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 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.
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin 78712.
B A
BA
Hill
H E
HE
Kleiner
E A
EA
Ryan
D M
DM
Dulik
T J
TJ
Monks
S S
SS
Lau
D000818:Animals; D001646:Bile / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004563:Electrochemistry; D005978:Glutathione / Q000378:metabolism; D006873:Hydroquinones / Q000032:analysis* / Q000378:metabolism / Q000633:toxicity; D066298:In Vitro Techniques; D007674:Kidney Diseases / Q000139:chemically induced; D007680:Kidney Neoplasms / Q000139:chemically induced; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013438:Sulfhydryl Compounds / Q000378:metabolism
D006873:Hydroquinones; D013438:Sulfhydryl Compounds; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione