Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A:Role of CYP2C9 and microsomal glutathione-S-transferase

Ochratoxin A (OTA], a mycotoxin that induces nephrotoxicity and urinary tra ct tumors, is genotoxic and can be metabolized not only by different cytoch romes P450 (CYP) but also by peroxidases involved in the arachidonic cascad e, although the exact nature of the metabolites involved in the genotoxic p rocess is still unknown. In order to establish the relation between OTA gen otoxicity and the formation of metabolites, we chose three experimental mod els: kidney microsomes from rabbit, human bronchial epithelial cells, and m icrosomes from yeast that specifically express the human cytochrome P450 2C 9 or 286 genes. OTA-DNA adducts were analyzed by P-32 postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phen obarbital (PB); (2) human pulmonary epithelial cells simultaneously pretrea ted (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 286 and 2C9. PB pretreatment significantly increa sed DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transfer ase, reduced DNA adduct level. DNA adducts were detected when OTA were incu bated with microsomes expressing human CYP 2C9 but not with those expressin g CYP 286. Several metabolites detected by HPLC were increased after PB tre atment. Some of them could be related to DNA-adduct formation. In conclusio n, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct Formation through pathways involving microsomal glutathion-S-transferase a nd CYP 2C9. (C) 2000 Wiley-Liss, Inc.