Potential Mechanisms Of Butylparaben-induced Cardiotoxicity In Zebrafish At Early Developmental Stages And The Protective Role Of EGCG

ObjectiveButylparaben(BuP),a typical endocrine chemical,has strong antibacterial effect.BuP exists widely in many areas,including cosmetics,personal care products,articles of everyday use,pharmaceutical industry.Previous study indicated that exposure to BuP could induce the potential embryonic toxicity.However,current evidence is unclear regarding the toxic mechanisms and treatment measure of BuP.The epigallocatechin gallate(EGCG)is a watersoluble monomer.EGCG is the most abundant polyphenolic flavonoid of green tea catechin compounds,which has higher antioxidant activity.The small size,external and rapid development,and optical transparency of zebrafish embryos are some of the advantages the zebrafish model system offers.There are two aims to this research,the first is to clarify the effects and mechanisms of BuP on zebrafish embryo development.The second aim is to clarify the protective effects and mechanisms of EGCG on BuP-induced embryonic toxicity.MethodsThe 3 hours post fertilization(hpf)embryos were randomly divided to blank control groups(H2O),solvent control group(0.01% DMSO),BuP groups(0.5,1,1.5,2 mg/L),EGCG groups(1 μM and 10 μM),R1 group(1.5 mg/L BuP and 1 μM EGCG co-exposure),R2 group(1.5 mg/L BuP and 10 μM EGCG coexposure),50 embryos per group and exposed from 3hpf to 96 hpf.There were three replicates for each group.The embryonic mortality,hatching rate and deformity rate were recorded per 12 hours.The larval body length,pericardial area,heart rate,sinus venosus and bulbus arteriosus(SV-BA)distance were recorded at 72 hpf.HE staining was used to observe pathological changes in the heart of 96 hpf zebrafish embryos.The 96 hpf larvae exposed to 1.5 mg/L BuP,R2 and control groups were performed to RNA sequencing(RNA-seq).Identificative of DEGs,GO analysis,KEGG analysis and protein-protein interactive network analysis were performed on RNA-seq data to further explore the mechanisms of abnormal embryonic development induced by BuP and the protective mechanisms of EGCG.ResultsThe 2 mg/L BuP caused significant increases in mortality rate as compared to the solvent controls(P<0.05).The hatching rates were significantly lower in the groups that received 1.5 and 2 mg/L BuP than in the control group(P<0.05).Compared with the solvent control,the larval body length was significantly decreased in 1.5 and 2 mg/L BuP group,while the deformity rates were significant increased(P<0.05).The pericardial edema was the most common malformation.1.5 mg/L BuP significantly decreased the heart rate and increased the pericardial area and SV-BA distance(P<0.05).Compared with the BuP group,the embryo/larval mortality was significantly decreased in R2 group at 48,72 and 96hpf(P<0.05).At 72 hpf,the EGCG-BuP co-exposure caused significant increases in heart rate and decreases in pericardial area and SV-BA distance as compared to the BuP exposure alone(P<0.05).The results of HE staining demonstrated that BuP induced the abnormal larval cardiac morphology as compared to the solvent controls,and the abnormal cardiac morphology was alleviated after 10μM EGCG intervention.RNA-seq indicated that BuP group was identified 1939 DEGs as compared to the solvent control,R2 group was identified 216 reversed DEGs as compared to the BuP group.In BuP group,GO analysis demonstrated that DEGs were significantly enriched in lipid metabolism, hormone metabolism,retinol metabolism and atrial cardiac muscle cell differentiation.KEGG analysis demonstrated that BuP exposure mainly affected retinol metabolic pathway,PPAR signaling pathway and MAPK signaling pathway.In R2 group,GO analysis demonstrated that DEGs were significantly enriched in retinol metabolism and regulation of hormone levels.KEGG analysis demonstrated that reversed DEGs mainly affect retinol metabolic pathyway.The changes of gene cyp26a1,cyp26b1,fabp2 were related to retinol metabolism,regulation of hormone and PPAR signaling pathway.The gene klb、prx、erbb4 played important roles in MAPK signaling pathway,and apoa4 b.2,elovl7 b,pla2g1b were related to lipid metabolism.According to PPIN,these genes were correlated.BuP-induced embryonic and cardiac toxicity were jointly regulated by lipid metabolism,retinol metabolism,PPAR signaling pathway and MAPK signaling pathway.ConclusionsBuP exposure could induce abnormal embryonic development and cardiac development through interfering with the expression of cyp26a1,cyp26b1,fabp2 in PPAR signaling pathway,and affecting retinol metabolism and lipid metabolism.EGCG can alleviate the BuP-induced toxicity of embryonic development and cardiac development through affecting the retinol metabolism.EGCG has preventive and therapeutic effects on embryonic and cardiac toxicity caused by BuP.