INHIBITION AND INACTIVATION OF CONSTITUTIVE CYTOCHROMES P450 IN RAT-LIVER BY PARATHION

Phosphorothioate pesticides, such as parathion (O,O-diethyl-O-4-nitrop henyl phosphorothioate), undergo enzymic oxidation to the active insec ticidal agents that are the analogous organophosphorus compounds. In h epatic microsomal fractions, the NADPH-mediated conversion of parathio n to paraoxon occurs with concomitant loss of cytochrome P450 (P450) a nd associated activities. In this study, the capacity of parathion to inactivate specific P450 enzymes was studied in rat hepatic microsomes . Parathion was a potent inhibitor of P450 3A2- and 2C11-mediated andr ost-4-ene-3,17-dione (androstenedione) 6beta- and 16alpha-hydroxylatio n (K(i) values of 13 +/- 2 and 2.3 +/- 0.1 mum, respectively, and K(m) /K(i) ratios of 1.4 +/- 0.2 and 11 +/- 1, respectively). After a 10-mi n preincubation between parathion and NADPH-supplemented microsomes, t o inactivate P450 before androstenedione hydroxylation was carried out , the corresponding K(m)/K(i) ratios were increased to 3.5 +/- 0.4 and 35 +/- 6, reflecting 2.5- and 3.2-fold enhancement of inhibition of P 450 3A2- and 2C11 -dependent activities. In contrast to these findings , P450 2A1/2-mediated androstenedione 7alpha-hydroxylation was refract ory to inhibition and P450 2C6-mediated progesterone 21-hydroxylation was inhibited but not inactivated by the pesticide. Further studies es tablished that androstenedione 6beta- and 16alpha-hydroxylation pathwa ys were inactivated with maximal half-times of 2.59 min and 1.72 min, respectively. Although the incubation of parathion (50 mum) with rat l iver microsomes for 1 0 min led to a 16% decrease in P450 estimated sp ectrophotometrically, immunoblot analysis revealed no change in the mi crosomal content of P450 2C11 apoprotein. Finally, NADPH-mediated meta bolism of parathion to paraoxon (by desulfuration) and 4-nitrophenol ( by oxidative cleavage of the phosphorothioate ester) occurred efficien tly in microsomes (4.32 and 4.35 nmol/min/mg of protein, respectively) . P450 loss was estimated under the same incubation conditions and, th us, 21 0 parathion molecules were oxidized for each molecule of holo-P 450 lost. These findings establish that parathion is a potent inhibito r and inactivator of the principal constitutive P450s, 3A2 and 2C11, i n rat liver, whereas the P450s 2A1 and 2A2 are refractory to either in hibition or inactivation. Another major constitutive enzyme, P450 2C6, is inhibited effectively by parathion but does not appear to be subje ct to inactivation.