Use of embryonic stem cell-derived cardiomyocytes to study cardiotoxicity of bisphenol AF via the GPER/CAM/eNOS pathway
Introduction
Bisphenols (BPs) are important chemical substances that are widely used in the synthesis of plastics, particularly polycarbonates and epoxy resins (Jin et al., 2018; Meng et al., 2019). For decades, BPs have been intensively introduced into the environment, which caused the exposure of animals and humans (Michalowicz, 2014). BPA is one of the most studied bisphenols; certain countries have restricted the import and sale of polycarbonate products containing BPA because of the documented adverse effects on terrestrial and aquatic wild life (Liu et al., 2019; Yin et al., 2015). Recent studies showed that BPA plays an important role in obesity, diabetes and numerous types of cancer (Duan et al., 2018; Ho et al., 2017; Pelch et al., 2019). Currently, bisphenol AF (BPAF) is a replacement of BPA and is widely used as a raw material in the plastic industry (Shi et al., 2016).
Widespread use of BPAF attracted considerable attention to the exposure levels and the ecological risk of BPAF. Recent studies in China and other countries detected BPAF in various environmental media(Li et al., 2018; Yu et al., 2015) and food(Cesen et al., 2016; van Leeuwen et al., 2019). Moreover, BPAF has been detected in various biological samples from various populations. Levels of BPAF in the blood of adults in China were from ND to 43 ng/l (Yang et al., 2019). The range of urinary levels of BPAF were from ND to 88.3 ng/l (Liu et al., 2019; Mokra et al., 2018) and from ND to 56 ng/l (Niu et al., 2017). The exposure level of BPAF in the population was at the low dose level. A study on the toxicity of BPAF, including an in vitro study, was limited due to BPAF being an environmental estrogen similar to BPA (Okazaki et al., 2018).
The chemical activity-structure correlation analysis indicates that the estrogen activity of BPAF is stronger than that of BPA (Maruyama et al., 2013). Similar to BPA, BPAF exerts its biological effects through estrogen receptors (ER). Moreover, BPAF is more estrogenic than BPA and has higher risk of adverse effects compared with that of BPA, including hormone-dependent breast cancer(Mesnage et al., 2017). Recent studies have shown that nongenomic effects of BPAF involve the members of the G protein-coupled receptor (GPER) pathway(Cao et al., 2017), and BPAF has substantially higher (ā¼9-fold) binding affinity to GPER than that of BPA.
The activation of GPER can rapidly activate the fluctuations in intracellular Ca2+ level in a concentration-dependent manner especially at the BPAF levels higher than 0.1 nM (Lei et al., 2019). A number of studies have reported that BPAF induces a remarkable increase in generation of reactive oxygen species (ROS) and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase, in the treatment groups compared with that in the control (Ding et al., 2017; Lee et al., 2013; Macczak et al., 2017). A recent study has shown that BPAF at the concentrations ranging from 0.005 to 5 Ī¼g/mL causes a significant reduction in the transmembrane mitochondrial function (Li et al., 2019; Mokra et al., 2015; Russo et al., 2019). Thus, it was suggested that BPAF may induce cardiotoxicity. Moreover, previous studies indicate that GPER are associated with the oxidative stress including reactive nitrogen species (RNS) (Chai et al., 2019; Lei et al., 2018). Exogenous substances can induce cardiotoxicity by upregulating the phosphorylation of endothelial nitric oxide synthase (eNOS) through GPER. Recent data of the literature indicated that an increase in the ratio of monomeric/dimeric eNOS promoted the production of O2Ā·ā and induced cardiotoxicity (Akolkar et al., 2017).
Heart disease is a high-risk disease. Recent studies have shown that incidence of heart disease remains stable and is higher in men than in women in recent years; however, heart disease remains the leading cause of death in men and women (Vikulova et al., 2019; Xu et al., 2018). Additionally, common risk factors of heart disease include estrogen-like substances (Brown et al., 2019; Schoenfeld et al., 2019). However, the effect of low doses of BPAF on cardiomyocytes has not been studied and whether BPAF can activate GPER signals to influence intracellular oxidative stress in cardiomyocytes remains unclear. Therefore, it is necessary to explore whether BPAF can cause cardiotoxicity.
In the present study, we hypothesized that the effects of BPAF on induction of cardiomyocyte hypertrophy may be due to nitrative stress via the GPER pathway. We used mouse embryonic stem cell-derived cardiomyocytes to determine the effects of BPAF on cardiomyocyte hypertrophy. The ratios of microfilaments/cell nucleus and microfilaments/mitochondrial length in cardiomyocytes were evaluated. The effects of BPAF without or with G15 (a GPER blocker) were examined. Finally, we compared the effects of BPAF on the crucial GPER downstream signaling molecule, eNOS. The data suggest that the activation of GPER and downstream signaling molecules by BPAF can result in cardiomyocyte hypertrophy.
Section snippets
Cell culture
Cardiomyocytes were derived from ESCs; ESC line R1 was used in the current study between passages 28 and 35. R1 stem cells were cultured in knockout Dulbeccoās modified Eagleās medium (KO DMEM, Gibco, USA), 10 % fetal calf serum (Sigma, USA), 2 mM l-glutamine (Gibco, USA), 0.1 mM nonessential amino acids (Gibco, USA), 1000 U/mL leukemia inhibitory factor (LIF, Millipore, USA), and 0.1 mM beta-mercaptoethanol (Gibco, USA). After 3 days of culture, 1000 R1 ESCs were cultured in 20 Ī¼L hanging
BPAF decreases cell viability and reduces the levels of MYH7, Gata4 and Mef2c in cardiomyocytes
To estimate the cytotoxic effect of BPAF, the Alamar blue assay was used to measure cell viability. Differentiated cells were treated with a series of concentrations of BPAF for 48 h. BPAF reduced the cell viability starting at the concentration of 5000 ng/ml. The viability of the cells exposed to 5000 ng/ml BPAF for 48 h was reduced to 26 % of that detected in the control. To confirm the differentiation of cardiomyocytes, MYH7, Gata4, and Mef2c were assayed after treatment with various
Discussion
BPA is banned in an increasing number of regions and fields; hence, BPAF is widely used. BPAF exposure has been associated with a number of diseases, such as fatty liver, endometriosis, anxiety, and diabetes (Duan et al., 2018; Jones et al., 2018; Meng et al., 2018b). However, it is not known whether BPAF can induce cardiotoxicity. In the present study, we used ESC-derived cardiomyocytes to examine the effects of BPAF exposure. There are some cell lines of cardiomyocytes, such as H9C2 cell
Conclusion
We used embryonic stem cells to induce cardiomyocyte differentiation. We found that BPAF can activate GPER, regulate PLCB1/IP3R/CAM/eNOS, and subsequently increase the ratios of microfilaments to mitochondrial length and the MYH7b levels. BPAF increases eNOS activity via GPER and promotes cardiomyocyte hypertrophy.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National key R&D program of China (2018YFC1602405), National Key R&D Program of China (2016YFC1000502), Shanghai Science & Technology Development Foundation (19140901200)ļ¼Research Program of Shanghai Collaborative Innovation Center for Translational Medicine (TM201716), China Postdoctoral Science Foundation (2018M632143), National Natural Science Foundation of China (21577091).
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