). Increasing substitu tion of the oxirane ring may prevent the approach of the oxirane to the active site of epoxide hydrase by steric hindrance. Interestingly, increasing substitu tion of the oxirane ring by electron-releasing substituents including alkyl groups facilitates chemical acid-catalysed conversion of oxiranes to vicinal diols (Prichard & Long, 1956) in distinct contrast to the enzyme-catalysed hydration of oxiranes. Studies on a large series of styrene oxides with various substituents on the aro matic ring, and epoxides with various substituents on the oxirane ring, as well as several other classes of compounds, showed structure-activity relationships which did not correlate well with what would be expected from a catalytic mechanism which initiates hydration of the oxirane ring by protonation of the oxygen as a consequence of an easily dissociable proton of the active site of the enzyme, nor from a mechanism which initiates the reaction by a nucleophilic attack of water (Oesch, et aL, 1971 c). SL 066160 Liver microsomes exhibit another enzymic activity towards certain oxirane substrates, namely cyclization of endogenous and of several foreign squalene oxides to lanosterol and lanosterol analogs (Clayton, van Tamelen & Nadeau, 1968; Corey, Lin & Jautelat, 1968). The underlying catalytic mechanism might conceivably be the same as the mechanism by which epoxide hydraae catalyses the hydration of arene and alkene oxides and the two reactions might be catalysed by the same enzyme. However, the cyclase activity has the reversed order of substitution requirements for optimal substrate activity (trisubstituted > Table 5. Effect of the substitution patters of the oxirane rise on the inhibitory potency of various styrene oxides towards hydration of [*H]styrene oxide. Data from Oeach (1973 b). Substrate (pHJstyrcne oxide) and inhibitor concn. 2 mM. The inhibitor was added in 20 pi acetonitrile at aero time with no preincubation. Thia amount of solvent had no significant affect on enzyme activity. Incubation mixture, conditions and assay as described (Oeach, at/., 1971 a). Inhibitor % Inhibition* of epoxide hydrate purified from liver of:_____________ ______________________________ Mon Guinea pig Rat SI 12 1 4911-5 52X2-2 ft 3214-1 3013-1 4014 3 2413 8 22 13-4 29 1 50 1912-7 18121 16 132 46 1 3 0 74 1 6 1 73 1 4 2 2l 18-1 161 32 1412-3 SL 066161 mrirane lene u, ..m ride u;dnac ns might be the reversed jbsdtuted > j, m inhibitory to oxide. \ concn. 2 mM. i bsdon. This .don mixture, 't i j i Ret 22 Inhibitor Epoxide Hydrascs 331 Table 5 (eontd.) % Inhibition* of tpoxidt hydrase purified from liver of: ___________ Mon (Stfinto pip Rot ns ns ns ns ns ns ns ns ns ns ns ns ! 40 243 29150 J6 3*2 14 123 ns ns ns Or*"5 ns ns ns "Significant effect with P< 0-001 (nsnot significant). traiu-d(substituted > cu-disubstituted > monosubstituted oxiranes) (Clayton, et a/., 1968 ; Corey, et al,, 1968) than the ones discussed above for the hydrase activity. Moreover, during purification of epoxide hydrase, squalene oxidocydase has been virtually completely removed (Oesch & Daly, 1971) clearly demonstrating that the cyclase is a different enzyme. Cydoalkene oxides and arene oxides behaved in a similar manner to acydic alkene oxides in that tri- or tctra-substitutcd oxiranes did not (or only marginally) interact with epoxide hydrase both as substrates or inhibitors. Among a variety of cydoalkene oxides, cydohexene oxide and 1,2,3,4-tetrahydronaphthalene 1,2-epoxide were outstanding in that they were potent inhibitors while at the same time they were very poor substrates. Kinetic analysis proved the inhibition to be aon-competitive with respect to the substrate, [*H]styrene oxide (Fig. 6), thus providing an explanation for the discrepancy between activities as substrate or as inhibitors (Oesch, et al., 1971 c). SL 066162 Table 6. Epoxide analogs ms inhibitors of purified epoxide hydrases with [*H]- atyrene oxide aa substrate Data from Oeach et al. (1971 c). Several 5- and 6>membered cyclic ethers had no significant effect on epoxide hydraae activity. Subatrite ([*HJsiyrene oxide) and inhibitor eoncn. 2 rim. The purified epoxide hydraae preparation (Oeach & Daly, 1971) was used (40 /d per incubation). The inhibitor was added in 20 pi of appropriate solvent (in moat cases acetonitrile) at aero time with no preincubation. The same amount of solvent eras added the controls. Incubation mixture, conditions and assay as described (Oeach, H al.. 1971 a). Inhibitor % Inhibition* Cr0 ns ns 41 o .NH O'* NH G3 yCHgOCONHa ns ns ns (Stimulotes 45) ns ns jcri, OgN ^ 3 43 Significant effect with P< 0*001 (ns --rvot significant). SL 066163 with pHJ- ten had no id inhibitor 1) wii used nt (in moot solvent was xd (Oesch, Epoxide Hydrates 333 Sulphur, nitrogen or carbon analogues of oxirane compounds proved either inactive or less potent than the parent epoxides (Table 6). Thus, cyclopropanes and aziridines had no inhibitory activity. Styrene sulphide was quite an active inhibitor (comparable to styrene oxide) whereas cyclohexene sulphide was in* active. The former is an analogue to a competitive epoxide inhibitor (styrene oxide) whereas the latter is an analogue to a potent non-competitive epoxide inhibitor (cyclohexene oxide), thus suggesting distinctly differing structural requirements for the sites involved in competitive and non-competitive inhibition. Interestingly, m-jV-methyl-p-nitrophenyloxaziridine was quite an active in hibitor whereas the trant-isomer was completely inactive (Oesch, et al., 1971 c). This behaviour closely parallels the dependency of activity on cis- versus transstereochemistry of 1,2-disubstituted oxiranes which has been discussed above. Styrene and 1,2-dialin, olefins corresponding to a potent competitive (styrene oxide) and non-competitive inhibitor (1,2,3,4-tetrahydro-naphthalene 1,2epoxide) had weak inhibitory activities (11 and 23%, respectively, at 2 x 10~* m). Hydration of these olefins to alcohols by the purified epoxide hydrase preparation in analogy to a bacterial hydrase which adds water both to epoxides and to olefins (Niehaus, et al., 1970) could not be detected (Oesch, et al., 1971 c). Inhibitors of epoxide hydrase m vivo could be useful for assessing the role of epoxides in drug toxicity and carcinogenicity and for delineating the role of epoxide hydrase in preventing mutagenic, carcinogenic or cytotoxic effects produced by several aromatic and olefinic compounds. The most promising candidates for in vivo inhibition at epoxide hydrase were the potent oxirane in hibitors which at the same time are very poor substrates for epoxide hydrase, 1,1,1-trichloropropene 2,3-oxide and cyclohexene oxide. Besides their potent inhibitory activity towards hydration of [*H]styrene oxide they are also very potent inhibitors towards hydration of naphthalene. 1,2-oxide and benzene oxide but have, on the other hand, no effect on the mono-oxygenase activity of liver, microsomes (determined with [4-aH]benzenesulphonanilide as substrate) (Oesch, et al., 1973 a). However, all attempts to inhibit epoxide hydrase in vivo have so far failed, even using very high (toxic) doses of these potent in vitro inhibitors. Studies on biopsy specimens showed that human liver also possesses the capability to convert epoxides enzymically to vicinal diols. Human epoxide hydrase was also localized exclusively in microsomal membranes. Structureactivity relationships for substrates and inhibitors of the purified human enzyme were qualitatively identical with the ones discussed above for the purified guineapig enzyme (Tables 4 and 5). The same was true for several other properties such as pH optimum, Michaelis constant and lack of requirement for low molecular weight co-factors (Oesch, 1973 a, b). However several quantitative differences were noted between preparations solubilized and purified simultane ously from liver microsomes of guinea-pig, rat and man (Tables 4 and 5). For a more complete discussion on these aspects see Oesch (1973 b) and Oesch, et al., (1973 c). Evidence for more than one hepatic epoxide hydrase About 30% of the activity of the epoxide hydrase preparation solubilized from guinea-pig liver microsomes was lost within 24-36 h after completion of the last purification step. This occurred whether the preparation was stored \ll. 2 A. SL 06616^ 334 F. Oesch frozen (-15) or between 0 and 5s. However the remaining activity was remarkably stable. In fact, almost full activity was recovered after heating at 60s for 3 min. Moreover, no decrease in activity was observed after several freeze-thaw cycles, after preincubation for 40 min at 37" or after storage at --15" for more than 6 months (Oesch & Daly, 1971 and unpublished observations). These differential stabilities suggested the presence of more than one liver enzyme capable of hydration of epoxides. The ratio of microsomal hydrase activity towards naphthalene oxide, benzene oxide and styrene oxide was ap proximately 5 : 2: 1 in the guinea-pig (Oesch, tt al., 1971 b) but in the rat 6 : 5 : 1 (Oesch, tt al., 1973 a), suggesting the possibility that a separate enzyme might be involved in the hydration of benzene oxide. Moreover, cyclohexene oxide, another oxirane fused to a six-membered earbocyde was much more potent an inhibitor towards hydration of benzene oxide than towards hydration of styrene oxide or naphthalene 2,3-oxide. Thus, hydration of benzene oxide was com pletely prevented by cydohexene oxide at a concentration one eighth of that of the substrate whereas equimolar concentrations of cydohexene oxide inhibited hydration of naphthalene oxide or styrene oxide only by about 50% (Oesch, tt al., 1973 a). More direct evidence for the existence of a separate benzene oxide hydrase was obtained from comparing the rate at which hydration of various epoxides proceeded with the crude homogenate (600 g supernatant) and with the purified preparation. The hydration of several structurally unrelated alkene oxides and arene oxides proceeded at an approximately 30-fold enhanced rate with the purified preparation (' purification factors * of 26-30) with the single exception of benzene oxide (` purification factor ' of ,4) (Oesch, tt al., 1971 b). Thus, it appeared that on the one hand either an epoxide hydrase with a broad substrate ` specificity' was present or several more specific epoxide hydrases bad been purified to the same extent, whereas on the other hand a separate ' benzene oxide hydrase ' had been largely removed by this purification procedure. Accordingly, benzene oxide does not inhibit hydration of styrene oxide even at concentrations four-fold that of styrene oxide while other arene oxides such as naphthalene oxide or phenanthrene oxide and other alkene oxides such as octene 1,2-oxide or phenoxy epoxypropyl ethers effectively inhibit hydration of styrene oxide at concentrations smaller than that of styrene oxide (Oesch, tt al., 1971 c). This lack of cross inhibition by benzene oxide was observed not only .with the purified epoxide hydrase preparation but also with microeomes and crude homo genates (from several species) suggesting that the different purification factor with benzene oxide was not due to alterations of the enzyme protein during solubilization but rather to the presence of at least two distinct hepatic epoxide hydrases, one with a rather broad substrate specificity and another with a rather narrow substrate specificity for benzene oxide and possibly some other closely related epoxides. Attempts to isolate ` benzene oxide hydrase * are now in progress. Evidence for a coupled mono-oxygtnase-epoxide hydrate complex Spectral studies with an olefin, 5a-androat-16-en-3a-ol, and a corresponding epoxide, 16a, 17o-epoxy-5a-androstan-3wK>l, showed that both steroids interacted with cytochrome P-450 with similar affinities {Kf, ~ 45 /at) (Von Bahr, Brandt & Gustafsson, 1972) implying that epoxide hydrase and cytochrome P-450 might g], 066165 -ctivity was heating at fter several tge at --15 servations). n ne liver nal hydrase de was ap: rat 6: 5 :1 ne might be xene oxide, e potent an n of styrene e was com>f that of the .e inhibited 3esch, et al., nzene oxide i of various md with the iated alkene hanced rate da the single P, 1971 b). vith a broad de hydrases i a separate n procedure, xide even tt ides such as ch as octcne m of styrene aL, 1971 c). nly with the rrude homo* ration factor Mein during >atic epoxide vith a rather Mher closely are now in ^responding ds interacted hr, Brandt & P-450 might Epoxide Hydrases 335 posaibly be the same molecular species. Yet no mono-oxygenase activity (determined with benzo(a)pyrene as substrate after attempted reconstitution with cytochrome c reductase and a lipid fraction) nor the carbon monoxide difference spectrum of reduced active or inactivated cytochrome P-450 at 450 or 420. nm, respectively, could be observed in the purified epoxide hydrase preparation, suggesting that the two activities, epoxide hydrase and mono-oxygenase, are not common to one protein (Oesch, et al,, 1972). However, a variety of other evidence suggests the presence of a relatively stable mono-oxygenase-epoxide hydrase multienzyme complex, besides a ' free ' 'epoxide hydrase in liver microsomes. Despite the property of arene oxides to rearrange quickly to the corresponding phenols substantial amounts of dihydrodiols can be isolated from the urine of animals treated with aromatic hydro carbons (Jerina, et al., 1970 b, and references therein). This suggests a close physical relationship of mono-oxygenase(s), responsible for the formation of arene oxides, and epoxide hydrase(s), responsible for their conversion to dihydrodiols (cf. section on the fate of oxiranes). Ifsuch a coupled mono-oxygenaseepoxide hydrase system catalyses the overall conversion of naphthalene via the obligatory intermediate naphthalene 1,2-oxide (see the section on biological formation of oxiranes) to naphthalene 1,2-dihydrodiol, radioactive naphthalene oxide, generated in situ from radioactive naphthalene, should not completely equilibrate with added non-radioactive naphthalene oxide. Indeed, the specific radioactivity of naphthalene dihydrodiol was markedly greater than that of naphthol after such an experiment with guinea-pig liver homogenate*. With the same enzyme preparation, 0*5 mM 1,1,1-trichloropropene 2,3-oxide effec tively inhibited dihydrodiol formation from (exogenous) naphthalene oxide (in hibition of ' free epoxide hydrase ') but not from naphthalene (no inhibition of ` coupled epoxide hydrase'). Accordingly, when radioactive naphthalene and non-radioactive naphthalene oxide were incubated together, addition of 1,1,1trichloropropene 2,3-oxide at a concentration which selectively inhibits the ` free epoxide hydrase * (0*5 mM) produced a marked increase in specific radioactivity of the product dihydrodiol. Moreover, dihydrodiol formation from naphthalene oxide was much more enhanced after pre-treatment of rats with phenobarbital than with 3-methylcholanthrene. For the formation of dihydrodiol from naph thalene the converse was true (Oesch & Daly, 1972, and unpublished results). Solubilized and purified cytochrome P-450 and cytochrome P-448 preparations from rat liver contained high levels of both epoxide hydrase and mono-oxygenase activity, whereas other microsomal enzymes, such as glucose-6-phosphatase or cytochrome c reductase, were absent (Oesch, et al., 1972). Thus, it appears that some mono-oxygenase(s) and epoxide hydrase(s) exist as a fairly stable multi enzyme complex which in contrast to * free epoxide hydrase ' is relatively in sensitive to inhibition by 1,1,1-trichloropropene 2,3-oxide and, again in contrast to free epoxide hydrase, is induced selectively by 3-methylcholanthrene. Moreover, this complex can be purified by procedures in which other microsomal enzymes are lost. With these purified cytochrome P-450 and cytochrome * Homogenates were used since the coupled mono-oxygenese-epoxide hydrase system did not reprodudbly survive the resuspension procedure after isolation of nucrosomss. Glutathione had been removed from the homogenates by titration with diethyl melcete prior to them experiments. SL 066166 7 v -mr 336 F. Oetch P-448 preparations it was, after reconstitution with cytochrome c reductase and a lipid fraction, possible to catalyse the following reactions : 1. The mono-oxygenase reaction, namely the molecular oxygen and NADPH dependent transformation or naphthalene to naphthalene oxide and to its isomerization product naphthol. The formation of naphthalene oxide was demonstrated by radiotracer trapping technique, isomerization of the isolated naphthalene oxide and re-isolation as 1-naphthol. 2. The epoxide hydrase reaction, namely transformation of synthetic naphthalene oxide to naphthalene dihydrodiol. 3. The mono-oxygenase and epoxide hydrase reactions coupled, namely the molecular oxygen and NADPH dependent transformation of naphthalene to naphthalene dihydrodiol (Oesch, *t aL, 1972). Mono-oxygenase activity towards naphthalene was similar in the reconstituted P-450 and P-448 systems whereas towards benzo(a)pyrene the mono-oxygenase activity was more than ten-fold higher in the reconstituted P-448 system. Interestingly, the ratio of dihydrodiol to naphthol after incubation of naph thalene was markedly higher with the reconstituted P-448 system (obtained from rats pretreated with 3-methylcholanthrene) as compared to the reconstituted P-450 system (obtained from rats pretreated with phenobarbital), although total levels of epoxide hydrase (determined with naphthalene oxide or styrene oxide as substrate) were higher in the cytochrome P-450 preparation (Oesch, et al., 1972). This is in good agreement with earlier observations, that 3-methylcholanthrene selectively induces the coupled mono-oxygenase-epoxide hydrase system (Oesch St Daly, 1972), whereas total levels of epoxide hydrase are much more induced by phenobarbital than by 3-methylcholanthrene (Oesch, vt a/., 1971 a, 1973 a. The figure ia mislabelled in the first publication but the results are stated correctly in the text). Such a coupled mono-oxygenase-epoxide hydrase enzyme system may be of great relevance to several problems such as the hepatoxidty of intermediate arene oxides derived from halobenzenes and the carcinogenic properties of inter mediate arene oxides derived from several polycyclic aromatic hydrocarbons, by circumventing these adverse effects of intermediate arene oxides by their rapid conversion to dihydrodiols. Thus, modestly decreased glycogen levels and moderate centrilobular necrosis of hepatocytes after treatment of 200-220 g male Sprague-Dawley rats with a moderate dose of chlorobenzene (600 mg/kg in cottonseed oil, injected intraperitoneally 24 h before sacrifice of the animals) was dramatically increased if the animals had been pre-trcated with phcnobarbital (0-2% in the drinking water for 3 days), a potent mono-oxygenase inducer (Remmer, 1959). However, pre-treatment of the animals with 3methylcholanthrcne (40 mg/kg in cottonseed oil, one intraperitoneal injection/day for 3 days), another potent mono-oxygenase inducer (Conney, Miller & Miller, 1956), did not produce an increase and in more than 90 per cent of the animals completely blocked the necrosis and glycogen depletion after the same dose of chlorobenzene (unpublished results). Similar effects of 3-methylcholanthrene pretreatment of rats on bromobenzene-induced hepatoxidty led Jollow, Zampaglione St Gillette (1971) to suggest, that this protection from hepatotoxicity by SL 066167 uctase and 1 l NADPH I ; and to its 1 oxide was he isolated 1> 1I aphthalene I tamely the 1 .ithalene to ( 11 I i constituted 1 -oxygenase 1 48 system. I i of naph1 ained from 1 constituted I mugh total 1 ne oxide as 1 1972). I . J^LnthreQe 1 tern (Oesch I j ' induced by ; 73 a* The . ,-d correctly ' imay be of ruermediate i ies of intert'' carbons, by their rapid levels and ! .; 200-220 g : (600 mg/kg the animals) j vith pheno-oxygcnase ils with 3t - ljection/day .*r & Miller, 1 the animals tme dose of holanthrene ow, Zampaotoxicity by t j1 ' j Epoxide Hydrates 337 3-methylcholanthrene may be afforded by induction of epoxide hydrase activity. However, the results presented above show that the total levels of epoxide hydrase are increased to a much greater extent by pre-treatment of rats with phenobarbital than with 3-methylcholanthrene, yet phenobarbital does not protect from chloro benzene-evoked hepatotoxicity but rather dramatically increases necrosis of hepatocytes. The enigma may be solved, by taking into account that, while dihydrodiol formation from naphthalene 1,2-oxide with liver preparations from rats pre-treated with phenobarbital was much more enhanced than after pre- treatment of animals with 3-methylcholanthrene, for dihydrodiol formation from naphthalene the converse was true, Le. the coupled mono-oxygenase- epoxide hydrase system Is selectively induced by 3-methylcholanthrene (Oesch & Daly, 1972). Observation of increased levels of dihydrodiol and catechol in the urine of animals pre-treated with 3-methylcholanthrene as compared to controls (Jollow, et al,, 1971) is consonant with this view. Thus, it now appears, that this coupled mono-oxygenase-epoxide hydrase multienzvme complex is of great importance for the metabolism of foreign aromatic and olefinic compounds, especially with respect to shortening the lifetime of intermediate oxiranes, con verting them rapidly to dihydrodiols before they react with nucleophilic moieties of cell constituents and thereby cause cytotoxic, mutagenic or carcinogenic effects. Selective inhibitors of this coupled system may provide insight into the mechanism of these adverse reactions, whereas selective activators or inducers may even prove useful in preventing them. References Al-Kassab, S., Boyland, E. St Williams, K. (1%3). Biochtm. J., 87, 4. Abies, B. N., Sims, P. Sc Groves, P. L. (1972). Science, N. Y., 176, 47. Anders, M. W. (1968). Biochtm. Pharmac., 17, 2367. Axelrod, J., Reichenthal, J. & Brodie, B. B. (1954). J. Pharmac. exp. Ther., 112, 49. Atengar, P. K*, Hayaischx, O., Naicajima, M. & Tomida, J. (1959). Biochim. biophyt. 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