Metabolism of ochratoxin A: Absence of formation of genotoxic derivatives by human and rat enzymes

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its made of carcinogenicity is not known. The metabolism and covalent binding o f OTA to DNA were investigated in vitro viith cytochromes P450, glutathione S-transferases, prostaglandin K-synthase, and horseradish peroxidase. Incu bation of OTA with rat or human liver microsomes fortified with NADPH resul ted 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 gluta thione conjugate formation with rat, mouse, or human kidney microsomes or p ostmitochondrial supernatants (S-9) [ < 5 pmol. min(-1) (mg of protein)(-1) ]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydro xyochratoxin 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 p roduct [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not for med with horseradish peroxidase. There was no evidence of DNA adduct format ion when [H-3]OTA was incubated with these enzyme systems in the presence o f calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes cat alyzed DNA adduct formation with the genotoxins aflatoxin B-1, 2-amino-3-me thylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There w as also no detectable [H-3]-OTA bound in vivo to kidney DNA of male Fischer -344 rats treated orally with [H-3]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposur e, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, P-32-postlabeling experiments did show evidence of DNA lesions wi th total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA base s. These results do not support the premise that OTA or metabolically activ ated species covalently bind to DNA and suggest that the P-32-postlabeled l esions are due to products derived from OTA-mediated cytotoxicity.