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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.

19F NMR spectroscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N alpha-acetyl-N epsilon-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by 19F and 13C NMR spectroscopy and mass spectrometry. N alpha-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation was greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However N alpha-acetyllysine, at concentrations of greater than 100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Both stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated nephrotoxicity.

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