Immunochemical detection of quinol--thioether-derived protein adducts

Glutathione (GSH) conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) produce severe renal proximal tubular necrosis in rats. Since the reactivity of quinones lies, in part, in their ability to alkylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone-thioether-mediated toxicity. An immunogen was synthesized by coupling 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-limpet hemocyanin (KLH). Anti-2-BrHQ-NAC-KLH antibodies were raised in rabbits and purified by affinity chromatography. Antibody binding to the 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ-NAC. Affinity-purified anti-2-BrHQ-NAC-KLH antibodies recognized adducted proteins in the kidneys of rats treated with HQ, 2-BrHQ, 2-bromo-bis(glutathion-S-yl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2, 5-bis(glutathion-S-yl)hydroquinone, and 2,3, 5-tris(glutathion-S-yl)hydroquinone. Immunoreactive proteins were found in all renal subcellular fractions of 2-BrHQ-treated rats, and the distribution of adducts was similiar to that obtained by quantifying 2-Br[14C]HQ covalent adducts. Western blot analysis revealed that three proteins, at 42, 46, and 79 kDa, 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.