INFLUENCE OF MODULATORS OF EPOXIDE METABOLISM ON THE CYTOTOXICITY OF TRANS-ANETHOLE IN FRESHLY ISOLATED RAT HEPATOCYTES

The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 x 10(-4) to 5 x 10(-3) M, with concentrations of 10(-3) M and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10(-4) M) and cyclohexene oxide (10(-3) M), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 x 10(-4) M). Cellular glutathione was depleted by diethyl maleate (5 x 10(-4) M), and its synthesis inhibited by 2.5 x 10(-3) M-L-buthionine (S,R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 x 10(-4) M) showed a rapid increase in cytotoxicity when 4-fluorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl maleate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.