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.