Neurodevelopmental Toxicity And Epigenetic Mechanism Of Bisphenols In Zebrafish Embryos

Bisphenols(BPs)are synthetic chemicals mainly used as raw material in the production of polycarbonate plastics and epoxy resins.As the restrictions and societal pressure on bisphenol A(BPA)grow on the basis of its associated health implications,the production and usage of BPA congener chemicals,including;bisphenol S(BPS),bisphenol F(BPF),and bisphenol(BPAF)hugely increased.The presumably safer alternatives of BPA;BPS,BPF,and BPAF are ubiquitously present in the natural environment resulting in a far-reaching and increased risk of human exposure.Despite the significant risk of human exposure to BPS,BPF,and BPAF,there is still a dearth of information about their toxicological properties,although such information is comprehensive for BPA.Moreover,epigenetic modifications through exposure to environmental toxicants have gained much consideration in understanding how contaminants could wield toxic effects,heightened at early stages of developmental susceptibility.The present study therefore seeks to investigates the toxicity of BPA and its commonly used alternatives;BPS,BPF,and BPAF on zebrafish embryos.Herein,zebrafish embryos were exposed to low concentrations(0.01,0.03,0.1,0.3,and 1 ?M)of BPA,BPS,BPF,and BPAF at4-hour post fertilization(hpf)until 120 hpf.To our knowledge,the bisphenol concentrations used in this are nearly environmentally relevant levels in assessing toxicological effects of bisphenol exposure.This study offers four main contributions,for which the procedures and contributions are briefly outlined below:(1)Developmental neurotoxicity of bisphenol A and S exposure in zebrafish embryos: To investigate the mechanisms mediating the developmental neurotoxicity,zebrafish embryos were treated with 0.01,0.03,0.01,0.3,1 ?M BPA and BPS.Also,we used transgenic zebrafish Tg(Hu C-GFP)to investigate whether BPA and BPS could exert effect on the neuron development.Reduction in body length,and increased heart rate were significant in 0.3 and 1 ?M BPA and BPS groups,while pericardial edema and spine malformations were remarkable in 0.3 and 1 ?M BPA groups compared with the control.The green fluorescence protein(GFP)intensity of Tg(Hu C-GFP)increased at 72 hpf and 120 hpf in BPS group,which was consistent with the increased m RNA expression of elval3 following BPS treatments,an indication of the plausible effect of BPS on embryonic neuron development.Additionally,the 0.3 and 1 ?M BPA and BPS treatments elicited hyperactivity and reduced static time in zebrafish larvae,suggesting behavioral perturbations.Moreover,q RT-PCR results showed that BPA and BPS could interfere with the normal expression of development-related genes vegfa,wnt8 a,and mstn1 at the developmental stages.The expression of neurodevelopment-related genes(ngn1,elavl3,gfap,?1-tubulin,mbp,and gap43)were significantly upregulated in BPA and BPS treatments,except for the remarkable downregulation of mbp and gfap elicited by BPA at 48(0.03 ?M)and 120 hpf(0.3 ?M)respectively;ngn1 at 48 hpf for 0.1 ?M BPS.Overall,the findings of this study highlighted that embryonic exposure to low BPA and BPS concentrations could be detrimental to the central nervous system development,and induce behavioral abnormalities in zebrafish at developmental stages.(2)Effects of bisphenol A and S on pancreatic ?-cell and DNA methylation in zebrafish:Herein,we investigated whether embryonic exposure to low BPA and BPS concentrations impair early pancreatic ?-cell differentiation as well as DNA methylation in its gene expression profile.BPA and BPS-induced effects on pancreatic-related genes,insulin gene,and DNA methylationassociated genes were assessed at developmental stages(24 – 120 hpf),while glucose level was measure at the 120 hpf.The insulin expression levels decreased at 72 – 120 hpf for 1 ?M BPA,while 0.32 and 0.24-folds changes of insulin expression were elicited by 0.3 and 1 ?M BPS,respectively at 72 hpf.Significant elevation of glucose levels;16.3%(for 1 ?M BPA),7.20%(for0.3 ?M BPS),and 74.09%(for 1 ?M BPS),compared to the untreated groups were observed.Further,pancreatic-related genes pdx-1,foxa2,ptfla,and isl1 were significantly interfered compared with the untreated group.Additionally,the maintenance methylation gene,dnmt1 was monotonically and significantly decreased at early stage of development following BPA exposure,but remained constant for BPS treatment relative to the control group.The de novo DNA methyltransferase genes(dnmt3,dnmt4,dnmt5,dnmt6,dnmt7,and dnmt8)were distinctively altered following BPA and BPS embryonic exposure.The data of this study revealed that embryonic exposure to low concentration of BPA and BPS can impair the normal expressions of pancreatic-associated genes and DNA methylation pattern of selected genes in zebrafish early development.(3)Effects of bisphenol F and AF on pancreatic ?-cell and DNMTs: This contribution of the study aimed to investigate the effect of low-dose BPF and BPAF on pancreatic ?-cell differentiation in zebrafish by altering DNA methylation and gene expression.The study reports the BPF and BPAF-induced effects on glucose level,as well as the transcription of genes related to the pancreas and DNA methylation.Furthermore,bisulfite sequencing PCR(BS-PCR)was performed to measure the methylation levels of promoter genes.The findings of the study reported a significantly increased glucose level elicited by 1 ?M BPF(28%),while a monotonic increase of 29%,55%,and 74% of glucose level was respectively observed in 0.1,0.3,and 1 ?M BPAF;concomitant with the decreased insulin gene expression.Furthermore,q RT-PCR results showed that BPF and BPAF upregulated pdx-1 expression,except for the suppressed pdx-1expression observed in 0.3 and 1 ?M BPF.While both BPF and BPAF upregulated ptf1 a expression throughout the developmental stages,foxa2 and isl1 m RNA expressions were dynamically and significantly altered during zebrafish development.Moreover,BPF and BPAF significantly decreased the expression of dnmt1 at 48 and 96 hpf,but was upregulated at 120 hpf.The de novo DNA methyltransferase genes(dnmt3,dnmt4,dnmt5,dnmt6,dnmt7,and dnmt8)were significantly and distinctively altered compared with the control group.It worth stating that,the methylation levels of pdx-1,the promotor gene,in both BPF and BPAF did not differ significantly relative to the control group.In summary,the results of the study demonstrated that exposure to BPF and BPAF in the early life stages wielded adverse effect on the normal expressions of pancreatic-associated genes and DNA methylation pattern of selected genes in zebrafish early development.(4)Developmental neurotoxicity of bisphenol F and AF and DNA methylation mechanism: This phase of the study seeks to assess the effect of low-dose BPF and BPAF on neurodevelopment in zebrafish by altering DNA methylation and gene expression.Zebrafish embryos were exposed to 0,0.01,0.03,0.1,0.3,and 1 ?M BPF and BPAF at 4-hour post fertilization(hpf)until 120 hpf.BPF and BPAF-induced effects on larvae behavior,transcription of genes related to neuron,and DNA methylation were assessed.In addition,bisulfite sequencing PCR was performed to measure the methylation levels of promoter genes at 24 hpf.The findings of this study reported that 0.3 and 1 ?M BPF and BPAF significantly inhibited hatch ratio at 48and72 hpf.Larvae body length was significant reduced in 0.3 and 1 ?M BPF;0.1,0.3,and 1 ?M BPAF at 72,96,and 120 hpf relative to the control group,and followed a concentration-dependent manner.The 1 ?M BPF,and 0.3 and 1 ?M BPAF reduced embryonic heart rate at 48 hpf,while0.3 and 1 ?M BPF and BPAF remarkably reduced larvae heart rate at 72 and 96 hpf.The ratio and extent of malformation(curved spin)increased with increasing BPF and BPAF concentration,with the highest percentage malformation of 15.34% observed in 1 ?M BPAF group.Also,BPF and BPAF markedly increased average speed,maximum acceleration,and mania time;while larvae static time markedly decreased.In addition,q RT-PCR results showed that BPF and BPAF could interfere with the normal expression of development-related genes vegfa,wnt8 a,and mstn1 at the developmental stages.Moreover,BPF and BPAF significantly increased the expression ngn1 and gfap m RNA throughout the early stages of zebrafish development,except for the downregulation of gfap at 48 hpf observed in BPAF groups,compared with the control.?1-tubulin and mbp were downregulated in BPF groups but upregulated in BPAF exposure groups.The elavl3 and gap43 m RNA were significantly and exclusively altered following exposure to BPF and BPAF.The transcription of dnmt1 was significantly suppressed,whereas the dnmt3,dnmt4,dnmt5,dnmt6,dnmt7,and dnmt8 were markedly and dynamically altered following embryonic exposure to BPF and BPAF.Noteworthily,the DNA methylation levels of ?1-tubulin,the promoter gene,in BPF and BPAF did not differ significantly relative to the control group.In summary,our study demonstrated that exposure to BPF and BPAF in the early life stages could elicit behavioral abnormalities,and exert deleterious effect on the epigenetic regulation of neurodevelopment of zebrafish.