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Oxidative Metabolism of 1-Nitropyrene by Rabbit Liver Microsomes and Purified Microsomal Cytochrome P-450 Isozymes¹

Department of Environmental Health Sciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Rabbit liver (male) microsomal metabolism of 10 µM [4,5,9,10-³H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 ± 0.45, 3.98 ± 0.19, 3.90 ± 0.16, and 3.75 ± 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored 1-nitropyren-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored 1-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and -naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (<1 µM), and an activator of form 3c at high concentrations. The presence of -naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54% of the metabolites were accounted for as 1-nitropyren-3-ol, while with form 3b, 73% of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.

¹ These studies were supported by NIH Grant ES03648 (P. C. H.) and Grant AA07219 from the National Institute of Alcohol Abuse and Alcoholism (D. R. K.).

² To whom requests for reprints should be addressed.

³ Recipient of Undergraduate Summer Fellowship Award.

Received 1/ 7/88. Revised 4/ 8/88. Accepted 4/25/88.