Mammalian Epoxide Hydrases: Inducible Enzymes Catalysing the Inactivation of Carcinogenic and Cytotoxic Metabolites Derived from Aromatic and Olefinic Compounds

Abstract

1. Several aromatic and olefinic compounds are converted to intermediate arene and alkene oxides by mammalian mono-oxygenases. Intermediate arene oxides rearrange non-enzymically to phenols. Arene and alkene oxides are converted by epoxide hydrases to vicinal diols and by glutathione S-epoxide conjugases to glutathione conjugates. Due to their high electrophilic reactivity, such oxiranes also bind to proteins, RNA and DNA. Mutagenic, carcinogenic and cytotoxic effects of several aromatic and olefinic compounds appear to be due to the formation of intermediate epoxides and their reaction with tissue constituents. 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 the corresponding phenol and on its chemical electrophilic reactivity.2. Epoxide hydrases, which convert such intermediate oxiranes to much less reactive vicinal diols, have been studied in greater detail. Epoxide hydrase 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 lung and not detectable in muscle, spleen, heart and brain. The enzyme is located exclusively in microsomal membranes. Epoxide hydrase activity is markedly increased after pretreatment of rats with phenobarbital or 3-methyl-cholanthrene and during maturation of rats. These increases are reminiscent of similar increases in microsomal mono-oxygenases. However, the extents of induction of total levels of these two enzymes or enzyme families are not comparable and are under separate genetic control.3. Several stereochemical properties of the reaction catalysed by epoxide hydrases have been studied with microsomal preparations. With styrene oxide and naphthalene oxide as substrates, attack by H218O occurs virtually exclusively at the 2-position. Product glycols which are stereochemically fixed by a ring structure invariably have the trans-configuration. Hydration of some acyclic alkene oxides has also been found to proceed via a trans-opening of the oxirane-ring. Cyclohexene oxide, benzene oxide and naphthalene 1,2-oxide are converted predominantly to 1R,2R-trans-diols, while in the case of phenanthrene 9,10-oxide the 1S,2S,-trans-diol predominates.4. Epoxide hydrase from guinea-pig liver microsomes was solubilized and purified, based on an assay with styrene oxide as substrate. The specific activity after the last purification step is about 40 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 is lost within 1–2 days. However, the remaining activity is remarkably stable. Gel electrophoresis of the final (stable) preparation shows 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 clearcut 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 KM with respect to styrene oxide as substrate is 2–8 × 10−4M and the apparent Vmax. 2–4 μmol product/mg N per 5 min. No metal ions or other low mol. wt. co-factors are necessary for 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 butoxide, α-naphthoflavone) do not influence epoxide hydrase activity, while sulphydryl reagents slightly, but significantly, inhibit the enzyme. Several alcohols, ketones and imidazoles stimulate the enzyme. Kinetic analysis of the activation by the potent stimulator, metyrapone, indicates negative co-operativity with the substrate.6. The active site of the enzyme readily accommodates, as substrates or competitive inhibitors, monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group, suggesting hydrophobic binding sites near the active site. With oxiranes having such a lipophilic substituent, the enzyme interacts with mono-, 1,1-di- and cis-1,2-disubstituted oxiranes, but not with trans-1,2-disubstituted oxiranes or tri- or tetra-substituted oxiranes, suggesting that increasing bulk around the oxirane ring prevents the approach of the oxirane to the active site. Several oxiranes fused to alicyclic rings (cyclohexene oxide, 1,2,3,4-tetrahydronaphthalene 1,2-epoxide) are potent inhibitors but very poor substrates. Kinetic analysis revealed non-competitive inhibition with respect to the substrate, styrene oxide. Styrene sulphide (an analogue of the competitive inhibitor, styrene oxide) but not cyclohexene sulphide (an