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Perspective Perspective: Ochratoxin A Is Not a Genotoxic Carcinogen Robert J. Turesky* Division of Environmental Disease Prevention, Wadsworth Center, NYS Department of Health, Albany, New York 12201 Received March 16, 2005
1. Introduction 2. Mutagenicity of OTA 3. Metabolism of OTA by Xenobiotic Metabolism Enzymes (XMEs) and Formation of Reactive Oxygen Species (ROS) 4. Pro-Oxidant and Biological Effects of OTA 5. DNA Adduct Formation of OTA 6. Conclusions
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1. Introduction Ochratoxin A (OTA)1 is a mycotoxin produced as a secondary metabolite by some Penicillium and Aspergillus fungal species (1), which may contaminate grains, bread, cereals, and other common food staples (2-5). OTA is nephritogenic, teratogenic, and immunosuppressive, and it is a potent renal carcinogen in experimental laboratory animals (3), which has led to regulatory control of this contaminant in foods. The toxicological data show large species and gender differences in OTAinduced nephrotoxicity and carcinogenicity (3, 6). The pig is the species that is most susceptible to the nephrotoxic effects of OTA, and the toxicity data of this species have been used to establish a provisional tolerable weekly human intake of 100 ng OTA/kg body weight, taking into account a 500-fold safety margin (7). OTA is also a potent renal carcinogen in the male rat with a no effect level of 21 µg/kg body weight (8, 9). Because of this potent carcinogenic effect, the proposed tolerable intake of OTA is controversial and continues to be a subject of debate. The daily human intake of OTA in Western European countries and North America has been estimated to range * To whom correspondence should be addressed. Tel: 518-474-4151. Fax: 518-486-1505. E-mail:
[email protected]. 1Abbreviations: AFB , aflatoxin B ; HRP, horseradish peroxidase; 1 1 IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; B[a]P, benzo[a]pyrene; DA, dark Agouti; OTA, ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy-3methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); OTB, ochratoxin B (N-{[(3R)-8-hydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); OTR, ochratoxin R [N-[(3R)-5-chloro-8hydroxy-3-methyl-1-oxo-7-isochromane-7-carboxylic acid]; OTQ, ochratoxin quinone, (N-{[(3R)-3-methyl-1,5,8-trioxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); OTA-GSH, ochratoxin A-glutathione conjugate; dG-C8-OTA, ochratoxin A-deoxyguanosine; PCBs, polychlorinated biphenyls; PCP, pentachlorophenol; ROS, reactive oxygen species; XME, xenobiotic metabolism enzyme.
from 0.7 to 4.7 ng/kg body weight, depending upon the country and upon seasonal variations (3, 4). OTA exposure has been linked to a fatal human kidney disease called endemic Balkan nephropathy (BEN), which is characterized by an increased incidence of tumors of the urinary tract (10, 11). Several DNA adducts of human kidney and bladder samples from BEN subjects display Rf values similar to those obtained from mouse kidney after treatment with OTA when assayed by 32P-postlabeling and led the investigators to postulate that OTA is involved in the etiology of kidney tumors in BEN subjects (11). However, other studies have concluded that OTA alone may not cause BEN, but it may act synergistically with other environmental toxicants and mycotoxins, heavy metals, and/or predisposing genotypes to provoke this disease. Alternatively, OTA may not be involved at all in BEN (5, 12, 13). IARC has classified OTA as a possible human carcinogen (group 2B) based on sufficient evidence for its carcinogencity in animal studies, although the evidence for the carcinogenicity of OTA in humans is inadequate (14). The mode of carcinogenic action of OTA is unknown, but the accumulation of R2uglobulin in kidney of male rats, which has been implicated in the higher susceptibility of male rats than female rats to some renal toxicants (15), does not appear to be involved in the carcinogenicity of OTA (16). Elucidation of the mechanism of OTA-mediated carcinogenesis in experimental animal models is important if we are to assesss the true human health risk of OTA.
2. Mutagenicity of OTA The literature on short-term bacterial mutagenicity tests on OTA is inconsistent. OTA is not mutagenic in most microbial and mammalian gene mutation assays, either with or without exogenous metabolic activation, and only medium derived from OTA-exposed rat hepatocyes was reported to provoke a mutagenic effect in the Ames reversion assay (17). However, another study was unable to reproduce this mutagenic effect (18). Very high concentrations of OTA (2-4 mM) were required to induce SOS DNA repair activity in Escherichia coli strain PQ37, in a study conducted in the absence of exogenous metabolic activation (19). The presence of Trolox C, a water soluble form of vitamin E, completely quenched the genotoxicity, suggesting that a free radical was the genotoxic intermediate. The investigators concluded that the C-5 chlorine atom of OTA was necessary for radical
10.1021/tx050076e CCC: $30.25 © 2005 American Chemical Society Published on Web 06/30/2005
Perspective
Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1083 Scheme 1. Chemical Structures of OTA and Its Major Metabolites
formation and DNA damage. OTA treatment also caused an induction of unscheduled DNA synthesis in rat hepatocytes, porcine urinary bladder epithelial cells, and primary human urothelial cells (20, 21), but whether these effects were due to OTA or OTA metabolites is not known. DNA strand breaks also occurred in spleen, kidney, and liver of mice given elevated concentrations of OTA (2.5 mg/kg body weight) (22) and similarly in kidney and liver of rats following treatment of OTA for 12 weeks at dose levels equivalent to 4 ppm (23). DNA strand breaks also formed in a dose-dependent manner in rat liver, kidney, and spleen, as assessed by the comet assay (0-2.0 mg/kg body weight for 2 weeks) (24), and the extent of DNA damage was higher in the presence of formamidopyrimidine-DNA glycosylase (Fpg), which converts oxidative and abasic site-damaged DNA into DNA strand breaks. Unexpectedly, the dechlorinated analogue ochratoxin B (OTB), which is considered to be much less toxic than OTA (3), also catalyzed oxidative DNA strand breakage at levels comparable to those induced by OTA. The overall conclusion from these studies is that OTA is not a mutagen, but it is clastogenic to mammalian cells (3, 18, 25, 26). Two recent studies have proposed that OTA is mutagenic in bacteria and mammalian cells, but there are controversies surrounding the data. OTA was reported to induce revertants in the Ames reversion assay using Salmonella typhimurium tester strains TA1535 and TA1538 with metabolic activation by mouse kidney but not liver microsomes fortified with arachidonic acid or NADPH as cofactors, leading to the conclusion that OTA undergoes bioactivation by both prostaglandin synthase (PGHS) and cytochrome P450s (P450) in kidney (27). However, rat kidney microsomes, derived from the most sensitive species and the target organ of OTA-mediated carcinogenesis, failed to induce revertants under the same conditions (28). de Groene and co-workers reported that OTA induced mutagenicity in NIH/3T3 cell lines expressing human P450s 1A1, 1A2, 2C10, and 3A4 and a shuttle vector containing the lacZ′ as the reporter gene, whereas neither vector-infected NIH3T3 cells nor P450 2D6- or 2E1-expressed cells displayed an increase in
mutation frequency (29). However, the level of P450catalyzed metabolism of OTA in these cells was very low, and there were no detectable differences between the various cell lines in the extent of OTA metabolism that could account for the observed differences in mutagenicity (de Groene, E. Personal communication). The analysis of the mutants revealed predominantly large deletions, a finding that again is consistent with the occurrence of single strand breaks and not DNA adducts (30).
3. Metabolism of OTA by Xenobiotic Metabolism Enzymes (XMEs) and Formation of Reactive Oxygen Species (ROS) A number of studies have been conducted on the metabolism of OTA to gain insight into whether the carcinogenicity of OTA is based upon the toxicant’s genotoxic properties and ability to covalently bind to DNA, following biotransformation by P450s and/or peroxidases. Several pathways of OTA metabolism have been identified in vitro, with rabbit, mouse, rat, and human liver microsomal samples and in experimental laboratory animals (Scheme 1). The P450-mediated oxidation of OTA occurs at very low rates with these microsomal samples (
