Immunochemical Analysis of Quinol−Thioether-Derived Covalent Protein Adducts in Rodent Species Sensitive and Resistant to Quinol−Thioether-Mediated Nephrotoxicity

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Abstract
2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ) is nephrotoxic in male Fischer 344 rats (20 μmol/kg) and albino guinea pigs (200 μmol/kg), but not BALB/c or B6C3F1 mice or Golden Syrian hamsters (200 μmol/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 ∼34 kDa (mitochondria), ∼35 kDa (nuclei) which comigrated with histone H1, and ∼73 kDa (urine) which comigrated with γ-glutamyl transpeptidase. These adducted proteins were not detected in other species. Qualitative differences in alkylated proteins may therefore contribute to species susceptibility to TGHQ.