A systematic comparison of the developmental vascular toxicity of bisphenol A and its alternatives in vivo and in vitro
Graphical abstract
Introduction
Bisphenols (BPs) are a class of compounds that are composed of a chemical structure of two hydroxyphenyl functionalities. As a variety of widely used materials in plastic containers, BPs are prevalent in daily life, such as in plastic bags, water bottles and cans (Fischnaller et al., 2016). Among them, bisphenol A (BPA) is a raw material widely used in the production of polyacrylates, polycarbonate plastics, polyester and phenol resins. Over the last decades, concerns about widespread human exposure and its adverse effects on health have led to regulations on the production and usage of BPA in several countries (Chen et al., 2016). In 2017, BPA was included in the REACH Substances of Very High Concern (SVHC) Candidate List for its reproductive toxicity and endocrine disrupting properties. Therefore, stringent restrictions and bans for BPA have been imposed in numerous countries. However, the chemical structure of BPA is irreplaceable in the plastic production industry; therefore, some bisphenol analogs, such as bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF), which have a similar structure to BPA, have been developed and used increasingly, becoming widely used as the primary substitutes of BPA (Pelch et al., 2019).
Unexpectedly, the speed of BPA replacement has exceeded people's expectations, and some of the concentrations of BPs are close to or even higher than those of BPA. For example, in water samples from Lake Taihu in China, the highest concentrations of BPA, BPF and BPS were 565, 1634 and 1569 ngL−1, respectively (Chen et al., 2017). In addition, BPAF was found to be more abundant than BPA in water (i.e., 0.90–246 ngL-1 versus 6.59–74.6 ng L−1) and in surficial sediments (i.e., 0.18–2010 ng g−1 versus 1.37–42.8 ng g−1) from Hangzhou Bay (China) (Yang et al., 2014). At present, the environmental standard of BPs in water environments is still limited. In 2009, Canada proposed a bill requiring that the effluent release target concentration for industries and paper recycling mills be 1.75 μg L−1 (Canada, 2020). In addition, the environmental protection department of Ontario, Canada, stipulated a water quality standard in 1994, in which the BPA limit was 5 μg L−1 (Genuis et al., 2012). Moreover, human exposure to BPs has also been reported. A multinational study involving seven Asian countries and the U.S. showed that BPS was detected in 81% of the urine samples, and the highest concentration was 21 ng mL−1 (geometric mean = 0.168 ng mL−1) (Liao et al., 2012). A recent study reported that the mean concentrations of BPS and BPF were greater than those of BPA in urine samples of young children in Switzerland (Lucarini et al., 2020). In human plasma from the Chinese general population, BPA, BPS, and BPAF were the predominant BPs, and the mean concentrations were 0.40, 0.15, and 0.073 ng mL−1, respectively (Jin et al., 2018).
Due to the structural similarity of these analogs to BPA and their ubiquitous presence in the environment, the question arises whether these alteratives are safer or more harmful. Recently, concerns have been raised regarding the adverse effects of BPA substitutes. In vitro and in vivo studies have shown that some BPA substitutes exhibit similar or even stronger endocrine disrupting effects, cytotoxicity and genotoxicity, reproductive toxicity, and neurotoxicity (Chen et al., 2016; Gu et al., 2019, 2020a, 2020b). For instance, Moreman et al. (2017) showed that these BPA substitutes induce toxic and estrogenic effects similar to those of BPA; of note, BPAF showed greater potency than BPA. However, to date, only a few studies are available on the issue of cardiovascular toxicity of BPA substitutes (Gao et al., 2015). Notably, our previous study showed that BPAF disrupts cardiac development processes in zebrafish, including increasing the apoptosis of larval hearts, reducing the number of heart endocardial cells and cardiomyocytes, and downregulating genes involved in cardiogenesis (Gu et al., 2020a).
In the present study, zebrafish, including transgenic Tg (fli1:EGFP) zebrafish, were used to compare the vascular toxicity of BPF, BPAF, BPS and BPA. Additionally, human umbilical vein endothelial cells (HUVECs) were employed to assess the tube-forming capabilities in three-dimensional (3D) matrices after BP exposure in vitro. To further confirm the potential mechanisms of BPs, oxidative stress was evaluated in zebrafish larvae and HUVECs. Collectively, our data provide insight into the environmental or human risk of BPA and its substitutes, and revelation of the potential adverse effects of BPA substitutes on vascular development.
Section snippets
Chemicals and reagents
BPA (purity: 99%, CAS: 80-05-7), BPF (purity: 99%, CAS: 620-92-8), BPAF (purity: 99%, CAS: 1478-61-1) and BPS (purity: 99%, CAS: 80-09-1) were purchased from J&K Scientific (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and the final concentration of DMSO used in each group did not exceed 0.1 mLL−1. MS-222 (3-ethyl aminobenzoate, methane sulfonate) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Metrigel was purchased from BD
Acute toxicity of BPs on zebrafish embryos
According to the 96-h acute toxicity test, all of the BPs could induce significant lethality in zebrafish embryos at the tested concentrations. The LC50 of BPs in zebrafish embryos is shown in Supplementary Table 1. The 96 h LC50 values for the four BPs in zebrafish were 6.81 mg L−1 (BPA), 7.13 mg L−1 (BPF), 2.60 mg L−1 (BPAF) and 300.8 mg L−1 (BPS). The mortality rate of the negative control group was less than 5%. The ranking of BPs toxicity was BPAF > BPA ≈ BPF > BPS.
BPs exposure hindered the growth of ISV
Having determined the
Discussion and conclusion
The vascular system is one of the first organs to develop in embryonic vertebrates; it supplies oxygen, nutrients, hormones and metabolites to tissues and removes waste products. Angiogenesis is complete in the early stage of embryogenesis, so it is key to initiating, maintaining, and regulating the cardiovascular system in vertebrates (Bautch and Caron, 2015; Majesky, 2018). Previous studies have shown that BPA can interfere with the angiogenic process in in vitro and in vivo fish models (
Credit author statement
Guixiang Ji: Conceptualization, Methodology, Investigation, Writing – review & editing. Jie Gu: Investigation, Writing – review & editing, Investigation, Formal analysis. Min Guo: Methodology, Investigation. Linjun Zhou: Methodology, Investigation. Zhen Wang: Methodology, Investigation. Lili Shi: Methodology, Investigation. Aihua Gu: Project administration, Funding acquisition, Writing – review & editing.
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 Central Scientific Research Projects for Public Welfare Research Institutes (GYZX200102), the National Key Research and Development Program (2019YFA0802701, 2018YFC1004203) , and the National Science Foundation of China (91839102, 91943301)..
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These authors contributed equally to this work.