The Toxic Mechanism Of Per-and Polyfluoroalkyl Substances To Model Animals,Plants,Microbes And Human Cells

Per-and polyfluoroalkyl substances（PFASs）,as a kind of typical emerging pollutants,were detected successively in various environmental media in our country,and the exposure level of soil and water was even higher than that of other countries.It brings potential risks to the environment and human health.In recent years,perfluorooctanoic acid(PFOA_C8),a typical long-chain perfluorooctanoic acid,and its salts have been banned in several countries,leading to a surge in the production and use of alternatives,but there is little research on the toxicological mechanisms of alternatives to PFOA_C8.At present,the biotoxic effects of hexafluoropropylene oxide dimer acid(Gen X_C6),the most typical short-chain substitute,are not clear.Therefore,it is urgent to further study the toxicity mechanism of PFOA_C8 and Gen X_C6.The main research contents and results are as follows:（1）Toxicity research in animals:Zebrafish embryos,a model animal,were used as experimental objects to study the mechanism of toxic effects of PFASs exposure on animals from the aspects of embryo morphology,cell apoptosis and molecular level.The results showed that exposure to PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6（v/v,1:1）could lead to embryo growth and organ dysfunction.The toxicity was PFOA_C8>PFOA_C8+Gen X_C6>Gen X_C6.The toxic effects of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 can also affect the incubation rate,mortality rate and deformity rate of embryos,and the combined exposure of PFOA_C8+Gen X_C6 may have additive teratogenic effect.The changes of these physiological indicators are positively correlated with the length of carbon chain of PFASs,exposure concentration and exposure time.This study revealed the mechanism of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 exposure leading to the dysfunction of embryonic growth and development:That is,by inhibiting the expression of genes related to apoptosis or autophagy,including atp1b2b,laptm4b,ctsbb,mt-co2,mt-co3 and mt-cyb,among which laptm4b and ctsbb are closely related to human tumors.（2）In vitro toxicity research of cells:Hep G2 cells of human hepatoma cell were used as the experimental object to study the mechanism of toxic action of PFASs exposure on cells from the cell morphology,molecular and protein levels.The results showed that exposure to PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6（v/v,1:1）would cause toxicity to human cells,and also lead to changes in gene diversity and gene expression level of Hep G2 cells.The toxicity might be PFOA_C8>Gen X_C6>PFOA_C8+Gen X_C6.In addition,exposure to PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6can cause changes in gene function and signaling pathway of Hep G2 cells at the gene level,and further affect protein interaction.Among them,TNF and Insulin resistance signaling pathway were significantly enriched in control（CK）vs PFOA_C8 group.（3）Toxicity research of plant seeds:The toxic mechanism of PFASs exposure on seed germination was studied from phenotype,absorption and transport levels,physiological and biochemical levels of model plants Nicotiana benthamiana（N.benthamiana）and Arabidopsis thaliana（A.thaliana）.Seed germination experiments showed that PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6（v/v,1:1）exposure would produce toxicity to plant seeds,indicating that seed germination of N.benthamiana and A.thaliana would be inhibited,and the toxicity was PFOA_C8>PFOA_C8+Gen X_C6>Gen X_C6.In addition,the bioaccumulation capacity of PFOA_C8 in N.benthamiana and A.thaliana was stronger than that of Gen X_C6,but Gen X_C6 had greater fluidity,and the enrichment and transformation modes of PFASs in N.benthamiana and A.thaliana were also different.Finally,the toxic effects of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 exposure had significant effects on the physiological and biochemical characteristics of N.benthamiana and A.thaliana seeds growth and development,and the degree of influence was positively correlated with exposure concentration,among which the antioxidant enzyme system was the most significant.（4）Pot toxicity research of plant seedlings:N.benthamiana and A.thaliana were used as experimental objects to study the toxic mechanism of exposure of PFASs on the growth and development of seedlings from plant morphology,absorption and transport levels,physiological and biochemical levels,and ultrastructure.The results were as follows:First,pot experiment showed that exposure of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6（v/v,1:1）could produce toxicity to the growth and development of plant seedlings,and the toxicity was PFOA_C8>PFOA_C8+Gen X_C6>Gen X_C6.Second,the transport and accumulation of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 in different parts of N.benthamiana and A.thaliana showed a tendency.For example,the accumulation of PFOA_C8 and Gen X_C6 in N.benthamiana organs was the highest in the root and the lowest in the seed.In A.thaliana organs,the accumulation of PFOA_C8and Gen X_C6 was the highest in roots,but the lowest in stems.Third,exposure to PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 can cause changes in trace elements,enzyme activities,growth cycle,yield and microstructure of different organs in N.benthamiana and A.thaliana,and the range of changes is closely related to exposure period,exposure duration and exposure concentration of PFASs.Fourth,the enrichment of PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6 in the mature seeds of N.benthamiana and A.thaliana would affect the seed germination of progeny,which may result in the generation separation toxicity effect.（5）Rhizosphere microbial toxicity research:The rhizosphere microorganisms of N.benthamiana and A.thaliana were subjected to PFASs for a long time.The toxic effects of PFASs on rhizosphere microorganisms were studied from the microscopic and molecular levels,and the potential of microbial remediation of contaminated soil was preliminarily explored.In this study,through the whole cycle pot experiment,it was found that PFOA_C8,Gen X_C6 and PFOA_C8+Gen X_C6（v/v,1:1）exposure would produce toxic effects on rhizosphere microorganisms,posing a threat to the species diversity,species abundance and species composition of the microbial community.In addition,the influence of PFOA_C8 exposure on rhizosphere microorganisms was significantly stronger than that of Gen X_C6 exposure,indicating that PFOA_C8 had greater toxicity to rhizosphere microorganisms.Then,Rhizobium daejeonense,Streptomyces coeruleofuscus and Sphingomonas mesophila were isolated and purified from long-contaminated rhizosphere soil.Finally,by inoculating resistant strain suspensions in pot experiments,it was found that inoculating microorganisms could not only promote the growth of N.benthamiana and A.thaliana,but also improve seed yield,and effectively reduce the transport and accumulation of PFOA_C8 in plants.It shows that microbial treatment has certain ecological restoration potential.However,these strains had no significant effect on the enrichment and transformation of Gen X_C6 in plants.In summary,this study studied the toxic effects of PFASs exposure on zebrafish embryos,human cells,two model plants and their rhizosphere microorganisms,and elucidated the toxic mechanism of PFASs on model animals,plants and human cells from the aspects of phenotype,physiological and biochemical indexes,microstructure,molecular and protein levels.The exposure of PFASs to aquatic animal populations and terrestrial plants were revealed in the hope of attracting human attention to PFASs pollution.In addition,this study found a new target for human tumor therapy,and also provided an important theoretical basis for microbial remediation of PFASs.