Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion.
1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide. 2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6beta-hydroxylations were similarly susceptible to inhibition by parathion (Km/Ki ratios 1.5-1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6beta-hydroxylation. Comparison of Km/Ki ratios indicated that 6beta-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (Km/Ki ratio of 8.0 versus 4.0). 3. The time-dependent loss of AD 6beta-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1 min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67 microM in microsomes from DEX-induced and control rats. 4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16beta-hydroxylation was inhibited potently in a reversible fashion by parathion (Ki = 0.37 microM; Km/Ki ratio about 73). Inhibition was not enhanced at parathion concentrations near the Ki by a preincubation step with NADPH. 5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (Ks=7.7 microM, deltaAmax=2.2 x 10(-2) a.u.nmol CYP-1; deltaAmax/Ks 2.86 x 10(3) a.u. nmol CYP(-1)/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively. 6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.
Murray
M
M
Faculty of Pharmacy, University of Sydney, NSW 2006, Australia. michaelm@pharm.edu.au
Butler
A M
AM
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006900:Hydroxylation; D066298:In Vitro Techniques; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010278:Parathion / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007306:Insecticides; D000735:Androstenedione; D009249:NADP; D010278:Parathion; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System