ALDRIN EPOXIDATION, A HIGHLY SENSITIVE INDICATOR SPECIFIC FOR CYTOCHROME-P-450-DEPENDENT MONO-OXYGENASE ACTIVITIES

  • 1 January 1979
    • journal article
    • research article
    • Vol. 7  (5) , 301-305
Abstract
Metabolic activation of lipophilic chemicals by hepatic monooxygenases plays a crucial role in the initiation of toxic processes like chemical carcinogenesis, bacterial mutagenesis and chemcially induced liver-cell necrosis. Aldrin epoxidation was studied in rat liver microsomes. The assay is very sensitive; amounts of less than 1 .mu.g 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 expoxidation 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 .mu.M. The Km in microsomes from PB-treated animals was 28 .mu.M. The same or similar form(s) of mono-oxygenase catalyze(s) the epoxidation in the 3 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 reudce 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 wk of age. The rate of benzo[a]pyrene hydroxylation remained unchanged during this period. Aldrin epoxidation offers a selective and sensitive assay for the activity of mono-oxygenases dependent on cytochrome P-450 forms.