IMMUNOCHEMICAL DETECTION OF QUINOL-THIOETHER-DERIVED PROTEIN ADDUCTS

Glutathione (GSH) conjugates of hydroquinone (HQ) and 8-bromohydroquin one (2-BrHQ) produce severe renal proximal tubular necrosis in rats. S ince the reactivity of quinones lies, in part, in their ability to alk ylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone-t hioether-mediated toxicity. An immunogen was synthesized by coupling 2 -bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-li mpet hemocyanin (KLH). Anti-2-BrHQ-NAC-KLH antibodies were raised in r abbits and purified by affinity chromatography. Antibody binding to th e 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immuno sorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ- NAC. Affinity-purified anti-2-BrHQ-NAC-KLH antibodies recognized adduc ted proteins in the kidneys of rats treated with HQ,2-BrHQ, 2-bromo-bi s(glutathion-S-yl)hydroquinone 2-(glutathion-S-yl)hydroquinone, 2,5-bi s(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroqu inone. Immunoreactive proteins were found in all renal subcellular fra ctions of 2-BrHQ-treated rats, and the distribution of adducts was sim iliar to that obtained by quantifying 2-Br[C-14]HQ covalent adducts. W estern blot analysis revealed that three proteins, at 42, 46, and 79 k Da, were adducted by all the compounds examined. The identification of these adducted proteins will be required to assess their significance in quinol-thioether-mediated nephrotoxicity.