Molecular Pharmacology, Vol 14, 323-336, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Cytochrome P-450-Mediated Covalent Binding of Hexachlorophene to Rat Tissue Proteins

¹ Department of Agricultural Chemistry and Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331

Enzyme-mediated binding of radioactivity from hexachlorophene (2,2'-[¹⁴C]methylenebis[3,4,6-trichlorophenol], [¹⁴C]HCP) to rat hepatic microsomes occurred in vitro in the presence of NADPH and oxygen, and was inhibited by nitrogen, reduced glutathione, cysteine, piperonyl butoxide, and 2-diethylaminoethyl 2,2'-diphenylvalerate HCl (SKF 525-A). Binding was increased 2-fold by treatment of animals with phenobarbital (80 mg/kg intraperitoneally, daily for 3 days) and appeared to be dependent on the production of an active metabolite that was formed oxidatively in liver microsomes by a cytochrome P-450 mixed-function oxidase. Increased microsomal binding was not observed in animals treated with 3-methylcholanthrene (20 mg/kg intraperitoneally, daily for 2 days). Thin-layer chromatography of dinitrophenyl derivatives prepared from Pronase-digested microsomal protein and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of solubilized microsomes indicated that the radiolabeled products were covalently bound to amino acid residues of microsomal protein. Raney nickel desulfunization of Pronase hydrolysates released several HCP metabolites that were apparently bound to sulfur-containing amino acids such as cysteine and homocysteine. The enhancement in binding of [¹⁴C]HCP to microsomal protein in the presence of epoxide hydrase inhibitors was consistent with the formation of HCP arene oxides or structurally related species. After intraperitoneal and oral administration of [¹⁴C]HCP (5 mg/kg) to male rats treated with phenobarbital, radiocarbon was covalently bound mainly to proteins of the liver, plasma, and kidney. Covalently bound radioactivity in the liver was associated primarily with proteins in the microsomal and 105,000 x g supernatant fractions. Lesser amounts were present in the nuclear-cell debris and mitochondrial fractions.