The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.

Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [ 14 C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N 2 -glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N 2 -glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N 2 -glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group. (Cancer Res 2006; 66(21): 10541-7)

The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in five human volunteers given a dietary equivalent of 14C-labeled MeIQx. The amount of the dose excreted in urine ranged from 20.2% to 58.6%, with unmetabolized MeIQx accounting for 0.7-2.8% of the dose. Five principal metabolites were detected in urine, and four of the derivatives were characterized by on-line UV spectroscopy and by HPLC-MS following immunoaffinity chromatography. Two metabolites were identified as the phase II conjugates N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid (MeIQx-N2-SO3(-)) and N2-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx-N2-Gl). Two other metabolites were the cytochrome P450-mediated (P450) oxidation products 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-MeIQx), and N2-(beta-1-glucosiduronyl)-N-hydroxy-2-amino-3,8-dimethylimidaz o[4,5-f]quinoxaline (NOH-MeIQx-N2-Gl). The latter product is a conjugate of the genotoxic metabolite 2-(hydroxyamino)-3,8-dimethylimidazo-[4,5-f]quinoxaline (NHOH-MeIQx). A large interindividual variation was observed in the metabolism and disposition of MeIQx; these four metabolites and unchanged MeIQx combined accounted for 6.3-26.7% of the total dose. The remaining principal metabolite found in all subjects accounted for 7.6-28% of the dose. It has not been previously identified in rodents or nonhuman primates, and its structure remains unknown. P450-mediated ring oxidation of MeIQx at the C-5 position, a major pathway of detoxication in rodents, was not detected in humans. Both 8-CH2OH-MeIQx formation and NHOH-MeIQx formation are catalyzed by P450 1A2 and may be useful biomarkers of P450 1A2 activity in humans. The levels of NHOH-MeIQx-N2-Gl found in human urine ranged from 1.4% to 10.0% of the dose, which is significantly higher than that formed in rodents and nonhuman primates undergoing cancer bioassays. Thus, bioactivation of MeIQx by P450-mediated N-oxidation is extensive in humans.