Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

• Published in: Biochemistry (Easton) 1987-04, Vol.26 (8), p.2280-2289
• Main Authors: Thomas, Paul E, Bandiera, Stelvio, Maines, Sarah L, Ryan, Dene E, Levin, Wayne
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies; Antigen-Antibody Complex; Biological and medical sciences; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Enzyme-Linked Immunosorbent Assay; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Immunodiffusion; Kinetics; Male; Microsomes, Liver - enzymology; Oxidoreductases; Oxidoreductases, N-Demethylating - isolation & purification; Oxidoreductases, N-Demethylating - metabolism; Rats
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00382a031

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.