Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAs, iAs, MMA, MMA, DMA, DMA, and DMMTA). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTA exhibited higher cellular uptake than pentavalent arsenicals. Except for MMA, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMA and DMMTA were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMA. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMA and DMA induced necroptosis. MMA, DMA, and DMMTA induced both cell death pathways. In summary, MMA exhibited the strongest cytotoxicity, followed by DMMTA, iAs, DMA, iAs, DMA, and MMA. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.