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Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10−25 pmol min^-1 (mg of protein)^-1]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min^-1 (mg of protein)^-1]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min^-1 (pmol of P450)^-1, respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min^-1 (pmol of P450)^-1]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min^-1 (mg of protein)^-1], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [³H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10⁹ DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [³H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [³H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10⁹ DNA bases), based upon liquid scintillation counting. However, ³²P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10⁹ DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10⁹ DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the ³²P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.

DNA adduct formation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in cynomolgus monkeys. The pattern and distribution of DNA adducts examined by ³²P-postlabeling were similar in all tissues 24 h after a single oral dose of IQ (20 mg/kg). The highest DNA adduct levels were found in the liver (3.67−11.19 adducts per 10⁷ bases), followed by kidney (0.53−1.16 adducts per 10⁷ bases), with comparable adduct levels detected in colon, heart, and pancreas (0.15−0.40 adducts per 10⁷ bases). Two 2‘-deoxyguanosine (dG) adducts accounted for approximately 90% of the observed lesions in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) was the major adduct and accounted for approximately 50−80% of the adducts, followed by 5-(deoxyguanosin-N²-yl)-amino-3-methylimidazo[4,5-f]quinoline (dG-N²-IQ) which accounted for 20−40% of the adducts. DNA adduct formation was also investigated in animals undergoing carcinogen bioassay with IQ administered at 10 or 20 mg/kg, 5 days per week for up to 9.2 years. In chronically treated animals, the DNA adduct levels in pancreas, kidney, and heart increased on average by 40- to 90-fold over those observed in animals given a single dose, while only 3- to 10-fold increases in adducts were observed in colon and liver. A sharp increase in the contribution of dG-N²-IQ to total DNA adducts occurred in all slowly dividing tissues during chronic treatment, and dG-N²-IQ became the predominant lesion. There was no preferential accumulation of dG-N²-IQ in the colon, a tissue with a high rate of cell division, and dG-C8-IQ remained the predominant lesion. These findings point to a preferential removal of the dG-C8-IQ adduct by enzyme repair system(s) in slowly dividing tissues. The respective roles of dG-N²-IQ and dG-C8-IQ, and the involvement of adduct repair in the potent hepatocarcinogenicity of IQ, merit further investigation.

The urinary excretion of the DNA alkylation product, 3-methyladenine (3-MeAde), was measured in human volunteers who were on controlled diets and consumed fresh fish, or frozen-stored fish that contained 50-fold higher levels of dimethylamine (DMA), with or without ingested nitrate. DMA potentially could react with nitrosating agents in the diet or within the body, and produce the potent carcinogen N-nitrosodimethylamine (NDMA), which can then react with DNA to form several adducts including 3-MeAde. Our findings show that there was no increase in urinary levels of 3-MeAde after consumption of fish preserved by frozen storage relative to levels after consumption of fresh fish. Furthermore, consumption of 225 mg sodium nitrate (equal to the nitrate content in a large glass of beet juice) at 1 h prior to consumption of the frozen-stored fish did not increase urinary 3-MeAde levels as would be expected if nitrate enhanced endogenous nitrosation of DMA. In contrast, urinary excretion of 3-MeAde from a volunteer who was a moderate cigarette smoker (11 cigarettes per day) was approximately 3- to 8-fold higher than dietary 3-MeAde intake. These findings indicate that consumption of high levels of DMA in fish does not result in detectable levels of NDMA formation and genetic damage as measured by the urinary biomarker 3-MeAde.