BPA-toxicity via superoxide anion overload and a deficit in β-catenin signaling in human bone mesenchymal stem cells

Abstract

Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine-disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose- and time-dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA-treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β-catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl₂ increased nuclear β-catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β-catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344-352, 2017.

Keywords: GSK3β; MnTBAP; bisphenol A; human bone mesenchymal stem cells; superoxide dismutase; β-catenin.

Figure 1

BPA induces cytotoxicity in hBMSCs in a time‐ and dose‐dependent manner. A: The dose‐dependent effect of BPA on cytotoxicity in hBMSCs. B: The time‐dependent effect of BPA on cytotoxicity in hBMSCs. Data are presented as the means ± SD. **denote differences at p < 0.01.

Figure 2

BPA induces cytotoxicity in hBMSCs via superoxide anion generation. A: Quantitative analysis of cellular MDA levels. B: Photomicrographs showing the suppressive effects of antioxidant mimetics on cytotoxicity in hBMSCs. C: Photomicrographs showing the suppressive effects of MnTMPyP, Allopurinol, and DPI on cytotoxicity in hBMSCs. Data are presented as the means ± SD. **denote differences at p < 0.01.

Figure 3

BPA reduces the accumulation of nuclear β‐catenin and expression of the β‐catenin/LEF pathway‐dependent target gene cyclin D1 through increased superoxide anion. A: Western blotting for nuclear β‐catenin and cyclin D1 in BPA‐treated hBMSCs. (a‐left): Photomicrographs showing nuclear β‐catenin and cyclin D1 immunoreactivity. (b‐right): Quantitative analysis of western blots (normalized to β‐actin). B: Western blotting for nuclear β‐catenin following pretreatment with MnTBAP in BPA‐treated hBMSCs. (a‐left): Photomicrographs showing nuclear β‐catenin immunoreactivity. (b‐right): Quantitative analysis of western blots (normalized to β‐actin). Data are presented as the means ± SD (n = 8 animals). **denote differences at p < 0.01. [Color figure can be viewed at wileyonlinelibrary.com.]

Figure 4

BPA‐induced deficits in β‐catenin signaling and cytotoxicity are alleviated by GSK3β inhibitor in hBMSCs. A: Western blotting for nuclear β‐catenin by pretreatment with LiCl₂ in BPA‐treated hBMSCs. (a) Photomicrographs showing nuclear β‐catenin immunoreactivity. (b) Quantitative analysis of western blots (normalized to β‐actin). B: Photomicrographs showing the suppressive effects of LiCl₂ on cytotoxicity in hBMSCs. C: Photomicrographs showing the suppressive effects of LiCl₂ and MnTBAP on cytotoxicity in hBMSCs. Data are presented as the means ± SD. * and ** denote differences at p < 0.05 and p < 0.01, respectively. [Color figure can be viewed at wileyonlinelibrary.com.]