Induction of unscheduled DNA synthesis in primary human urothelial cells by the mycotoxin ochratoxin A

Ochratoxin A (OTA) is a widespread contaminant in human staple food. Exposu re of humans to this mycotoxin is a matter of concern because OTA is a know n rodent carcinogen. As the urothelium is one target tissue of this mycotox in, primary cultured human urothelial cells (HUC) from adults and children were used to analyze the induction of unscheduled DNA synthesis (UDS) by OT A. HUC were isolated from the ureters or renal pelves of two nephrectomized adults and of two children with ureteropelvic junction stenosis and cultur ed under serum-free conditions. After a confluency of 70-80% was reached, c ell proliferation was suppressed by arginine-deficient medium (ADM), and UD S was assessed autoradiographically by H-3-thymidine incorporation upon exp osure to OTA (10-2000 nM), ethyl methanesulfonate (EMS, 5 mM, positive cont rol), or dimethyl sulfoxide (DMSO, 0.2%, solvent control). In control cultu res the level of UDS was low. Exposure to EMS resulted in an induction of U DS (2-to 5-fold compared to control), thus allowing the sensitive detection of repair resulting from induction of DNA lesions in all four specimens, a nd demonstrating that repair of EMS-induced DNA lesions can take place unde r the chosen culture conditions. In two HUC cultures derived from adults, a significant induction of UDS was observed in the concentration range of 50 -500 nM OTA. The highest fraction of cells in repair (CIR) was found at 50 nM OTA for the HUC from the older male (50% CIR). The maximum response in t he other specimens from the adult female and the 7-year-old boy were seen a t OTA concentrations of 500 and 250 nM, respectively. In contrast to all ot her specimens, no significant induction of UDS by OTA was found in the KUC cultures derived from an infant's urothelium. Signs of cytotoxicity were ob served above 500 nM OTA in all cultures. The varying susceptibility toward OTA observed in vitro may hint at varying predispositions of individuals in vivo.