Ochratoxin A (OTA) is a widespread contaminant in human staple food. Exposure of humans to this mycotoxin is a matter of concern because OTA is a known rodent carcinogen. As the urothelium is one target tissue of this mycotoxin, primary cultured human urothelial cells (HUC) from adults and children were used to analyze the induction of unscheduled DNA synthesis (UDS) by OTA. HUC were isolated from the ureters or renal pelves of two nephrectomized adults and of two children with ureteropelvic junction stenosis and cultured under serum-free conditions. After a confluency of 70-80% was reached, cell proliferation was suppressed by arginine-deficient medium (ADM), and UDS was assessed autoradiographically by 3H-thymidine incorporation upon exposure to OTA (10-2000 nM), ethyl methanesulfonate (EMS, 5 mM, positive control), or dimethyl sulfoxide (DMSO, 0.2%, solvent control). In control cultures the level of UDS was low. Exposure to EMS resulted in an induction of UDS (2-to 5-fold compared to control), thus allowing the sensitive detection of repair resulting from induction of DNA lesions in all four specimens, and demonstrating that repair of EMS-induced DNA lesions can take place under 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 the other specimens from the adult female and the 7-year-old boy were seen at OTA concentrations of 500 and 250 nM, respectively. In contrast to all other specimens, no significant induction of UDS by OTA was found in the HUC cultures derived from an infant's urothelium. Signs of cytotoxicity were observed above 500 nM OTA in all cultures. The varying susceptibility toward OTA observed in vitro may hint at varying predispositions of individuals in vivo.
A cell culture system with human-derived urothelial cells was established based upon previous experience with cultures of porcine urinary bladder epithelial cells. Human tissue specimens used were derived from urinary bladders (n = 17) or ureters (n = 50) of patients undergoing urological operations. The epithelial origin and differentiation status was evaluated by an immunohistochemical staining for cytokeratins 7, 8, 18, 19, and 20 for isolated and cultured cells. Specimens from human ureters were better suited for primary cell cultures of the urothelium than specimens from human urinary bladders. Successful attachment and proliferation were reached by 98% of the ureter specimens (urinary bladder: 71%) and confluency was reached by 78% of the ureter cultures (urinary bladder: 18%). In the first 14 d of culture the cytokeratin patterns of cultured cells were comparable to those of native mucosa cells. During prolonged cell culture the cytokeratin patterns of the human urothelial cells (HUC) changed into a beginning dedifferentiation: Cytokeratin (CK) 18 was only detectable in cell cultures cultured for more than 29 d, whereas CK 19 was not detectable at d 29. Cell cultures of primary human urothelial cells may be used for in vitro testing of cytotoxic or genotoxic effects.
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