Studies on the mechanism of denaturation of cytochrome P-450 by cyclophosphamide and its metabolites.

Several lines of investigation were pursued to understand mechanisms involved in the in vivo depression of rat hepatic microsomal mixed function oxidase by cyclophosphamide, an important anti-cancer and immunosuppressive agent. Essentially exclusive metabolism-dependent binding to microsomal proteins of 14C from [4-14C]cyclophosphamide, compared with 3H from [chloroethyl-3H]cyclophosphamide, suggests the binding of the metabolite acrolein. Of the various metabolites and analogs of cyclophosphamide tested (which did not contain a peroxy or a hydroperoxy group), only acrolein and 4-hydroxycyclophosphamide (which releases acrolein in solution) caused denaturation of microsomal cytochrome P-450 in vitro; this denaturation was identical with that produced by sulfhydryl reagents. Of the various chemicals tested, only those which contained either a free amino group (except lysine) and/or a free sulfhydryl group (e.g. semicarbazide, cysteine, glycine, glucosamine) effectively blocked (40-80%) the binding of 14C as well as protected against acrolein-induced denaturation of cytochrome P-450. These data further suggested interaction of cyclophosphamide metabolite with free amino and/or free sulfhydryl groups in proteins. However, comparison with [3H]aflatoxin B2a which interacts with free protein amino groups via the formation of Schiff bases, clearly attributed the preferential binding of 14C to cysteine sulfhydryl groups in these proteins. Studies on chemical models derived from reaction between acrolein and cysteine also supported this suggestion. When microsomes isolated from incubations metabolizing [4-14C]cyclophosphamide were subjected to gel electrophoresis, the major radioactive band detected by autoradiography was associated with a cytochrome P-450 band at 55,000 daltons, the major band induced by phenobarbital in the rat. All these results taken together strongly point to the possibility that acrolein is the cyclophosphamide metabolite responsible for the depression of the mixed function oxidase activities. Acrolein most likely produces this effect by alkylation of the sulfhydryl group(s) in the active site of cytochrome P-450.