Mode Of Action Exploration Of Bisphenol A Based On The Strategy Of Toxicity Testing In The 21st Century

In the past,chemical toxicity testing and risk assessment mainly relied on animal experiments and “apical endpoints”.This approach was expensive and not complied with the “3R” principle,and the relevance to humans was questioned.To overcome these shortcomings,the National Research Committee of the United States published a research report entitled “Toxicity Testing in the 21 st Century:A Vision and a Strategy” in 2007,proposing that it should be based on in vitro medium and high throughput human-derived cells（lines）or cell components tests,as well as computational toxicology and systems biology models,to construct toxic pathway-based toxicity testing methods,which promotes the application of toxic pathways into chemical risk assessment.Mode of action（MOA）refers to a sequence of key events（KEs）supported by the weight of evidence and related to toxicity endpoints.The application of MOA for next-generation risk assessment can simplify the elaboration of the mechanism of chemical toxicity,concentrate on the key toxicity pathways related to the endpoints of the risk assessment,and conduct extrapolation based on the pathway changes in the early stage of the adverse effects.Bisphenol A（BPA）is a typical chemical contaminant in food.In the past several decades,with ignoring of the early pathway changes,the risk assessments of BPA were based on the traditional animal experiments and “apical endpoints”,which introduced substantial uncertainties.Based on the strategy of Toxicity Testing in the 21 st Century,this study firstly conducted a systematic review to describe the adverse health effects of BPA,and then public database-based bioinformatics analysis and quantitative weight of evidence method were employed to establish and evaluate the MOAs of the possible adverse effects,and the corresponding human cell lines and high-throughput method were selected to verify and complement the MOAs of BPA-induced reproductive toxicity.This study provides a reference for the application of Toxicity Testing in the 21 st Century and serves as basis for the next-generation risk assessment and MOA-based health-based guidance values of BPA.Part 1: A systematic review of the adverse health effects of bisphenol AObjective: To systematically describe the hazard of BPA on the basis of the European Food Safety Authority（EFSA）risk assessment report published in2015,and identify the most likely adverse health effects caused by BPA,to provide basis for the establishment of MOA.Methods: The risk assessment of EFSA in 2015 systematically searched the English literatures published before 2014.This part conducted a systematic review of English literatures published after 2014（until September 9,2019）and all Chinese literatures that did not be searched by EFSA.Search databases included the Pub Med,Web of Science,Embase,Toxline,Scopus,CNKI and CQVIP.All literatures were screened via the title,abstract,and full-text reading.Results: After screening,233 articles were finally included.In terms of the reproductive and developmental toxicity,neurological and neurodevelopmental toxicity,immunotoxicity,cardiovascular toxicity,metabolic effects and carcinogenic effects,the results of human epidemiological studies of BPA are inconsistent,and the number of high-quality studies is small.In animal studies,The NOAEL of BPA to adult rats and mice is currently considered to be 5mg/kg.bw/d.BPA oral exposure during development and adulthood could affect the reproductive and developmental ability of animals,and the learning and memory abilities of offspring.The results of animal studies on immunotoxicity,cardiovascular toxicity,and metabolic effects are not consistent.The possibility of genotoxicity of BPA is rated as “unlikely”.There is not enough evidence to show that BPA has carcinogenic effects on animals,but BPA exposure may cause animal mammary epithelial hyperplasia and prostate hyperplasia leading to an increased risk of mammary gland and prostate cancer.Conclusions: BPA oral exposure during development and adulthood may affect the animal reproductive and developmental ability and nervous system development,and may cause the animal mammary gland hyperplasia and prostate hyperplasia and increase the sensitivity of breast and prostate cancer.Part 2: Establishment of mode of actions of bisphenol A based on bioinformatics analysis and a quantitative weight of evidence methodObjective: To map the potential MOAs based on the adverse health effects identified in the part 1.Methods: A BPA-related literature search was performed in the Comparative Toxicogenomics Database（CTD）.After excluding of the irrelevant literatures,the main characteristics of different target organs（i.e.,embryo,female gonad,male gonad,neuro,breast and prostate）were summarized,including target genes and phenotypes,etc.The DAVID online analysis tool was further used to conduct pathway enrichment analysis based on the included genes（count³2）to establish the MOA hypothesis.A quantitative weight of evidence method was used to evaluate the established MOA hypothesis.Results: The results of the CTD database search showed that the common target genes of BPA on embryo,female gonad,male gonad,neuro,breast and prostate mainly include ESR1,ESR2,MAPK1,MAPK3,BCL2,CASP3,BAX,AR and AKT1.The common phenotypes mainly include apoptosis process,cell proliferation,testosterone biosynthetic process,and estrogen biosynthetic process.The common KEGG pathways include estrogen signaling pathway and pathways in cancer.After evaluation using a quantitative weight of evidence method,it is thought that the MOA of BPA male reproductive toxicity includes the molecular initiating event（MIE）—binding of BPA to G-protein coupled estrogen receptor（GPR30）and/or estrogen receptor（ER）α/b,KE1—activation of mitogen activated protein kinase（MAPK）,serine/threonine kinase（Akt）,protein kinase G（PKG）and protein kinase A（PKA）signaling pathways,KE2—cell dysfunction in testis,and KE3—testis dysfunction,with an MOA score of 54.The MOA of BPA developmental neurotoxicity includes MIE—binding of BPA to ERα/b,KE1—activation of the extracellular regulated protein kinase（ERK）signaling pathway,KE2—activation of the N-methyl-D-aspartic acid receptor（NMDAR）,KE3—hippocampal neuron dysfunction,and KE4—learning-memory deficit,with an MOA score of 59.Research on BPA female reproductive toxicity,developmental toxicity,and promotion of prostate hyperplasia or increased sensitivity of prostate cancer are not enough to establish the MOAs,and MOA of promotion of breast hyperplasia or increases breast cancer sensitivity is under development.Conclusions: Two MOAs of BPA induced male reproductive toxicity and developmental neurotoxicity were established: BPA can activate the MAPK,Akt,PKG,and PKA signaling pathways through binding to GPR30 and/or ERα/b,leading to a testis dysfunction,and activate ERK and NMDAR signaling pathways via binding to ERα/b to cause learning-memory deficit.However,the confidence of the two MOAs is moderate,and with the current limited human data,the relevance to human is insufficient.Part 3: Mode of action of bisphenol A-induced damage on human normal ovarian epithelial cellsObjective: To explore the MOA of BPA-induced damage on human normal ovarian epithelial cells use the human-derived cell line（human normal ovarian epithelial cell IOSE80）based on the strategy of Toxicity Testing in the 21 st Century.Methods: A physiologically based pharmacokinetic（PBPK）model was used to calculate the concentration of the female gonads.Human normal ovarian epithelial cells IOSE80 were administrated for 28 days with BPA groups,a negative control of 0.1% dimethyl sulfoxide（DMSO）and a positive control of 10 n M 17β-estradiol（E2）.An absolute quantitative transcriptome sequencing and analysis was used to identify the possible key pathways that BPA leaded to the damage of IOSE80 cells,and qPCR test was employed to verify the mRNA expression level of the possible key pathways.On this basis,ER and ERK inhibitors were administrated to measure the expression of the proteins of possible key pathways and cell cycles using Western blot test and flow cytometry,respectively,to identify the potential KEs.The Bayesian benchmark dose（BBMD）software was used to analyze the concentration-response relationship,and the benchmark dose lower confidence limit（BMDL）values.Results: Based on the maximum concentration（Cmax）value of female gonad through oral exposure,nine BPA concentrations were designed for the CCK-8 test,and four BPA concentration groups were set based on the results of the CCK-8test.The results of absolute quantitative transcriptome sequencing showed that after a 28-day exposure to BPA in IOSE80 cells,the differentially expressed genes were mainly enriched in the KEGG pathway of ribosome biogenesis in eukaryotes,RNA transport,cell cycle,cellular senescence,progesterone-mediated oocyte maturation,etc.which were possible key signaling pathways.The qPCR results showed that compared with the negative control group,the relative mRNA expression of MAPK3 and CDKN3 increased significantly,and the relative mRNA expression of CDC20 decreased significantly,after being exposed to BPA for 28 days.Western blot results showed that the relative expression levels of phosphorylated ERK and CDKN3 proteins increased with increasing BPA concentration and could be partially inhibited by ERK inhibitor U0126.The cell cycle measurement results showed that,compared with the negative control group,the 28-day exposure of BPA can significantly reduce the proportion of G0/G1 phase and increase the proportion of S phase.All KEs included in BBMD analysis consist of relative mRNA expression of MAPK3 and CDKN3,and the proportion of G0/G1 phase and S phase.Among all the BMDL values,the BMDL value of MAPK3 mRNA expression was the smallest,and the BMDL₅ and BMDL₁₀ were 77.188 and 107.040 nM,respectively.Conclusions: Under the present experimental conditions,the MOA of RWPE-1 cell damage caused by the actual human exposure levels of BPA may include: KE1-activation of the ERK pathway,KE2-increased expression of CDKN3,and KE3-cell cycle disorder（the decreased proportion of G0/G1 phase and increased proportion of S phase）.Part 4: Mode of action of bisphenol A-induced damage on human normal prostate epithelial cellsObjective: To explore the MOA of BPA-induced damage on human normal prostate epithelial cells use the human-derived cell line（human normal prostate epithelial cell RWPE-1）based on the strategy of Toxicity Testing in the 21 st Century.Methods: A PBPK model was used to calculate the concentration of the male gonads.Human normal prostate epithelial cells RWPE-1 were administrated for 28 days with BPA groups,a negative control of 0.1% DMSO and a positive control of 103 n M E2.An absolute quantitative transcriptome sequencing and analysis was used to identify the possible key pathways that BPA leaded to the damage of RWPE-1 cells,and qPCR test was employed to verify the mRNA expression level of the possible key pathways.On this basis,ER and GPR30 inhibitors were administrated to measure the expression of the proteins of possible key pathways and cell cycles using Western blot test and flow cytometry,respectively,to identify the potential KEs.The BBMD software was used to analyze the concentration-response relationship and the BMDL values.Results: Based on the Cmax value of female gonad,nine BPA concentrations were designed for the CCK-8 test,and four BPA concentration groups were set based on the results of the CCK-8 test.The results of absolute quantitative transcriptome sequencing showed that after 28 days of exposure to BPA,the differentially expressed genes of RWPE-1 cells were mainly enriched in the endocytosis,cell cycle,cellular senescence,MAPK signaling pathway,TNF signaling pathway,which are possible key signaling pathways.The results of qPCR verification showed that,compared with the negative control group,the mRNA expression of MAPKAPK2,MAP2K3,JUN,FOS,CDKN1 A and CCND1 increased significantly,and the mRNA expression of TP53 and CDC25 C decreased significantly,after being exposed to BPA for 28 days.Western blot results showed that the relative expression levels of phosphorylated MAPKAPK2 and c-fos protein increased with increasing BPA concentration.Compared with the negative control group,the 28-day exposure to BPA can significantly increase the proportion of G0/G1 and S phase,and reduce the proportion of G2/M phase in RWPE-1 cells significantly.All KEs included in BBMD analysis consist of relative mRNA expression of MAPKAPK2,JUN,CDKN1 A,CDC25C and CCND1,and the proportion of G0/G1 phase,S phase and G2/M phase.Among all the BMDL values,the BMDL value of the G2/M phase ratio was the smallest,and the BMDL₅ and BMDL₁₀ were 110.580 and 175.862 n M,respectively.The mRNA expression of CDKN1 A was also relatively small,and the BMDL₅ and BMDL₁₀ were 142.394 and 207.648 nM,respectively.Conclusions: Under the present experimental conditions,the MOA of RWPE-1 cell damage caused by the actual human exposure levels of BPA may include: KE1-MAPK pathway activation,KE2-increased expression of CCND1 and CDKN1A,and decreased expression of CDC25 C,KE3-cell cycle disorder（the increased proportion of G0/G1 phase and S phase,and decreased proportion of G2/M phase）.SummaryBased on the Strategy of Toxicity Testing in the 21 st Century,this study firstly conducted a systematic review to identify the most likely adverse health effects of BPA,and then the MOAs of BPA male reproductive toxicity and developmental neurotoxicity were established using bioinformatics analysis and quantitative weight of evidence.Finally,two human-derived cell lines and high-throughput sequencing methods were used to explore the MOAs of BPA-induced damage on human normal ovarian epithelial cells and human normal prostate epithelial cells under actual human exposure levels（PBPK model）.This study could serve as accumulation data for risk assessment of BPA,and provide a reference for the establishment of MOAs of chemicals and the application and promotion of Toxicity Testing in the 21 st Century in risk assessment.