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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.

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ) is nephrotoxic in male Fischer 344 rats (20 micromol/kg) and albino guinea pigs (200 micromol/kg), but not BALB/c or B6C3F1 mice or Golden Syrian hamsters (200 micromol/kg). Since quinones are known to alkylate proteins, and because such macromolecular damage may play a role in cytotoxicity, we investigated the covalent binding of TGHQ to kidney (target tissue) and liver (nontarget tissue) of rodents "sensitive" or "resistant" to the nephrotoxic effects of TGHQ. Immunohistochemical staining of tissue obtained 2 h after administration of TGHQ, with rabbit anti-2-bromo-N-(acetyl-L-cystein-S-yl)HQ antibodies, correlated with the subsequent region of necrosis observed 19 h after dosing in Fischer 344 rats and guinea pigs. Immunohistochemical staining was localized to the S3 segment of the renal proximal tubules, at the corticomedullary junction along the medullary rays, and in the outer stripe of the outer medulla. Immunostaining was also observed in the same region in hamsters, but subsequent necrosis did not develop. In contrast, no immunostaining was observed in mice. Moreover, immunostaining was not detected in the livers of any species. Western blot analysis revealed numerous immunoreactive renal proteins in TGHQ-treated animals. The most distinctive immunostaining renal proteins were observed in Fischer 344 rats at approximately 34 kDa (mitochondria), approximately 35 kDa (nuclei) which comigrated with histone H1, and approximately 73 kDa (urine) which comigrated with gamma-glutamyl transpeptidase. These adducted proteins were not detected in other species. Qualitative differences in alkylated proteins may therefore contribute to species susceptibility to TGHQ.

2,3,5-(Triglutathion-S-yl)hydroquinone [2,3,5-(triGSyl)HQ] is a potent nephrotoxicant when administered to male rats. We now report that significant species differences exist in susceptibility to 2,3,5-(triGSyl)HQ-mediated nephrotoxicity. Metabolism of glutathione conjugates involves cleavage of teh glutamate and glycine moieties by gamma-glutamyltranspeptidase (gamma-GT) and dipeptidases, respectively, and the nephrotoxicity of 2,3,5-(triGSyl)HQ can be prevented by the inhibition of renal gamma-GT. The resulting cysteine conjugate exhibits a balance between N-acetylation, and N-deacetylation of the mercapturic acid biosynthesis in various species contribute to species susceptibility to 2,3,5-(triGSyl)HQ. Renal gamma-GT activity toward 2,3,5-(triGSyl)HQ was highest in the rat (Fischer 344 and Sprague-Dawley) and consistent with the sensitivity of this species to 2,3,5-(triGSyl)HQ (20 micromol/kg iv)-mediated nephrotoxicity. The gamma-GT-mediated hydrolysis of 2,3,5-(triGSyl)HQ was similar in B6C3F1 and BALB/c mice and guinea pigs. In these species, the gamma-GT activity ranged between 30-45% of the activity measured in rats. Although, the activity of gamma-GT was similar in mice and guinea pigs, only guinea pigs were susceptible to 2,3,5-(triGSyl)HQ (200 micromol/kg iv)-induced renal necrosis. The gamma-GT-mediated hydrolysis of 2,3,5-(triGSyl)HQ was lowest in the hamster, and this species were not susceptible to the renal toxicity of this conjugate. Thus, factors in addition to gamma-GT activity probably contribute to species susceptibility to 2,3,5-(triGSyl)HQ nephrotoxicity. The kinetics of the AT-125-mediated inhibition of gamma-GT differed between species, indicative of potential differences in the regulation of gamma-GT. Consistent with this view, the ratio between the hydrolysis and transpeptidation of 2,3,5-(triGSyl)HQ varied 10-fold between the species examined, and was highest in the guinea pig (0.48) and lowest in the hamster (0.05). Guinea pigs also exhibited the highest renal cytosolic N-deacetylase activity and the lowest N-acetylase activity. The ratios of N-deacetylation to N-acetylation in guinea pigs, BALB/c mice, B6C3F1 mice, hamsters, Fischer 344 rats, and Sprague-Dawley rats were 4.57, 0.16, 0.14, 0.04, 0.03, and 0.02, respectively. Because quinol-cysteine conjugates seem to undergo oxidation more readily than the corresponding mercapturates, the balance of N-deacetylase and N-acetylase in the guinea pig may contribute to the susceptibility of this species to 2,3,5-(triGSyl)HQ nephrotoxicity.

Chemical reaction of 1,4-benzoquinone with GSH gives rise to several multisubstituted hydroquinone (HQ)-GSH conjugates, each of which causes renal proximal tubular necrosis when administered to male Sprague-Dawley rats. In addition, HQ has recently been reported to be nephrocarcinogenic following long-term exposure in male rats. Since neither the mechanism nor the extent of HQ oxidation and thioether formation in vivo is known, we have assessed both the qualitative and quantitative significance of HQ-thioether formation in vivo and in vitro. HQ (1.8 mmol/kg, ip) was administered to AT-125-pretreated male Sprague-Dawley rats, and bile and urine samples were analyzed with a HPLC-coulometric electrode array system (CEAS) and by liquid chromatography (LC)/continuous-flow fast atom bombardment (CF-FAB) mass spectroscopy. Five S-conjugates of hydroquinone were identified in bile, and one S-conjugate was identified in urine. The major biliary S-conjugate identified was 2-glutathion-S-ylhydroquinone [2-(GSyl)HQ] (18.9 +/- 2.7 mumol). Additional biliary metabolites were 2,5-diglutathion-S-ylhydroquinone [2,5-(diGSyl)HQ] (2.2 +/- 0.6 mumol), 2,6-diglutathion-S-ylhydroquinone [2,6-(diGSyl)HQ] (0.7 +/- 0.3 mumol),2,3,5-triglutathion-S-ylhydroquinone [2,3,5-(triGSyl)HQ] (1.2 +/- 0.1 mumol), and 2-(cystein-S-ylglycyl)hydroquinone. 2-(N-Acetylcystein-S-yl)HQ was the only urinary thioether metabolite (11.4 +/- 3.6 mumol) identified. The quantity of S-conjugates excreted in urine and bile within 4 h of HQ administration [34.3 +/- 4.5 mumol (4.3 +/- 1.1% of dose)] appears sufficient to propose a role for such metabolites in HQ-mediated nephrotoxicity and nephrocarcinogenicity. Rat liver microsomes catalyzed the NADPH-dependent oxidation of HQ (300 microM), in the presence of GSH, to form 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ. A fraction of the microsomal oxidation of HQ appears to be catalyzed by cytochrome(s) P450, although the exact amount remains unclear. 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ (300 microM) also underwent NADPH-dependent oxidation and GSH conjugation in liver microsomes. The extent of the nonenzymatic oxidation of HQ and its GSH conjugates correlated, approximately, with their half-wave oxidation potentials.