Determination of cytochrome P-448 activity in biological tissues.
Three enzymes used for the determination of cytochrome P-448 activity, namely aryl hydrocarbon hydroxylase, biphenyl 2-hydroxylase and ethoxyresorufin O-de-ethylase, were evaluated with respect to their specificity, sensitivity and inducibility. Purified cytochrome P-448 (LM4), but not cytochrome P-450 (LM2), catalysed the O-de-ethylation of ethoxyresorufin in a reaction that was markedly inhibited by 9-hydroxyellipticine. After the administration of 3-methylcholanthrene to rats all three activities were induced, the extent of induction being highest for ethoxyresorufin O-de-ethylase. Administration of very small doses of benzo[a]pyrene (50 micrograms/kg) to rats to induce cytochrome P-448 specifically increased only the O-de-ethylation of ethoxyresorufin. 3-Hydroxybenzo[a]pyrene, the major metabolite determined by the aryl hydrocarbon hydroxylase assay, undergoes further NADPH-dependent oxygenation leading to loss of fluorescence. On the basis of these observations and those by other workers, we conclude that ethoxyresorufin O-de-ethylase provides the most specific, sensitive and reproducible means of determining cytochrome P-448 activity.
D000818:Animals; D001564:Benzo(a)pyrene; D001580:Benzopyrenes / Q000494:pharmacology; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003580:Cytochromes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004611:Ellipticines / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008722:Methods; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D001580:Benzopyrenes; D003580:Cytochromes; D004611:Ellipticines; D010078:Oxazines; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; C007649:ethoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C008009:9-hydroxyellipticine; D006899:Mixed Function Oxygenases; C017170:Cyp1a2 protein, rat; D019388:Cytochrome P-450 CYP1A2