METABOLIC N-OXIDE FORMATION BY RABBIT-LIVER MICROSOMAL CYTOCHROME-P-4502B4 - INVOLVEMENT OF SUPEROXIDE IN THE NADPH-DEPENDENT N-OXYGENATIONOF N,N-DIMETHYLANILINE

NADPH-sustained N-oxygenation of N,N-dimethylaniline (DMA) was investi gated with the aid of a reconstituted membranous cytochrome P4502B4 sy stem. The N-oxidative process did not appear to be supported by hydrox yl radicals or products arising from lipid peroxidation. However, supe roxide dismutase was a very potent scavenger of N-oxide formation, whi le catalase was ineffective. Superoxide by itself did not bring about N-oxygenation of DMA. Therefore, O2- was presumed to serve as a source of the actual proximate oxidant. The reconstituted hemoprotein system catalyzed N-oxygenation of DMA when excess H2O2 substituted for NADPH /O2. This 'peroxygenase' process was entirely dependent on the presenc e of native enzyme and was not inhibited by CO or metyrapone. By contr ast, cyanide severely blocked metabolic transformation. Among some oth er hemeproteins tested, only horseradish peroxidase was efficient in p roducing appreciable amounts of N-oxide in the presence of H2O2. Perox idatic DMA N-oxygenation in intact liver microsomes fortified with cum ene hydroperoxide was 2-fold stimulated by pretreatment of rabbits wit h phenobarbital, whereas administration of 3-methylcholanthrene or eth anol decreased turnover. Studies with uninduced hepatic microsomes, in which the activity of the flavin-containing monooxygenase had been pa rtially suppressed by thermal treatment, revealed pronounced susceptib ility of the NADPH-dependent N-oxide formation to the inhibitory actio n of both superoxide dismutase and antibody to NADPH-cytochrome P-450 reductase. These findings were interpreted to mean that at least 23% o f the total amount of N-oxide produced in these preparations resulted from superoxide-dependent conversion of DMA by the P-450 system.