Rat liver glutathione S-transferase-catalyzed conjugation of glutathione to the endogenous epoxides of oleic acid and cholesterol

cis-9,10-Epoxy-octadecanoic acid (oleic acid epoxide, OAE) and 5 alpha,6 alpha-epoxy-cholesterol (ChE) are endogenous epoxides. Unlike other epoxides, the oxirane groups of OAE and ChE are relatively stable against nucleophiles. OAE lacks toxicity and mutagenicity, while ChE is considered harmful, mutagenic and cancerogenic to animals. In humans, ChE is associated with cancer. The metabolism of OAE and ChE includes hydrolysis by cytosolic and microsomal hydrolases to their diols and glutathione (GSH) conjugation by GSH S-transferases (GST) to form the GSH conjugates (R-SG; R, residue). The GST-catalyzed GSH conjugation of OAE and ChE is poorly investigated. This article reports on the GSH conjugation of OAE, its methyl ester (OAEMe) and of ChE by rat liver homogenate GST. The GSH conjugates of OAE, OAEMe and ChE, i.e., OAE-SG, OAEMe-SG and ChE-SG, respectively, were determined by pre-column derivatization with o-phthaldialdehyde (OPA)/2-mercaptoethanol, high-performance liquid chromatography (HPLC) and fluorescence detection. Complex biphasic kinetics were observed with substrate inhibition of GST activity by OAE, OAEMe and ChE, an optimum pH of about 8.3 for OAE, and no measurable chemical GSH conjugation, underlying the importance of GST for the biotransformation of these epoxides. The results confirm the substrate concentration-dependent kinetic mechanism of GST isoforms first reported by William B. Jakoby (J. Biol. Chem. 1974) for exogenous electrophiles including the epoxide 1,2-epoxy-3-(p-nitrophenoxy)propane and the organic nitrates. This mechanism allows for maximal GST activity that can be achieved under given concentrations of GSH, epoxides and other electrophiles.