Epoxide Hydrolysis in the Cytosol of Rat Liver, Kidney, a N D Testis

-The hydrolysis of transand cts-stilbene oxide and benzo[a]pyrene-4,5-oxlde was measured m cytosol and mlcrosomes of liver, kidney, and testis of control and clofibrate-fed rats Significant levels of nonprotein sulfhydryls were detected in cytosol from hver (4.6 mM) and testis (1 5 mM) Glutathxone was moderately stable m these fractions and Interfered with the partmon assays as conjugates were retained in the aqueous phase along with diols When the products were separated by thin-layer chromatography, slgmficant amounts of glutathione-conlugates were found to have been formed in the cytosol of liver and testis Overnight dialysis or premcubatmn of cytosol with 0 5 mM &ethylmaleate ehmlnated conjugate formation without affecting diol production In dialyzed cytosol from clofibratefed rats (0 5%, 14 days), the rates of hydrolysis of trans-stdbene oxide were 506, 171, and 96% of controls for hver, kidney, and testis, respectively, and 126% of controls m hver mlcrosomes Rates of hydrolysis of cts-stdbene oxide were 149, 172, and 96% of controls m mlcrosomes and 154, 124, and 91% of controls m cytosols from livers, kidneys, and testis of clofibrate-fed rats respectwely Hydrolysis of benzo[a]pyrene-4,5-oxlde was similar to that of cls-stflbene oxide ConJugation of the cts-stllbene oxide with glutathlone was detected m cytosols from all three t~ssues w~th lesser amounts m the mlcrosomes from hver and kidneys After clofibrate treatment, the rates of th~s activity were 200. 173, and 95% of controls m cytosol from hver, kidneys and testis, and 203 and 202% of controls In mlcrosomes from liver and kidneys respectively These results indicate that epoxlde hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other ewdence which suggests that hydrolysis of ctsand trans-stdbene oxides m cytosol IS catalyzed, in part. by &stmct enzymes Hydrolysis of the oxlrane (epoxide, arene oxide) moiety is an important degradat lve route of metabohsm for this potential ly toxic functional group. Three distinct epoxlde hydrolases ( E H t , E C 3.3 2.3, epoxide ether hvdrolases) have now been described in mammal ian tissue Two are predominant ly located in the mlcrosomal cell fraction and Include a broadrange xenoblot lc metabolizing E H referred to as microsomal E H ( m E H ; [1-5]), and a second enzyme which appears specific for delta-5-sterolds [6, 7]. The third enzyme is also a broad-range xenoblot ic metabohzing E H predominant ly localized in the cell sap and referred to as cytosohc E H (cEH; [4, 5]). Prehminary evidence has been presented from a variety of sources [8-11] that another E H with some similarities to m E H exists within the cytosol of mammahan cells. Besides hydrolysis, epoxldes also can be degraded enzymatically by reduct ion and conjugat ion with glu* Address correspondence to Davtd E Moody, Ph D , Department of Entomology, Umverslty of Cahfornm, Davis, CA 95616 + Abbrewatlons EH, epoxlde hydrolase, cEH, cytosohc epoxlde hydrolase, mEH, mlcrosomal epoxlde hydrolase, GST. glutathlone S-transferase, CSO, c~s-stllbene oxide, TSO, trans-stllbene oxide, BPO. benzo[a]pyrene-4,5oxide, GSH, glutathmne, NPS, nonprotein sulfhydryl, and DEM. &ethylmaleate tathlone (GSH) The former pathway has only been described for a limited number of epoxldes. The latter activity is associated with a family of Isozymes with broad substrate specificity [12] referred to as glutathlone S-transferases (GST, E C 2 5 1.18). Recent ly radlometr lc parti t ion [13-15] and contlnuous spectrophotometr lc [16] assays have been developed that can distinguish be tween hydrolysis and glutathlone conjugat ion of the same epoxldes In cytosohc fractions. When trans(TSO) and ctsstllbene oxide (CSO) are used, c E H and m E H also can be distinguished by their respective substrate speclfiCXtles [14, 15] As the majori ty of reduced GSH is present in the cytosollc cell fraction, it is concelvable that conjugat ion with GSH or potentially other nonprotein sulfhydryls (NPS) could compete with hydrolysis of epoxldes as a degradatlve pathway within this cell fraction Tests for this competi t ion have seldom been carried out [13, 16], but they may be of importance when conducting m vttro assays for E H , particularly if significant G S H levels are preserved in the cytosohc cell fraction in question This compet i t ion could be of particular Importance when comparing E H actlwtles in different tissues or in tissues from control versus t reated animals Several stu&es have shown that m E H and GST can be induced by t rea tment with several diverse xenobIotlcs, which in general also induce the microsomal polysubstrate-m~xed-functlon-oxldase system