Chem. Res. Toxicol. 2001, 14, 25-33
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Carcinogenicity of a Nephrotoxic Metabolite of the “Nongenotoxic” Carcinogen Hydroquinone Serrine S. Lau,*,† Terrence J. Monks,† Jeffrey I. Everitt,‡ Elena Kleymenova,§ and Cheryl L. Walker§ Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712-1074, Chemical Industry Institute for Toxicology, Research Triangle Park, North Carolina 27709-2233, and Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957 Received July 31, 2000
Hydroquinone (HQ) is a potential human carcinogen to which many people are exposed. HQ generally tests negative in standard mutagenicity assays, making it a “nongenotoxic” carcinogen whose mechanism of action remains unknown. HQ is metabolized to 2,3,5-tris(glutathion-Syl)HQ (TGHQ), a potent toxic and redox active compound. To determine if TGHQ is a carcinogen in the kidney, TGHQ was administered to Eker rats (2 months of age) for 4 or 10 months. Eker rats carry a germline mutation in the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene, which makes them highly susceptible to the development of renal tumors. As early as 4 months after the initiation of treatment (2.5 µmol/kg, ip), TGHQ-treated rats developed numerous toxic tubular dysplasias of a form rarely present in vehicle-treated rats. These preneoplastic lesions are believed to represent early transformation within tubules undergoing regeneration after injury by TGHQ, and adenomas subsequently arose within these lesions. After treatment for 10 months (2.5 µmol/kg for 4 months followed by 3.5 µmol/kg for 6 months), there were 6-, 7-, and 10-fold more basophilic dysplasias, adenomas, and renal cell carcinomas, respectively, in TGHQ-treated animals than in controls. Most of these lesions were in the region of TGHQinduced acute renal injury, the outer stripe of the outer medulla. Loss of heterozygosity (LOH) at the Tsc-2 locus was demonstrated in both the toxic tubular dysplasias and tumors from rats treated with TGHQ for 10 months, consistent with TGHQ-induced loss of tumor suppressor function of the Tsc-2 gene. Thus, although HQ is generally considered a nongenotoxic carcinogen, our data suggest that HQ nephrocarcinogenesis is probably mediated by the formation of the quantitatively minor yet potent nephrotoxic metabolite TGHQ, which induces sustained regenerative hyperplasia, loss of tumor suppressor gene function, and the subsequent formation of renal adenomas and carcinomas. In addition, our data demonstrate that assumptions regarding mechanisms of action of nongenotoxic carcinogens should be considered carefully in the absence of data on the profiles of metabolites generated by these compounds in specific target organs for tumor induction.
Introduction Hydroquinone (HQ) is used as a developer in the photographic industry, as an antioxidant in the rubber industry, and as an intermediate in the manufacturing of food antioxidants. HQ is also an important metabolite of benzene (1, 2). HQ has been identified in relatively high concentrations in the smoke of unfiltered cigarettes (up to 155 µg per cigarette) (3) and was chosen for study by the National Cancer Institute because it is produced
in large quantities, humans are frequently exposed to it, and there are little adequate carcinogenicity data on it (4). Smoking unfiltered cigarettes and long-term cigarette smoking (g30 years) correlate with a high incidence of renal cell carcinoma (RCC) in men (5-7). Although the basis for the increased incidence of renal tumors in cigarette smokers is not known, cigarette smoke contains high concentrations of oxidants and free radicals, the principal radical in the tar phase being the 1,4-benzoquinone/hydroquinone redox couple (8).
* To whom correspondence should be addressed: Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074. Phone: (512) 471-5190. Fax: (512) 471-5002. E-mail:
[email protected]. † The University of Texas at Austin. ‡ Chemical Industry Institute for Toxicology. § The University of Texas M. D. Anderson Cancer Center. 1 Abbreviations: BrdU, bromodeoxyuridine; γ-GT, γ-glutamyl transpeptidase; OSOM, outer stripe of the outer medulla; Tsc-2, tuberous sclerosis 2; PCR, polymerase chain reaction; LOH, loss of heterozygosity; RCC, renal cell carcinoma; VHL, von Hippel-Lindau; HQ, hydroquinone; TGHQ, 2,3,5-tris(glutathion-S-yl)HQ; ip, intraperitoneal; HPLC, high-pressure liquid chromatography; GAP, GTPase activating protein; H&E, hematoxylin and eosin.
Although HQ is generally not mutagenic in short-term bacterial mutagenicity assays (9, 10), and no mutagenic activity has been found in mouse cells in vivo (11), HQ causes base pair changes in the TA1535 Salmonella typhimurium test strain (12) and is mutagenic in oxidantsensitive (TA104 and TA2637) Salmonella test strains (13), consistent with the mutagenicity of 1,4-benzoquinone in several Ames bacterial test strains (14). HQ is also clastogenic and induces sister chomatid exchange (4, 12, 15), catalyzes the in vitro formation of 8-oxode-
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10.1021/tx000161g CCC: $20.00 © 2001 American Chemical Society Published on Web 12/14/2000
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Chem. Res. Toxicol., Vol. 14, No. 1, 2001
oxyguanosine (16, 17), and causes single-strand DNA breaks in isolated hepatocytes (18). In addition, HQ causes renal tubular cell degeneration in the renal cortex of male Fischer 344 rats and, at high doses, markedly increases the number of tubular cell adenomas (4). Renal cell tumors also arise in animals exposed to HQ, but only at nephrotoxic doses and only in male rats (4, 19) and mice (19). HQ also acts as a tumor promoter, in a manner depending on the target organ and on the initiation protocol used (20). Neither the mechanism of HQ-mediated nephrocarcinogenicity in male rats nor the basis for the species and sex differences is known. The acute nephrotoxicity of HQ is dependent upon the activity of γ-glutamyl transpeptidase (γ-GT), which indicates that HQ’s toxicity is dependent upon the formation of metabolites that are substrates for this enzyme (21). Consistent with this view, GSH conjugates of HQ, in particular 2,3,5-tris(glutathion-S-yl)HQ (TGHQ), are potent nephrotoxicants (21, 22), and HQ is metabolized in vivo to GSH conjugates in amounts sufficient to support their role in the acute nephrotoxic effects of HQ (23). Spontaneous RCC is rare in rats, occurring in most strains with a frequency of
