Abstract
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.
Highlights
Several studies have implicated cytochrome P-450/448 in the Essentially exclusive metabolism-dependent binding oxidative detoxification and activation of carcinogens, teratoto microsomal proteins of 14C from [4-’4C]cyclopho~- gens, mutagens, and toxicants
Since cytochrome P-450 is composed of an apoprotein and heme embedded in a lipophilic environment, interaction of the reactive intermediates with either of these components may lead to inactivation
Some of the suggested mechanisms include: generation of free radicals which initiate peroxidative destruction of the cytochrome as in the case of carbon tetrachloride [15, 16], epoxide formation from double and triple bonds leading to the production in vitro as well as in viva of “green pigments” via alkylation of the heme moiety of the cytochrome [4, 6,7,8,9, 47], chelation of heme iron by metal chelators [5], covalent binding of the cytochrome apoprotein sulfhydryl groups by elemental sulfur produced during the metabolic desulfuration of sulfur-contaming chemicals [10,11,12,13,14]
Summary
Comparison ofr3HJ-and [14C]Cyclophosphamide Binding to Microsomal Protein-Hepatic microsomes were incubated for 1h, in the presence or absence of NADPH, with [chloroe t h ~ l - ~ Ho]r- [4-14C]cyclophosphamideand covalent binding of the isotope to the microsomes and the radioactivity remaining in the aqueous phases after chloroform extraction were estimated. Formation of Schiff base between dialdehyde cleavage products of aflatoxin BZaand free amino groups in proteins has been reported [42] Since both acrolein and aldophosphamide would contain I4C derived from cyclophosphamide isotopically labeled with I4Cin the ring (Fig. I),these metabolites may beinvolved in the microsomal binding of 14C if the formation of Schiff bases is involved. R~~~~~ 4 ~ , 3 ~Picomoles metabolite/ml As % of total in aqueous phase aqueous phase
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