Vol. 293, Issue 3, 1112-1120, June 2000

Inactivation of Hepatic CYP2E1 by an Epoxide of Diallyl Sulfone¹

Peter D. Premdas, Raymond J. Bowers and Poh-Gek Forkert

Departments of Anatomy and Cell Biology (P.D.P, P.G.F.) and Chemistry (R.J.B.), Queen's University, Kingston, Ontario, Canada; and Colour Your Enzyme (R.J.B.), Bath, Ontario, Canada

Diallyl sulfone (DASO₂) inhibits hepatic CYP2E1. In this investigation, we have tested the hypothesis that an epoxide formed from DASO₂ is responsible for inactivation of hepatic CYP2E1 in mice. An epoxide of DASO₂ (1,2-epoxypropyl-3,3'-sulfonyl-1'-propene; DASO₃) was synthesized and conjugated to glutathione (GSH) to produce the conjugates S-(1R,S-[[ 1-hydroxymethyl-2,3'-sulfonyl]-1'-propenyl]ethyl)glutathione (diastereomers) and S-(1-[[2R,S-hydroxypropyl]-3,3'-sulfonyl]-1'-propenyl)glutathione (diastereomers). Their identities were confirmed by ¹H NMR analysis, and these were used as analytical standards. HPLC analysis revealed a major peak for the GSH conjugates that eluted at 20.5 min. This peak was detected in liver microsomal incubations performed with DASO₂ in the presence of NADPH. A similar peak also was detected in incubations of CYP2E1-expressed lymphoblastoid microsomes, NADPH and DASO₂. The generation of the epoxide-derived GSH conjugates in the microsomal incubations was concentration-dependent, and reached saturation at 0.75 to 1.0 mM DASO₂. Formation of the conjugates was also time-dependent and peaked at 2.0 h after DASO₂. Levels of DASO₃ formed from DASO₂, as estimated by production of a 4-(p-nitrobenzyl)pyridine derivative, were maximal at 1 mM DASO₂ at 30 min. CYP2E1-dependent p-nitrophenol hydroxylase activity was decreased in microsomes incubated with DASO₂, with alterations that were proportional to the concentration of DASO₂ (0.25-1.0 mM) used. Dose-dependent decreases in hydroxylase activity also were found in microsomes from mice treated in vivo with DASO₂ (25-200 mg/kg). These DASO₂-induced decreases corresponded with reduced amounts of immunodetectable CYP2E1. Levels of spectrally detectable P450 and heme were both diminished by DASO₂. These results supported the contention that an epoxide formed from DASO₂ mediates the inactivation of hepatic CYP2E1.