Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization.
The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.
Voorman
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Department of Biochemistry, Michigan State University, East Lansing 48824.
Aust
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D000818:Animals; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction; D006207:Half-Life; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D011075:Polybrominated Biphenyls / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012451:Safrole / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000378:metabolism
D011075:Polybrominated Biphenyls; D000072317:Polychlorinated Dibenzodioxins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase; C032790:3,3',4,4',5,5'-hexabromobiphenyl; D012451:Safrole; C015959:isosafrole