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A Case for the Genotoxicity of Ochratoxin A by Bioactivation and Covalent DNA Adduction Richard A. Manderville* Department of Chemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada Received March 8, 2005
1. 2. 3. 4. 5. 6.
Introduction Bioactivation of OTA DNA Adduction Model for OTA-Mediated Genotoxicity Opportunities for Future Studies Conclusions
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1. Introduction Ochratoxin A (OTA;1 see Scheme 1) is a mycotoxin that originates from several species of Aspergillus and Penicillium fungi and is a common contaminant of foodstuffs (1-4). OTA is nephrotoxic, hepatotoxic, teratogenic, and immunotoxic to several species of animals. It also causes kidney tumors in rats (5-7). In humans, OTA is classified by the International Agency for Research on Cancer (IARC) as a possible carcinogen (group 2B), based on sufficient evidence for carcinogenicity in animal studies and inadequate evidence in humans (8). OTA is associated with Balkan endemic nephropathy (BEN), which is a chronic kidney disease characterized by renal failure and high incidences of urinary tract tumors; BEN is restricted to areas of the Balkans where high levels of OTA are found in food (9, 10). In Europe, estimates of daily mean dietary human intake of OTA range from 0.15 to 2.51 ng/kg of body weight from food analyses, with cereal products being the main contributor (11). Because of the persistence of OTA in the food chain, exposure to the toxin is a potential human health hazard and this has prompted adoption of regulatory limits in several countries (3). The Joint FAO/WHO Expert Committee on Food Additives and Contaminants has set a provisional tolerable weekly intake of OTA at 100 ng/kg body weight (12). However, this assessment used nephrotoxic effects in pigs as the end point and the acceptable intake levels of OTA are hotly debated because of the growing concern about OTA-mediated genotoxicity and its mechanism of action as a carcinogen. Two general hypotheses have been advanced concerning the mechanisms of genotoxic activation. The first notion suggests that OTA induces genotoxicity by initiat* To whom correspondence should be addressed. Tel: 519-824-4120 ext. 53963. Fax: 519-766-1499. E-mail:
[email protected]. 1Abbreviations: OTA, ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy3-methyl-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); OTHQ, ochratoxin hydroquinone (N-{[(3R)-5,8dihydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl- L alanine); OTQ, ochratoxin quinone (N-{[(3R)-3-methyl-1,5,8-trioxo-7isochromanyl]carbonyl}-3-phenyl-L-alanine); 4-(R)-OH-OTA, 4-(R)hydroxy-OTA; 4-OH-OTB, 4-hydroxy-OTB; OTR, ochratoxin R [(3R)5-chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carboxylic acid]; OTAGSH, ochratoxin A-glutathione conjugate; OTA-dG, carbon (C)-bonded ochratoxin A-deoxyguanosine adduct; AMS, accelerator mass spectrometry; PCP, pentachlorophenol.
ing a flux of reactive oxygen species (ROS) that promotes oxidative DNA damage and oxidative stress. In this model, the toxin does not interact with DNA directly and formation of ROS need not involve bioactivation of OTA as the initiation event. The second notion suggests that OTA undergoes bioactivation to form electrophilic species that react with DNA directly to generate covalent DNA adducts. While unequivocal proof of OTA-mediated DNA adduction is still lacking, the recent characterization of several biomarkers for OTA bioactivation and covalent reaction with DNA supports the argument that DNA adduction contributes to OTA-mediated genotoxicity. These advances are presented in this perspective, which makes a case for the genotoxicity of OTA by bioactivation and covalent DNA adduction.
2. Bioactivation of OTA The metabolism of OTA has been studied using in vitro and in vivo assays in both liver and kidney (13-27). As outlined in Scheme 1, major metabolites include the following: hydroxylated derivatives 4(R)-, 4(S)-, and 10hydroxyochratoxin A (see Scheme 1 for numbering in OTA) that are most likely formed by P450 (13, 16, 20, 22) or possibly peroxidases [prostaglandin-H-synthase (PGHS) and/or lipoxygenases] present in kidney (18, 21); OTR (22), which stems from cleavage of the peptide bond in OTA by carboxypeptidase A (28). The nonchlorinated analogue ochratoxin B (OTB) (17-21, 27) has also been identified in metabolic studies. How this metabolite is derived from OTA has not been established, although El Adlouni et al. have noted that OTB formation is related to P450 2C9, which is closely linked to OTA genotoxicity (20). The yields of these metabolites {OTR (1%), 4-(R)hydroxy-OTA [4-(R)-OH-OTA] (2.5%), and OTB (2%)} are low (18), and in general, they are more rapidly eliminated than OTA, which provides one explanation for their lower toxicity (22, 23). Trace amounts of the hydroquinone analogue ochratoxin hydroquinone (OTHQ) have also been detected in urine of rats treated with OTA (2 mg/ kg body wt) for 2 weeks (27). Pentose and hexose conjugates (22, 23) and a lactone-opened ring, which have also been detected in hepatocytes and urine of OTAtreated animals, form from hydrolysis of the lactone moiety (23). The metabolism of OTB also generates hydroxylated species such as 4-hydroxy-OTB (4-OH-OTB) (24). On the basis of structure-activity relationships, Malaveille and co-workers proposed that the presence of the C-5 chlorine atom in OTA is one determinant of its genotoxic action (29, 30). We speculated that the parachlorophenolic moiety of OTA would undergo oxidative dechlorination to afford the quinone species ochratoxin quinone (OTQ) (see Scheme 2), which in the presence of
10.1021/tx050070p CCC: $30.25 © 2005 American Chemical Society Published on Web 07/01/2005
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Scheme 1. Metabolism of OTA
Scheme 2. Proposed Pathways for the Bioactivation of OTA
reducing agents would yield OTHQ (Scheme 1). Electrochemical methods (31), treatment of OTA with a biomimetic iron-oxo system (32), or redox active transition metals (33) have provided indirect evidence for the intermediacy of OTQ in OTA oxidation. Further evidence was derived from characterization and identification of ochratoxin A-glutathione conjugate (OTA-GSH) (Scheme 2) that is produced at a rate of ∼1-3 pmol min-1 (mg of protein)-1 from treatment of 100 µM OTA in the presence of 5 mM GSH following incubation for 1 h with rat liver microsomes (34). Using our MS data, Mally et al. have confirmed that OTA-GSH is detectable by LC-MS/MS following in vitro activation of OTA in the presence of GSH (27). Our laboratory has also examined the photoreactivity of OTA, and this has provided insights into OTA chemistry (35-37). Photoirradiation of OTA generates the nonchlorinated OTB derivative, especially in the presence of an H-donor, such as a sugar (35) or thiol (36). In the presence of O2, photooxidation of OTA generates OTQ in analogy to photooxidation of halogenated phenols that yield benzoquinone derivatives (38). These results differ from the oxidative chemistry of OTA (32-34) that does
not yield OTB even in the presence of thiols (33). Photophysical measurements (36, 37) suggest that the interaction of OTA with a solvated electron initiates C-Cl bond homolysis to afford an OTA carbon-centered radical and Cl-. Interaction of the carbon-centered radical with an H-donor provides a route to the nonchlorinated OTB derivative (36). This reaction is akin to reductive dehalogenation of alkyl halides and aryl halides that are catalyzed by P450s (39-41). As pointed out by Guengerich (39), ferrous P450 should be a good 1e reducing agent, if a substrate can bind and receive electrons, and transfer of an electron to a substrate may be competitive with oxidation even in the presence of O2. One view posits that all OTA metabolites identified in animals are formed at very low rates and their structures and mechanisms of formation do not suggest reactive intermediates; at best, reactive metabolites are only formed in very low concentrations in rodents (22, 24, 27). However, the chemistry of OTA suggests that at least three OTA-derived electrophilic species are anticipated (Scheme 2). Electrochemical measurements show that OTA undergoes a 1e oxidative process in aqueous media to form the phenoxyl radical at E1/2 ) 1.04 V vs NHE
Perspective
(31). The phenoxyl radical of OTA (Scheme 2) should undergo a redox-cycling mechanism by H-atom abstraction from essential thiols, such as GSH and protein sulfhydryls, to yield thiyl radicals; this repeatedly generates the parent OTA, which gives the appearance that OTA is poorly metabolized. That OTA depletes levels of GSH in hepatocytes and mammalian cell lines (26, 42) is consistent with OTA phenoxyl radical as the initiation event (43). The benzoquinone OTQ reacts with GSH to yield OTA-GSH (27, 34), or it is reduced to OTHQ, which is detected in urine of rats fed OTA (27). That only trace levels of OTHQ have been detected in urine of rat is to be expected since chlorinated phenols undergo P450catalyzed oxidation by direct formation of a benzoquinone metabolite without the obligatory intermediacy of the hydroquinones, which are generated from reduction of the benzoquinone (44). Our chemical studies have shown OTQ to be a highly reactive species, and so, the amount of OTHQ detected does not reflect the total amount of OTQ produced from oxidative metabolism of OTA. The third electrophilic species is the carbon-centered OTA radical (36) that is anticipated from reductive dehalogenation (39-41) of OTA to yield the nonchlorinated OTB derivative. Unlike the resonance-stabilized OTA phenoxyl radical, this carbon-centered radical would react with H2O to yield OTB and hydroxyl radical (HO•). Collectively, phenoxyl radicals (45, 46), benzoquinone electrophiles (47), and carbon-centered radicals (39, 48, 49) are known to react directly with DNA to form covalent DNA adducts. Oxidative biotransformation of OTA would also be expected to contribute to ROS production. In the presence of GSH, redox cycling of the OTA phenoxyl radical with thiyl radical generation will yield a GSH disulfide anion radical that can reductively activate O2 to generate the superoxide radical anion (O2•-) and hence H2O2, which in the presence of free Fe2+ would generate HO• by the Fenton reaction; a pathway regarded as a contributor to peroxidase-driven toxic effects of phenolic xenobiotics (43). Redox cycling of the OTQ/OTHQ redox couple would also contribute to O2•- formation, while H-atom abstraction of H2O by the carbon-centered radical would lead to OTB and HO• directly. A number of reports have provided evidence for the involvement of ROS by OTA treatment leading to an increase in 8-oxo-dG levels, DNA strand scission (15, 42, 50-54), and other biomarkers for oxidative stress (55-57). The pathways outlined in Scheme 2 are consistent with these findings. While enzymatic bioactivation of OTA represents one model for ROS production, Hoehler et al. have shown that OTA still causes generation of ROS in bacteria that have very low P450 or peroxidases (58, 59). They propose that OTA increases the permeability of the cell to Ca2+ and the presence of the pro-oxidant OTA uncouples oxidative phosphorylation resulting in the increased leakage of electrons from the respiratory chain producing O2•- and free Fe2+ (58, 59). Thus, perturbation of Ca2+ homeostasis by OTA results in enhanced ROS production in a manner analogous to those proposed with other pro-oxidants, such as tert-butyl hydroperoxide, and does not involve enzymatic bioactivation of OTA. Interestingly, this nonenzymatic pathway for ROS production by OTA is also expected to liberate free Fe2+. Thus, the interaction of OTA with Fe2+ may play a key role in the bioactivation of OTA to electrophilic species capable of reacting covalently with DNA.
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Figure 1. Chemical structures of authentic OTA-DNA adduct standards.
3. DNA Adduction On the basis of the 32P-postlabeling method, OTA forms DNA adducts in mice, rats, pig, and human (18-21, 6065). In the first report (60), DNA was isolated from liver, kidney, and spleen excised from mice 24, 48, and 72 h after oral treatment with OTA at 0.6, 1.2, and 2.5 mg/kg body weight. Adducts detected were not the same in the three organs examined, which was ascribed to differences in OTA metabolism. Adducts formed in a time- and dosedependent manner; at high dose, 40 adducts/109 nucleotides were found in kidney DNA and 7 adducts/109 nucleotides were found in liver after 72 h. A follow-up study (61) on DNA adduct levels with a high dose of OTA (2.5 mg/kg body weight) over a 16 day period showed total DNA adduct levels to reach a maximum at 48 h with 103, 42, and 2.2 adducts/109 nucleotides in kidney, liver, and spleen, respectively. In the kidney, the adduct labeled no. 1 was dominant and represented 36% of the total at 72 h; this adduct reached its maximum level at 72 h and was present for the duration of the experiment with 7.7 adducts/109 nucleotides detected in the kidney after 16 days. A relation between OTA dose and DNA adduct levels in kidney of fisher rats has also been described (63). Treatment of mother mice with OTA (0.5 and 2 mg/kg bw) also induced DNA adducts in both mother and progeny with the high dose OTA generating the greatest yield of adduct (64). Tumor tissue from kidney and bladder of Bulgarian patients undergoing surgery for cancer has also been analyzed for OTA-mediated DNA adducts, and comparison to mice kidney adducts showed that the major adduct no. 1 comigrated with the major adduct detected in Bulgarian tumor samples (65). Mechanistic studies on DNA adduction by OTA show that pretreatment with antioxidants (vitamin E, vitamin C, and superoxide dismutase + catalase) (67) or indomethacin (17, 67), an inhibitor of PGHS, dramatically decreases DNA adduct levels in cell culture or in the kidney and urinary bladder of mice treated with OTA. Modulators of glutathione pathways also modify the OTA-mediated DNA adduct pattern observed in 32Ppostlabeling experiments (15, 52, 66). In vitro experiments using kidney microsomes, or horseradish peroxidase (HRP), show that OTA forms guanine specific DNA adducts (68, 69). This prompted our laboratory to use the photochemistry of OTA to assess its reactivity toward deoxyguanosine (dG) (70). These studies led to the isolation and identification of the carbon-bonded C8-OTAdG adduct (OTA-dG) shown in Figure 1. With an authentic sample of OTA-dG in hand, it was then demonstrated by LC-MS/MS analysis using negative ionization electrospray mass spectrometry (ES-) that the same species could be formed upon activation of OTA with
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HRP/H2O2, Fe2+, and Cu2+ ions, with Fe2+ activation generating the highest levels of OTA-dG (70). The OTA photochemistry was also used to generate authentic C8 OTA-3′-monophosphate-dG (3′-dGMP) adducts for use as cochromatographic standards for 32Ppostlabeling detection of OTA-mediated DNA adduction in the kidney of rat and pig (71). In addition to the anticipated C-OTA-3′-dGMP adduct, the oxygen (O)-OTA3′-dGMP adduct shown in Figure 1 was isolated and characterized by LC/MS with in-line UV and electrospray ES- detection. Because the photoreaction of OTA is known to yield the OTA phenoxyl radical (37) and we have established that chlorophenoxyl radicals yield C8dG-O adducts (45, 46), the O adduct of OTA stems from the intermediacy of the OTA phenoxyl radical in covalent bond formation. The C-bonded adduct may stem from ambident phenoxyl radical [O vs C attachment (45)] and/ or the carbon-centered radical shown in Scheme 2. 32 P-postlabeling experiments demonstrated that the C-OTA-3′-dGMP adduct comigrated with the major lesion detected in the kidney of rat following chronic exposure [fed three times a week for their lifespan (∼2 years)] to OTA and with one of four adducts detected in the kidney of pig following subacute exposure (pig fed OTA continuously for a 3 week period), while the O-OTA-3′-dGMP adduct was shown to coelute with a lesion detected in rat kidney (72). The amount of adduct comigrating with C-OTA-3′-dGMP was ∼16 adducts/109 nucleotides (A. Pfohl-Leszkowicz, personal communication). This major lesion had the same chromatographic properties as the adduct no. 1 detected in the original 32P-postlabeling experiments (60, 61) that was shown to be present in diseased kidney and bladder tissue of Bulgarian subjects (65) and in French patients undergoing surgery for kidney and bladder tumors (62). These results are highly suggestive that OTA-dG forms in the kidney of rat and pig, which is consistent with the chemistry of OTA and the established ability of phenoxyl radicals (45, 46) and carbon-centered radicals (39, 48, 49) to react covalently with the C8 site of dG. Inspection of OTA-mediated tumorigenesis in Lewis and dark Agouti (DA) rats shows that induction of renal tumors by OTA is sex and strain specific in DA and Lewis rats, with DA males being most responsive and DA females being resistant (7, 72). In the initial study by Castegnaro et al. (7), DNA adducts by 32P-postlabeling were significantly correlated with renal carcinogenicity of OTA, being highest in DA males and lowest in DA females, suggesting that OTA acts as a direct genotoxic carcinogen. In a follow-up study by Pfohl-Leszkowicz et al. (72), the expression of P450s (1A, 2A, 2B, 2C, 2D, and 3A) in kidney and liver microsomes of untreated and OTA-treated DA and Lewis rats (both sexes) was determined by western blot analysis. These studies demonstrated that sex differences were linked to P450 2C11 expression in the livers and kidneys of male rats (72), providing a rationale for the greater susceptibility of males than females to OTA-induced renal carcinogenicity and DNA adduction. Despite positive 32P-postlabeling evidence for DNA adduction by OTA, other researchers question the ability of OTA to act as a direct genotoxic carcinogen. Mutagenicity assays for OTA have not provided definitive answers. OTA induced revertants in the Ames reversion assay using S. typhimurium tester strains TA1535 and TA1538 with activation by mouse kidney microsomes (73)
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and produced a mutagenic response in NIH/3T3 cell lines stably expressing human P450s 1A1, 1A2, 2C10, and 3A4 using a shuttle vector containing the lacZ′ gene (74). However, rat kidney microsomes, the most sensitive species and target organ of OTA-mediated carcinogenesis, failed to induce revertants (75) and OTA is not mutagenic in most microbial and mammalian gene mutation assays (76). Many positive genotoxicity studies with OTA in mammalian cells have been performed in systems that have no or very low capacity for oxidative biotransformation (29, 30). Using [3H]OTA and treating rats with a single oral dose of 0.2 mg/kg of body weight for 24 h, Schlatter and co-workers were unable to detect OTAmediated DNA adducts by liquid scintillation counting with a limit of detection of 1.3/1010 DNA bases (77). The Turesky laboratory (25) also found no evidence for DNA adduction by OTA in male rats following treatment with 1 mg/kg [3H]OTA body weight for 24 h. In rat and human primary hepatocytes, Gross-Steinmeyer et al. also detected no DNA adducts by 0.1-10 µM [3H]OTA over a 8 h period (26). Because no DNA adducts were detected using the more specific [3H]OTA/liquid scintillation counting method, it has been suggested that most if not all of the DNA adducts attributed to OTA by 32P-postlabeling do not contain an OTA moiety, and the lesions may be the result of OTA-mediated cytotoxicity (25). These findings coupled with the recent report by the Dekant laboratory (27) that treatment of male Fisher 344 rats with 14C-labeled OTA (0.5 mg/kg body weight) fails to yield detectable levels of adduct (
