Characteristics of the active oxygen in covalent binding of the pesticide methoxychlor to hepatic microsomal proteins

Abstract

This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the Superoxide anion (O − 2) or a species generated from O − 2 was examined with the help of Superoxide dismutase (SOD) and with an SOD-mimetic agent, CuDIPS [Cu 2+(3,5-diisopropylsalicyclic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn 2+(3,5-diisopropylsalicyclic acid);], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O 2 − was involved in covalent binding, but was not accessible to SOD. Additional support for O 2 − involvement stems from the observation that a-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H 2O 2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H 2O 2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe 2− needed for generation of OH is much more easily obtained from Fe 3+-EDTA than from Fe 3+-desferal, which resists reduction. The inhibitory effect by DETAPAC may be due to chelation of another metal which is needed for the reaction. Lastly, certain scavengers of singlet oxygen inhibited covalent binding with little effect on the formation of polar metabolites of methoxychlor. In conclusion, these studies support the involvement of OH and singlet oxygen, possibly derived from O − 2, in the formation of the reactive methoxychlor intermediate. However, the less likely possibility that these compounds, used to probe for active oxygen, merely inhibit the activity of a cytochrome P-450 which specifically catalyzes the formation of the reactive methoxychlor intermediate but not of the demethylation of methoxychlor cannot be ruled out.