Extrapolating from experimental to human studies via the in vitro paradigm

Despite being the “gold standard” for toxicity testing, rat in vivo studies are estimated to be only 37-50% accurate at predicting xenobiotic toxicity in humans. If validated, in vitro systems could be incorporated into toxicity testing, allowing direct comparisons to be made between humans and rats and facilitating a reduction of in vivo testing. In this project, a normal rat urothelial (NRU) cell culture system was developed and optimised for comparison against an established normal human urothelial (NHU) cell culture system. NRU cells had a reduced lifespan in culture compared to NHU cells, leading to an investigation of the pathways regulating cellular proliferation. The PI3K/Akt pathway was found to be active in proliferating NRU cells, whereas the EGFR/MAPK and β-catenin pathways were confirmed to regulate NHU cell proliferation. Inhibition of the PI3K/Akt pathway enabled NRU cells to be subcultured to passage 2, most probably by enabling EGFR/MAPK and β-catenin pathway activation, although this requires experimental confirmation. To further increase cell lifespan, the proto-oncogene BMI1 was overexpressed in NRU and NHU cells, with the effects on lifespan and differentiation compared against both human telomerase reverse transcriptase (hTERT) overexpressing and control (empty vector) cells. NRU cell lifespan was not increased by BMI1 or hTERT overexpression. BMI1 overexpression increased NHU cell lifespan in culture, comparable with hTERT overexpression, but further work is required to determine the extent of the effects on lifespan and phenotype beyond passage 12. Finally, the metabolic competence of urothelial cells was explored, with a focus on the cytochrome P450 2B (CYP2B) family of metabolic enzymes. Xenobiotic induction of CYP2B expression by rat urothelium was demonstrated in vivo but not in NRU and NHU cells. Xenobiotic-induced CYP2B protein expression was achieved in a rat ex vivo organ culture model. Together these results identify a previously unknown metabolic capability of urothelial cells that can be modelled ex vivo, and demonstrate fundamental differences in rat and human urothelial cell physiology that support the validation of human tissue-specific in vitro models for assessment of xenobiotic toxicity.