Cultured porcine urinary bladder epithelial cells as a screening model forgenotoxic effects of aromatic amines: Characterisation and application of the cell culture model

Isolated epithelial cells from porcine urinary bladders were maintained in dividing long-term monolayer cultures, and were used as a model system for the urinary bladder in toxicological studies in vitro. To examine the state of differentiation during the culture period, the culture system was chara cterised morphologically by light and transmission electron microscopy and by immune fluorescence labelling with antibodies against cytokeratins 7,13 and pan. The cultured cells were identified as urothelial epithelium by the ir polarised structure, and by their expression of several uroepithelial sp ecific morphological features, such as fusiform vesicles, tight junctions a nd an asymmetric apical cell membrane. Additionally, the cells were labelle d with anti-cytokeratin 7, 13 and Dan antibodies, and negatively with anti- vimentin antibodies. The maintenance of suitable culture conditions was sho wn by the stable enzyme activities of gamma -glutamyltranspeptidase, alkali ne phosphatase and acid phosphatase over a culture period of 4 weeks. A goo d viability of the cultured cells under the chosen culture conditions was s hown by the presence of low amounts of lactate dehydrogenase (less than or equal to 5%) in the culture medium. The activities of the chosen marker enz ymes For cell differentiation (gamma -glutamyltranspeptidase), lysosomes (a cid phosphatase) and luminal membranes (alkaline phosphatase) were relative ly stable over the observed culture period. Enzyme activities involved in m etabolism of xenobiotics were determined, to define the ability for metabol ism in cultured cells compared with bladder tissue in situ. Several constit utive phase I and II enzyme activities were found to be stable during the c ulture period, indicating that the cultured cells should be able to metabol ise xenobiotics in a comparable manner to the urothelium in vivo. The cytot oxic effects of xenobiotics were investigated and IC50 values were determin ed by means of lactate dehydrogenase leakage and inhibition of neutral red uptake. The induction of sister chromatid exchanges was used as a parameter for the genotoxic effects of several xenobiotics. This cell culture system was found to be a very good screening system for the testing of substances that affect the bladder, especially aromatic amines.