HEPATIC BIOTRANSFORMATION OF PARATHION - ROLE OF CYTOCHROME-P450 IN NADPH-MEDIATED AND NADH-MEDIATED MICROSOMAL OXIDATION IN-VITRO

In vitro studies have established that cytochrome P450 (P450) is deact ivated by the electrophilic sulfur atom released during the enzymic ox idation of parathion to paraoxon. However, in vivo studies in rats hav e been unable to demonstrate significant P450 loss. This study evaluat ed the possibility that there may be alternate pathways of parathion b iotransformation in liver, other than those mediated by P450 and suppo rted by NADPH. Initial experiments confirmed that parathion administra tion did not decrease microsomal P450 or testosterone hydroxylation ac tivities. Subsequent in vitro experiments identified an NADH-dependent pathway of parathion biotransformation, and MS was used to confirm th at paraoxon and 4-nitrophenol were the products of both the NADH- and NADPH-dependent reactions. The Michaelis constants of the NADH-depende nt formation of paraoxon and 4-nitrophenol (26 +/- 6 mu M and 53 +/- 1 0 mu M, respectively) were approximately 3-fold greater than those for the NADPH-supported reactions (9 +/- 1 mu M and 18 +/- 3 mu M, respec tively). Induction of male rats with phenobarbital and dexamethasone, but not P-naphthoflavone, produced similar increases in the rates of N ADH- and NADPH-mediated parathion metabolism. Rates of NADH- and NADPH -dependent metabolism were highly correlated in linear relationships. An anti-NADPH-cytochrome P450 reductase (NADPH-P450 reductase) antibod y partially inhibited microsomal parathion oxidation mediated by eithe r cofactor, and the P450 inhibitor clotrimazole was similarly effectiv e against the NADH- and NADPH-supported oxidation of parathion. Finall y, a reconstituted system containing P450 2B1, NADPH-P450 reductase, a nd phospholipid supported parathion oxidation mediated by NADH. Michae lis constants for NADH-supported parathion metabolism to paraoxon and 4-nitrophenol were almost an order of magnitude greater than those far NADPH-supported metabolism Consistent with this finding, P450 inactiv ation by NADH was observed, but only at higher cofactor concentrations (>2 mM), which suggests that the same mechanism may be operative in t hionosulfur oxidation mediated by both cofactors. Considered together, these findings indicate that NADH and NADPH are both cofactors for th e P450-mediated metabolism of parathion in rat hepatic microsomes. Rat liver reportedly contains higher concentrations of NAD(H) than NADP(H ), but these are lower than the concentrations required for P450 loss. Thus, it is conceivable that the NADH-mediated reaction may function in vivo and serve to protect P450 from deactivation.