Abstract
Here, we used female adult rat adipose-derived stem cells (rASCs) and human adipose-derived stem cells (hASCs) to compare the toxicities and potencies of several widespread environmental toxins that may be endocrine-disrupting chemicals, including bisphenol A (BPA), and the newer BPA alternatives bisphenol S (BPS), bisphenol AF (BPAF), and tetramethyl bisphenol F (TMBPF). Adult stem cells were cultured to 80% confluency in vitro and then exposed to BPA (1 and 10 µM), 17β-estradiol (E2; 10 µM), BPS (1 and 10 µM), BPAF (3 × 10-3-30 µM), TMBPF (0.01-50 µM), or control media alone (with 0.01% ethanol) for varying time intervals from 20 min to 5 hrs. Using several cellular assays, the levels of cell death, apoptosis, caspase-6 activation, and potencies were compared across chemical treatments and vehicle-treated controls. There was significantly decreased cell viability and increased apoptosis in rat and human stem cells treated with each BPA analog, as early as 20 min of exposure, and at low doses. With higher magnification, higher resolution imaging it was evident that in many of the BPA analog-treated cells, the Apopxin Deep Red dye indicative of apoptosis was localized to the cytoplasmic compartments of cells, while the nuclear green DCS1 dye indicative of late-stage apoptosis and necrosis was localized to the nuclei of cells. Notably, BPAF and TMBPF showed cytotoxicity in a dose-dependent manner (BPAF LC50 = 0.014 µM (rASCs) and 0.009 µM (hASCs); TMBPF LC50 = 0.88 µM (rASCs) and 0.06 µM (hASCs); lethal concentration with 50% survival). The rank order of potency was BPAF>TMBPF>BPA>BPS. The majority of cell death was due to apoptosis as indicated by high levels of activated caspase-6 in the cytoplasm of almost 100% of cells treated with the BPA analogs. This data allows for further confirmation of caspase-6-mediated apoptosis using higher magnification imaging that definitively demonstrate the cytotoxic and apoptotic effects of these BPA analogs. For a complete description, interpretation, and discussion of the data refer to the article in press [1].