Aldrin epoxidation, a highly sensitive indicator specific for cytochrome P-450-dependent mono-oxygenase activities.
Aldrin epoxidation was studied in rat liver microsomes. The assay is very sensitive; amounts of less than 1 microgram of microsomal protein were sufficient for activity determination. The very low background, stability of the metabolite, and the complete separation of substrate and metabolite permit estimation of mono-oxygenase activities of less than 1 pmol per mg of protein per min by a simple procedure. Pretreatment of animals with the mono-oxygenase inducer phenobarbital (PB) increased the epoxidation rate 3-fold, whereas 3-methylcholanthrene (MC) treatment markedly depressed enzyme activity. Induction with MC did not change the apparent Km of the reaction, which was 18 muM. The Km in microsomes from PB-treated animals was 28 muM. These data suggest that the same or (a) similar form(s) of mono-oxygenase catalyze(s) the epoxidation in the three different microsomal preparations. SKF 525-A, metyrapone, and 7,8-benzoflavone were almost similarly active as inhibitors in microsomes from control and inducer-treated rats. Sensitivity to these inhibitors was low; 0.7 mM SKF 525-A and 0.4 mM 7,8-benzoflavone were necessary to reduce enzyme activity by 50%, whereas 0.5 mM metyrapone caused an inhibition of 10-45%. The activity of aldrin epoxidation in untreated rats increased almost parallel to the activity of ethylmorphine demethylation between 3 and 10 weeks of age. The rate of benzo[a]pyrene hydroxylation remained unchanged during this period. The results demonstrate that aldrin epoxidation offers a selective and sensitive assay for the activity of mono-oxygenases dependent on cytochrome P-450 forms.
D000375:Aging; D000452:Aldrin / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D004852:Epoxy Compounds / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000254:growth & development; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013997:Time Factors
D004852:Epoxy Compounds; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D000452:Aldrin