Characterization of Rat Cytochrome P450 Isozymes Involved in the Covalent Binding of Cyclosporine A to Microsomal Proteins

Abstract

Cyclosporin A (CsA) is an immunosuppressant drug which is extensively metabolized by the hepatic microsomal monooxygenases. Among other toxicities, CsA is nephrotoxic and hepatotoxic. In the present study, the NADPH-dependent cytochrome P450-supported metabolism of CsA to reactive metabolite(s) capable of covalently binding to proteins was studied. The covalent binding was inhibitable in vitro with classical cytochrome P450 inhibitors. The covalent binding of CsA metabolite(s) was induced six- to eightfold in liver microsomes from rats of both sexes treated with dexamethasone, suggesting that a P450 3A-related protein was involved in the covalent binding of CsA metabolite(s). However, the isozyme responsible was not P450 3A1 or 3A2, since inhibitory monoclonal antibodies to these isozymes did not inhibit the covalent binding. The binding was, however, inhibitable in vitro with cytochrome P450 3A substrates and inhibitors such as erythromycin and triacetyloleandomycin. Greater amounts of CsA covalent binding occurred in liver microsomes from adult uninduced female rats than males or immature rats of either sex. Therefore, a female-specific isozyme of P450 present in adult female rat liver microsomes, which may or may not be identical to a dexamethasone-inducible isozyme, is also involved in the metabolism of CsA to form covalent binding metabolites. The covalent binding of CsA was 50% inhibited by glutathione. However, mannitol and superoxide dismutase did not affect the binding. This suggested that at least some of the metabolites of CsA involved in covalent binding were electrophilic in nature; however, hydroxyl radicals and superoxide anion radicals were not involved.