Major Isozymes of Rat Liver Microsomal Cytochrome P-450 Involved in the /V-Oxidation of AMsopropyl-a-(2-methylazo)-p-toluamide, the Azo Derivative of Procarbazine

Seven isozymes of cytochrome P-450 were tested to establish whether they could W-oxidize azoprocarbazine to form the two isomerie azoxy metabolites after optimizing the reconstitution of various purified isozymes with regard to substrate concentration, exogenous lipid, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c (P-450) reducÃ-aseconcentration. Two isozymes, cytochromes P-450PB-c (an isozyme present in un treated rats or in rats treated with phénobarbitalor 0-naphthoflavone) and P-450„NF-B (the major 0-naphthoflavone-induced iso zyme), had appreciable turnover numbers for the W-oxidation reaction. The product ratio [A/-isopropyl-«-(methyl-ONN-azoxy)p-toluamide formation relative to A/-isopropyl-«-(methyl-NNOazoxy)-p-toluamide formation] obtained with cytochrome P450pB-c was nearly identical to those values obtained with liver microsomes from untreated and phenobarbital-treated rats. In addition, cytochrome P-450„NF-B and liver microsomes from ßnaphthoflavone-treated rats had nearly identical product ratios. Specific inhibitory antibodies to four purified isozymes were used to titrate the A/-oxidase activity of liver microsomes from un treated, phénobarbital-, pregnenolone-16a-carbonitrile-, or ßnaphthoflavone-treated rats. Anti-cytochrome P-450PB-cglobulin inhibited more than 70 to 90% of the formation of W-isopropyl«-(methyl-ONN-azoxy)-p-toluamide in microsomes from un treated, phénobarbital-, and pregnenolone-16a-carbonitriletreated rats, respectively, but only 20 to 50% of A/-isopropyl-a(methyl-NNO-azoxy)-p-toluamide formation. A small amount (25 to 30%) of inhibition was observed with anti-cytochrome P-450pB/ PCN-E globulin. Anti-cytochrome P-450„NF-B globulin inhibited more than 85% of the synthesis of either azoxy isomer catalyzed by liver microsomes from /3-naphthoflavone-treated rats. These re sults demonstrate that two isozymes are responsible for the oxidative metabolism of azoprocarbazine and can account for the major portion of this A/-oxidase activity in liver microsomes from untreated and phénobarbital-, pregnenolone-16«-carbonitrile-, or ß-naphthoflavone-treated rats.