Development And Immunotoxicity Of 6:2 FTSA To Zebrafish

Per-and polyfluoroalkyl substances(PFASs)are a class of anthropogenically synthesized economically important persistent organic pollutants that are ubiquitous in the environment and pose a threat to biological health.6:2 fluorotelomer sulfonic acid(6:2 FTSA)is a new alternative to Perfluorooctane sulfonic acid(PFOS),a typical representative of PFASs,which has been widely used in recent years and the concentration/frequency of environmental detections has increased significantly.However,there is a lack of studies on the biotoxicity of this alternative and the toxicological information needs to be improved.In this thesis,we firstly investigated the effects of PFOS and its substitute 6:2 FTSA on the developmental phenotype and immune-related indexes of zebrafish larvae at different concentrations,and explored the developmental and immunotoxicity of the two substances on zebrafish at the individual level.The effects of PFOS and its substitute 6:2 FTSA on gene transcription and metabolite expression of zebrafish larvae at the same concentrations were then investigated using transcriptomics and non-targeted metabolomics techniques,and correlation analysis was performed to reveal the mechanisms of developmental and immunotoxicity induced by the two substances at the molecular level.The results of this research will provide a theoretical basis for the accurate evaluation of the ecological risk of 6:2 FTSA in the environment,and the main results are as follows:(1)In the morphological observation experiment section,zebrafish larvae were acutely exposed(2-5 dpf)to 6:2 FTSA and PFOS at 0,0.5 and 1.5 mg/L,and malformation,body length,survival rate,heart rate,and cell apoptosis of zebrafish larvae were counted and recorded at the end of the exposure.Both low concentrations(0.5 mg/L)6:2 FTSA and PFOS exposures resulted in a significant increase in malformation and cell apoptosis in zebrafish larvae,in addition to a developmental delay(significant reduction in body length)in the PFOS treatment group.Exposure to high concentrations(1.5 mg/L)of 6:2 FTSA and PFOS further increased the malformation rate of zebrafish larvae,and both showed developmental delay,reduced heart rate and cell apoptosis,in addition to a significant reduction in survival in the PFOS treatment group.This study suggests that both 6:2 FTSA and PFOS can induce developmental toxicity in zebrafish larvae with a dose-effect relationship.(2)In the immune index assay section,a blank control(culture medium),6:2 FTSA(0.5mg/L)and PFOS(0.5 mg/L)treatment groups were set up for acute exposure(2-5 dpf)of zebrafish larvae,and immune-related biochemical indexes(CAT,SOD,MDA,NO,TNOS,i NOS,LZM,ACP,AKP,IL-1β,TNF-α,NF-κB,TLR4)were measured at the end of the exposure.All the indicators(TNOS,i NOS,ACP,AKP activity and MDA,IL-1β,TNF-α,NF-κB,TLR4 content)increased significantly under 6:2 FTSA exposure,except for SOD and LZM activity,which decreased significantly,and NO content which did not change significantly.All the indexes(CAT,SOD,TNOS,i NOS,ACP,AKP activity and MDA,IL-1β,TNF-α,NF-κB,TLR4 content)increased significantly under PFOS exposure,except for LZM activity which decreased significantly and NO content which did not change significantly.This study suggests that both6:2 FTSA and PFOS induce immunotoxicity in the organism by inducing oxidative stress and inflammatory responses,and that this process may be regulated by the TLR-NF-κB signaling pathway.(3)In the transcriptomics experiment section,a blank control(culture medium),6:2 FTSA(0.5 mg/L)and PFOS(0.5 mg/L)treatment groups were set up for acute exposure(2-5 dpf)of zebrafish larvae,and differential genes were screened by RNA-seq at the end of the exposure.Expression of genes on steroid biosynthesis,cytokine-cytokine receptor interactions and TLR/NOD-MAPK signaling pathway(e.g.,cyp24a1,cxcl8 b,irf3,map3k8,stat1 b,il1b,tnfb,hsp70)were significantly increased under 6:2 FTSA exposure.PFOS exposure significantly increased the expression of genes on steroid biosynthesis,unsaturated fatty acid biosynthesis,fatty acid chain elongation system,α-linolenic acid metabolic signaling pathway(e.g.,cyp51,cyp24a1,msmo1,fads2,elovl2,pla2g3),as well as on cell adhesion molecules and Ig A production pathway of the intestinal immune network(mhc2dab and si:busm1-266f07)expression was significantly reduced.This study shows that both 6:2 FTSA and PFOS affect the developmental process and induce immunotoxicity by changing the expression of genes in metabolic,transcriptional and immune-related pathways in zebrafish larvae,with 6:2 FTSA mainly activating the inflammatory response and PFOS not only activating the inflammatory response but also inhibiting the production of Ig A antibodies in the intestine.The mechanism of immunotoxicity of 6:2 FTSA and PFOS on zebrafish larvae is different.(4)In the metabolomics experiment section,a blank control(culture solution),6:2 FTSA(0.5 mg/L)and PFOS(0.5 mg/L)treatment groups were set up for acute exposure(2-5 dpf)of zebrafish larvae,and differential metabolites were screened by non-targeted metabolomics techniques at the end of the exposure.6:2 FTSA treatment group was dominated by carboxylic acids and their derivatives(e.g.,pantothenic acid,threonine,alanylglutamine)differential metabolites predominate,mainly annotated to pathways related to metabolism(e.g.,amino acid metabolism,lipid metabolism).The PFOS treatment group is dominated by fatty acyl(e.g.,arachidonic acid,γ-linolenic acid,5-keto)and glycerophospholipid(e.g.,lysophospholipids,glycerophosphatidylcholine,phosphatidylcholine)differential metabolites,mainly annotated to pathways related to metabolism(e.g.,lipid metabolism)and immune(e.g.,Ig A production in the intestinal immune network,Fc γ R-mediated phagocytosis)related pathways.Furthermore the results of association analysis further confirmed that both 6:2 FTSA and PFOS induce developmental and immunotoxicity in zebrafish larvae mainly by affecting metabolism related pathways.This study suggests that both 6:2 FTSA and PFOS induce organismal toxicity by changing the expression of metabolism related metabolites and signaling pathways in zebrafish larvae,but the organismal responses of both are not completely identical.