IDENTIFICATION OF MULTI-S-SUBSTITUTED CONJUGATES OF HYDROQUINONE BY HPLC COULOMETRIC ELECTRODE ARRAY ANALYSIS AND MASS-SPECTROSCOPY

Chemical reaction of 1,4-benzoquinone with GSH gives rise to several m ultisubstituted hydroquinone (HQ)-GSH conjugates, each of which causes renal proximal tubular necrosis when administered to male Sprague-Daw ley rats. In addition, HQ has recently been reported to be nephrocarci nogenic following long-term exposure in male rats. Since neither the m echanism nor the extent of HQ oxidation and thioether formation in viv o is known, we have assessed both the qualitative and quantitative sig nificance of HQ-thioether formation in vivo and in vitro. HQ (1.8 mmol /kg, ip) was administered to AT-125-pretreated male Sprague-Dawley rat s, and bile and urine samples were analyzed with a HPLC-coulometric el ectrode array system (CEAS) and by liquid chromatography (LC)/continuo us-flow fast atom bombardment (CF-FAB) mass spectroscopy. Five S-conju gates of hydroquinone were identified in bile, and one S-conjugate was identified in urine. The major biliary S-conjugate identified was 2-g lutathion-S-ylhydroquinone [2-(GSyl)HQ] (18.9 +/- 2.7 Amol). Additiona l biliary metabolites were 2,5-diglutathion-S-ylhydroquinone [2,5-(diG Syl)HQ] (2.2 +/- 0.6 mumol), 2,6-diglutathion-S-ylhydroquinone [2,6-(d iGSyl)HQ] (0.7 +/- 0.3 Amol), 2,3,5-triglutathion-S-ylhydroquinone [2, 3,5-(triGSyl)HQ] (1.2 +/- 0.1 mumol), and 2-(cystein-S-ylglycyl)hydroq uinone. 2-(N-Acetylcystein-S-yl)HQ was the only urinary thioether meta bolite (11.4 +/- 3.6 mumol) identified. The quantity of S-conjugates e xcreted 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 nephrocarcinoge nicity. Rat liver microsomes catalyzed the NADPH-dependent oxidation o f HQ (300 muM), in the presence of GSH, to form 2-(GSyl)HQ, 2,5-(diGSy l)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 amo unt remains unclear. 2-(GSyl)HQ, 2,5-(diGSyl)HQ, and 2,6-(diGSyl)HQ (3 00 muM) also underwent NADPH-dependent oxidation and GSH conjugation i n liver microsomes. The extent of the nonenzymatic oxidation of HQ and its GSH conjugates correlated, approximately, with their half-wave ox idation potentials.