Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450.
The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [( chloroethyl-3H]CP) and [4-14C]cyclophosphamide [( 4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar (Km, 0.44 and 0.42 mM; Vmax, 4.8 and 7.0 nmol of polar metabolites formed/min/nmol of cytochrome P-450 for [4-14C]CP and [chloroethyl-3H]CP, respectively). The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The order of inhibition by various mixed-function oxidase inhibitors was SKF greater than alpha- and beta-naphthoflavones greater than metyrapone. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations.
D000171:Acrolein / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D003520:Cyclophosphamide / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D000447:Aldehydes; D002250:Carbon Radioisotopes; D014316:Tritium; D000171:Acrolein; D003520:Cyclophosphamide; D012343:RNA, Transfer; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases