Covalent Protein Adducts of Hydroquinone in Tissues from Rats: Identification and Quantitation of Sulfhydryl-Bound Forms

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Abstract
The Michael-type addition of sulfhydryl groups to benzoquinone (BQ) or substituted benzoquinones is proposed as the primary mechanism by which these electrophilic intermediates react with either cellular glutathione or protein sulfhydryls. This reaction constitutes a reductive alkylation with a substituted hydroquinone (HQ) derivative resulting from the addition. In the case of HQ, oxidative conversion of the parent material to BQ followed by conjugation with glutathione leads to metabolic activation, producing intermediates which are nephrotoxic as well as having other proposed biological activities. Chemically, BQ may react with more than 1 equiv of glutathione (or other sulfhydryl reagents) to produce HQ derivatives substituted with up to four sulfhydryl groups. Similarly, multiply substituted protein−S adducts of HQ were anticipated to occur in vivo following administration of this material. In the current studies, sulfhydryl-bound HQ protein adducts were detected and quantitated in protein isolated from rats using a modification of the alkaline permethylation procedure of Slaughter and Hanzlik [(1993) Anal. Biochem.208, 288−295]. In particular, total protein−S adducts to HQ in kidney or blood reached a level of 420 or 80 pmol/mg of protein, respectively, 6 h following a single gavage dose of 100 mg/kg HQ. Measured half-lives of protein−S adducts in kidney and blood were 23.9 and 36.0 h, respectively. The applicability of protein−S adducts as a tissue dosimeter for HQ is discussed.