INCREASE IN LIVER MICROSOMAL GLUTATHIONE-S-TRANSFERASE ACTIVITY BY PHENOBARBITAL TREATMENT OF RATS - POSSIBLE INVOLVEMENT OF OXIDATIVE ACTIVATION VIA CYTOCHROME-P450

The possible involvement of oxidative activation of liver microsomal glutathione (GSH) S-transferase by the cytochrome P450 system was investigated. When rats were given phenobarbital (PB) intraperitoneally for 3 days, liver microsomal GSH S-transferase activity was stimulated 1.3-1.4-fold and the effect of PB on the transferase was potentiated by combination with a catalase inhibitor, 3-amino-1,2,4-triazole. Immunoblotting of microsomal proteins from PB-treated rats with anti-microsomal GSH S-transferase antibody after SDS-PAGE showed the presence of a dimer of the transferase. When microsomal suspensions prepared from PB-treated rats were placed on ice without GSH, the microsomal GSH S-transferase activity gradually increased with time and reached 200% of the initial level at 3 hr when activation of the transferase by N-ethylmaleimide was lost. The time-dependent increase in GSH S-transferase activity in PB-treated microsomes was prevented by addition of 0.1 mM GSH. The increase in microsomal GSH S-transferase activity by NADPH was depressed by cytochrome P450 inhibitors such as SKF 525-A (2-diethylaminoethyl-2,2-diphenylvalerate), metyrapone or isoniazid in agreement with the concomitant decrease in generation of hydrogen peroxide in microsomes. These results indicate that the increase in GSH S-transferase activity in liver microsomes by PB treatment of rats is due to the oxidative modification of the enzyme by reactive oxygen species which are concomitantly increased following induction of cytochrome P450.