title: Metabolism-mediated cytotoxicity of ochratoxin A.
abstract: Ochratoxin A (OTA) is produced by various strains of Aspergillus and Penicillium and is a common contaminant of food commodities. OTA is metabolised by cytochrome P450 (CYP450) enzymes resulting in hydroxylated metabolites, 4R-OH-OTA and 4S-OH-OTA, and possibly in other minor metabolites including OTA-quinones. However, until now conflicting data have been presented regarding the role of biotransformation products in the adverse effects of OTA. Hence, the aim of this study was to further assess the metabolism-mediated cytotoxicity of OTA in an in vitro model encompassing NIH/3T3 cells, stably expressing the human CYP450 enzymes CYP2C9 and CYP3A4, respectively. In addition, modulation of the cellular glutathione (GSH) content was used to identify a role of GSH in OTA-induced cytotoxicity. Following exposure to OTA, cells expressing CYP2C9 showed a significant reduction in neutral red (NR) uptake but not in Alamar blue (AB) reduction, as compared to the control LNCX cells which do not express CYP450 enzymes. CYP3A4-expressing cells showed no difference in viability from control LNCX cells. When pre-treated with l-buthionine S,R-sulphoximine (BSO) to deplete GSH, CYP2C9-expressing cells showed also a loss of cell viability as compared to LNCX cells, although to a lesser extent as compared to non-depleted CYP2C9-expressing cells. Data presented in this study support previous findings, indicating that different biotransformation pathways contribute to the cytotoxicity induced by OTA.
authors.affiliation: Department of Veterinary Pharmacy, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 16, 3584 CM, Utrecht, The Netherlands.
authors.forename: A Y
authors.initials: AY
authors.lastname: Simarro Doorten
authors.forename: S
authors.initials: S
authors.lastname: Bull
authors.forename: M A M
authors.initials: MA
authors.lastname: van der Doelen
authors.forename: J
authors.initials: J
authors.lastname: Fink-Gremmels
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D016923:Cell Death; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D041681:NIH 3T3 Cells; D009499:Neutral Red; D009793:Ochratoxins / Q000633:toxicity*; D010078:Oxazines; D014162:Transfection; D014966:Xanthenes
chemical_list: D007202:Indicators and Reagents; D009793:Ochratoxins; D010078:Oxazines; D014966:Xanthenes; C025589:ochratoxin A; C005843:resazurin; D009499:Neutral Red; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione