Exposure of primary porcine urothelial cells to benzo(a)pyrene: in vitro uptake, intracellular concentration, and biological response

Abstract

More than 90% of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5μM BaP for up to 24h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70μM in cells exposed to 0.5μM BaP and from 29.9 to 406.64μM in cells exposed to 10μM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.