The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

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Abstract
The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5‐oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg‐yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in sifu in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5‐oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30–50% and towards androstene oxide 60–90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg‐yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibiton of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes. Kinetic evidence strongly suggests that at least two different binding sites on the enzyme molecule mediate the observed effects: the substrate‐binding site (competitive inhibition) and a further binding site distinct from the substrate‐binding site. Moreover, the effects of the four modulators on microsomal epoxide hydrolase from rat, mouse, rabbit and man were similar, indicating that the major functions of the enzyme (at least those investigated in this study) were quite well conserved between these species during evolution.

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