Endocrine And Metabolic Disrupting Effects Of Bisphenol A And Its Substitutes

At present,a large number of domestic and foreign studies show that human beings are frequently exposed to endocrine disrupting chemicals(EDCs).Therefore,EDCs with clear toxic effects and serious threats to human health have been controlled or banned worldwide,such as bisphenol A(BPA).Because BPA was banned,many bisphenol analogs have flooded into the market,including bisphenol AF(BPAF),bisphenol F(BPF)and bisphenol S(BPS).However,more and more studies show that these substitutes,whoes structures are similar to BPA,also have endocrine disrupting effects.Therefore,it is of great significance to conduct a comprehensive evaluation of endocrine disrupting effects and a more in-depth health risk assessment on the substitutes.In this paper,BPA and its typical substitutes BPAF,BPF and BPS were studied.On the one hand,H295R cell line was used as the in vitro model,and UPLC-MS/MS technique was used to evaluate the synthesis and secretion of bisphenol A and its typical substitutes.We established a method for simultaneous determination of multiple hormones and provided the technical support for finding safer substitutes.On the other hand,HepG2 cell line was used as the in vitro model,and two high-throughput techniques,~1H-NMR and PCR Array,were combined to explore the metabolic perturbation effects and possible metabolic regulation mechanisms of bisphenol A and its substitutes.We maked up for the gap in metabolomics research of bisphenol A and its substitutes,and provided a new method to evaluate the toxicity of compounds and their substitutes.The research content and results mainly include the following two parts:(1)UPLC-MS/MS technique was used to establish a method for simultaneous determination of 9 steroid hormones,and H295R cells were used as the model to study the effects of BPA,BPAF,BPF and BPS exposure on extracellular hormone secretion level,so as to evaluate the endocrine disrupting effects of BPA and its substitutes.The results showed that the UPLC-MS/MS method had good linear(r~2>0.99),specificity,accuracy and accuracy.The LODs and LOQs were?1 ng/mL,the LOQs of progesterone,androstenedione and testosterone were even?0.05 ng/mL.The matrix effect was between 83.87-119.46%,and the recovery was within the range of80.03-113.32%.In addition,the determination of extracellular steroid hormone levels in H295R cells showed that high concentrations(10~-66 M)of BPA,BPAF,BPF,and BPS up-regulated the level of 17β-estradiol,while down-regulated levels of androstenedione and testosterone.Therefore,BPAF,BPF and BPS have endocrine disrupting effects similar to BPA,and all of them have estrogenic effects and androgen inhibiting effects.(2)~1H-NMR and PCR Array were used to study the effects of BPA,BPAF,BPF and BPS exposure on the expression levels of extracellular metabolites and related intracellular metabolites of HepG2 cells,and to systematically explore the possible risks of BPA and its substitutes on human health and the molecular mechanism of toxic effects from the perspective of genes and metabolism.The results showed that BP analogues resulted in disturbances in 7-15 metabolites classified as amino acid(alanine,glutamine,glutamate,etc.),intermediates and end-products in the glycolysis and the tricarboxylic acid cycle(acetate,lactate,pyruvate,etc.).These metabolites were associated with 3-8 disrupted pathways,which included two common pathways(pyruvate metabolism,alanine,aspartate and glutamate metabolism)and indicated enhanced glycolysis.Their rank in order according to the number of metabolites and pathways was BPF>BPA>BPAF>BPS.The following glycometabolism PCR Array analysis suggested that BPAF has increased the pyruvate kinase(PKLR)expression level while the other three BP analogues decreased the expression level of glucokinase(GCK)that indicating glucose sensing impairment.Our results demonstrated the potential for using metabolomic and PCR array to understand the underlying action of mechanisms and identify the potential targets for future targeted risk assessment.