Characterization of a novel cytochrome P450 from the transformable cell line, C3H/10T1/2.
The cytochrome P450 in the transformable C3H/10T1/2 (10T1/2) cell line has been characterized and compared to the major polycyclic aromatic hydrocarbon (PAH)-inducible hepatic form, cytochrome P450IA1 (P450IA1). The mouse hepatoma cell line, Hepa-1, was used as an in vitro model for P450IA1 expression and regulation by PAH. Microsomes from uninduced and benz[a]anthracene (BA)-induced 10T1/2 cells provided PAH mono-oxygenated product profiles that were totally different from metabolite profiles produced by microsomes from uninduced and BA-induced Hepa-1 cells even though total activities were similar. The proximate carcinogen, 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) was a major product for the 10T1/2 microsomes, while Hepa-1 formed less than 2% of this metabolite. Hepa-1 converted benzo[a]pyrene (BP) to BP-4,5-diol and DMBA to 7-hydroxymethyl-12-methyl-BA, while 10T1/2 did not produce either product. Polyclonal antibody to rat hepatic P450IA1 did not inhibit metabolism of either PAH substrate by 10T1/2 microsomes, but totally inhibited such metabolism by Hepa-1 microsomes. Western immunoblot analysis of BA-induced 10T1/2 microsomes showed that less than 1% of total P450 was P450IA1. The PAH-metabolizing activity of 10T1/2 microsomes was highly inducible (14-fold) by pre-treatment of non-confluent intact cells with BA, but was only half as inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, the P450IA1 activity of Hepa-1 cells was highly inducible by both compounds. The distinct metabolite profiles, antibody inhibition data and lack of immunoreactivity all indicate that PAH metabolism in 10T1/2 cells is catalyzed by a form of P450 distinct from P450IA1. The anomalous induction patterns suggest that this novel isozyme is predominantly regulated by a mechanism other than the Ah receptor.
Pottenger
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Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Jefcoate
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D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000818:Animals; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D001564:Benzo(a)pyrene / Q000378:metabolism; D002461:Cell Line, Transformed; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D004851:Epoxide Hydrolases / Q000494:pharmacology; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification*; D051379:Mice; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000502:physiology
D001551:Benz(a)Anthracenes; D007527:Isoenzymes; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D001564:Benzo(a)pyrene; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; C030935:benz(a)anthracene; D004851:Epoxide Hydrolases