Major isozymes of rat liver microsomal cytochrome P-450 involved in the N-oxidation of N-isopropyl-alpha-(2-methylazo)-p-toluamide, the azo derivative of procarbazine.

Seven isozymes of cytochrome P-450 were tested to establish whether they could N-oxidize azoprocarbazine to form the two isomeric 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) reductase concentration. Two isozymes, cytochromes P-450PB-C (an isozyme present in untreated rats or in rats treated with phenobarbital or beta-naphthoflavone) and P-450 beta NF-B (the major beta-naphthoflavone-induced isozyme), had appreciable turnover numbers for the N-oxidation reaction. The product ratio [N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide formation relative to N-isopropyl-alpha-(methyl-NNO-azoxy)-p-toluamide formation] obtained with cytochrome P-450PB-C was nearly identical to those values obtained with liver microsomes from untreated and phenobarbital-treated rats. In addition, cytochrome P-450 beta NF-B and liver microsomes from beta-naphthoflavone-treated rats had nearly identical product ratios. Specific inhibitory antibodies to four purified isozymes were used to titrate the N-oxidase activity of liver microsomes from untreated, phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats. Anti-cytochrome P-450PB-C globulin inhibited more than 70 to 90% of the formation of N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide in microsomes from untreated, phenobarbital-, and pregnenolone-16 alpha-carbonitrile-treated rats, respectively, but only 20 to 50% of N-isopropyl-alpha-(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 beta NF-B globulin inhibited more than 85% of the synthesis of either azoxy isomer catalyzed by liver microsomes from beta-naphthoflavone-treated rats. These results demonstrate that two isozymes are responsible for the oxidative metabolism of azoprocarbazine and can account for the major portion of this N-oxidase activity in liver microsomes from untreated and phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats.