Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades and bisphenol-F (BPF) and -S (BPS) have been suggested as “safer” alternatives. In the present study we used hepatocyte-like cells (HLC) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system, and, during their transition to HLCs, they were exposed to BPA, BPF and BPS for 8 days. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as down- regulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity. Human embryonic stem cells (hESCs) derived hepatic progenitor cells were mature into HLC in the presence of different concentrations of bisphenol A (BPA), -F (BPF) and -S (BPS) for 8 days to determine the CYP3A IC50 value for each cell line and compound. Analysis of Cyp3A P450 activity upon exposure to BPA revealed that both Man12 and H9 HLCs responded in similar fashion, with IC50 values of 20 μM and 19 μM respectively. However, cell line differences were observed upon exposure to BPF and BPS. Man12 HLCs were more sensitive to these compounds when compared to H9 HLCs. BPF- and BPS-exposed Man12 HLCs displayed IC50 values of ~10 μM and 25 μM respectively, whereas this was increased in H9 HLCs to ~29 μM and 31 μM respectively. Genome-wide gene expression profile and PCR array to examine key genes involved in cell viability and apoptosis were performed in HLCs exposed to the IC50 concentration of the bisphenols for CYP3A function. Five technical replicates were prepared for each condition. In replicate E of the Man12 cell line untreated control, vehicle control and BPS treated cells died prematurely in culture. Therefore, no microarrays were generated for these experiments. Since the limma analysis was performed paired against the untreated control, Man12 replicate E was excluded from the analysis.