Study On Toxicity And Mechanism Of BPA And Its Substitutes On Pharyngeal Cartilages Development Of Zebrafish (Danio Rerio)

Background: As one of the most common endocrine-disrupting chemicals(EDCs),bisphenol A(BPA)is a threat to human beings.The adverse effects of BPA on human health and wild populations have been well explored.Craniofacial deformity is a common defect in humans,which are known to be caused by genetic and environmental factors;environmental causes of craniofacial abnormalities are still under investigation.Since craniofacial skeletal responds to changes in hormones,BPA exposure could potentially influence skeletal health in humans.However,little is known about the effects of BPA on craniofacial skeletal development in humans.Therefor,we used the extensively studied zebrafish(D.rerio)to to examine to what extent low-dose BPA exposure affects craniofacial cartilage development.Materials and methods: Mature wild-type AB line zebrafish,all the fish were maintained in a recirculation system.Experimental design:(1)Parental-embryonic BPA exposure strategy:embryos from parental BPA exposure)1 ?M)were collected and continuously exposed to different BPA concentrations(0.05,0.1,1 and 10 ?M),larvae were collected for alcian blue,Haematoxylin-eosin(HE),TUNEL staining or global transcriptome analysis at 120 h post-fertilization(hpf).(2)Embryos from parental BPA exposure(1 ?M)were collected and maintained in vehicle control until 4.5 or 120 hpf before being collected for gene expression analysis.(3)Eggs were exposed to four increasing concentrations of BPA(0.0038,0.05,0.1,and1 ?M)until 120 hpf.(4)Eggs were exposed to BPA/BPS/BPAF until 120 hpf,histomorphometrical and transcriptomic essay was applied to investigate the effects of BPA/BPS/BPAF exposure on pharyngeal cartilages development of zebrafish offspring and its underlying toxic effects mechanism.Results and discussion: PART ONE: Parental BPA exposure affected hatchability and heart rate of F1 progeny induced subtle pharyngeal cartilage and gross craniofacial malformation.Parental BPA exposure altered gene expression involved in defense responses,ROS metabolic process,apoptosis,p53 and MAPK signaling pathway.These newly identified gene expression patterns,pathways and gene networks of offspring after early life exposure may lead to long-term adverse morphological and functional consequences,including the observed pharyngeal cartilages and craniofacial defects.PART TWO: Parental BPA exposure changed gene expression of offspring embryos related to epigenetic regulation,highlight that epigenetic inheritance may involve in the transgenerational effects of BPA.PART THREE: Embryonic BPA exposure at environmentally relevant concentrations during the early-life stage is able to affect pharyngeal cartilage and craniofacial structures development in zebrafish.We also confirm that BPA exerts its effects via classic nuclear estrogen receptors,estrogen-related receptors,as well as androgen receptor.PART FOUR: BPA alternatives BPS and BPAF exposure during early stages of development is able to affect pharyngeal cartilage and craniofacial structures.Conclusion: BPA exposure affected chondrogenesis in the viscerocranium of zebrafish,via classical nuclear estrogen receptor,estrogen-related receptor,as well as androgen receptor.Apoptosis,p53 signaling pathway and MAPK signaling pathway were closely associated with the toxicity of BPA exposure.Epigenetic inheritance may involve in the transgenerational effects of BPA.Aternatives BPS and BPAF also caused changes in the angles and lengths of craniofacial cartilage elements.