Neurotoxicity And Immunotoxicity Of Perfluorooctanoic Acid On Juvenile Cynoglossus Semilaevis Through Transcriptome Analysis

Perfluorooctanoic acid?PFOA?is an organic environmental pollutant that can be detected in waters,soil,and living organisms extensively.It is persistent and bioaccumulative in the environment and can be toxic to organisms.Half smooth tongue sole?Cynoglossus semilaevis?is a warm temperatural and benthic marine fish and it is sensitive to marine environmental pollutants.It can be used as a model species in the study of ecotoxicology to monitor marine environmental pollution.In this study,5-month-old half smooth tongue soles was used as the test species and exposed to 50mg/L PFOA water body at different time periods to observe the tissue lesions and count the number of deaths,Using Illumina Novaseq?6000 transcriptome sequencing platform for high-throughput sequencing and gene data mining of entire tissues.The results of this study are as follows:1.Compared with the control group of half smooth tongue sole,the tissue damage and death of half smooth tongue sole exposed to PFOA were more serious.From the 5th day of half smooth tongue sole exposed to PFOA,blisters appeared in intestinal tissues.On the 7th day,liver turned yellowish and gallbladder turned swollen and black.The apparent symptoms on the 10th day were similar to those on the 7th day.On the 14th day,the visceral mass was swollen and the liver and brain had hyperemia.The cumulative mortality in the test group began to increase sharply from the 9th day of exposure on half smooth tongue sole.On the 14th day,the cumulative mortality rate of the experimental group reached 56.4%,while the mortality rate of the control group was 17.6%.2.There were 33 transcriptomes were obtained by transcriptome sequencing was performed on half smooth tongue sole exposed to PFOA for 0,3d,5d,7d,10d,and 14d.The length of each transcriptome is between 39,743,324 and 55,918,860 bp,consisting of1,663,039,762 reads in total.After filtering out the unqualified sequences in raw reads by Cutadapt,the valid reads of all samples accounted for more than 94.00%of the total raw reads and the GC content of valid reads was more than 47%?including 47%?.The number of mapped reads that can be compared to the genome is more than 91%.The valid reads are spliced into 24044 genes.3.Differential expression analysis of 24044 genes?|log₂ fold change|?1,P<0.05?showed that the number of significantly differential expressed genes varied with the exposure time of PFOA.There were 2497,4634,915,1178 and 1560 significantly differentially expressed genes on the 5th day,7th day,10th day and 14th day respectively.Among which the number of differentially expressed genes on the 5th day of PFOA exposure was the largest,the number of differentially expressed genes on the 7th day was the smallest.Among the five times exposed to PFOA,there were 688,1664,480,749,and1295 differentially expressed genes that were up-regulated,and 1809,2970,435,429 and265 differentially expressed genes that were down-regulated,respectively?P<0.05?.A further analysis of all differentially expressed genes at five exposure time showed that there were 8 genes that were significantly differentially expressed at each exposure time,namely,vwa5a,gata2,transcription factor Sp8-like,myoglobin-like,plasminogen activator inhibitor 1-like,epigen-like,uncharacterized protein LOC112488231 and uncharacterized protein LOC103384322.The abnormal expression of these genes is involved in immune deficiency,hyperosteogeny,tumor,cancer,leukemia and other diseases.The quantitative real-time PCR was used to verify the genes gata2,LOC103383752,LOC103394625 and vwa5a.The results showed that the gene expression trends in the quantitative PCR results were consistent with the gene expression trends in the transcriptome sequencing results,but the gene expression levels were different.4.During the five time points of PFOA exposure to half smooth tongue sole,the differential expression of genes related to GO function annotations such as nervous system development,axonogenesis,brain development,neuron projection,and dendrite were significant.Metabolic pathways associated with synaptic vesicular circulation,retrograde neural signaling,glutamate synapses,gamma-aminobutyric synapses,axon guidance,dopaminergic synapses,and calcium signaling pathways were significantly different.It shows that PFOA can influence the nervous system of half smooth tongue sole.5.During the five time points of PFOA exposure to half smooth tongue sole,the differential expression of genes related to GO function annotations such as cell adhesion,homophilic cell adhesion via plasma membrane adhesion molecules and ATP binding were significant.Metabolic pathways associated with Cell adhesion molecules?CAMs?,phagosome,pathogenic Escherichia coli infection,adrenergic signaling in cardiomyocytes,p53 signaling pathway and complement and coagulation cascades were significantly different.It shows that PFOA can influence the immune system of half smooth tongue sole.This study explored the toxic mechanism of PFOA on the half smooth tongue sole from the transcriptome level.Through the comprehensive analysis of differentially expressed genes,GO function annotations and KEGG metabolic pathways,it was revealed that PFOA mainly damages the immune system and nervous system of half smooth tongue sole.Combined with the obvious phenomena of liver damage and cerebral hemorrhage that observed in the juvenile of half smooth tongue sole during infection period,it can be concluded that half smooth tongue sole may produce toxic symptoms after long-term exposure to PFOA.This study provided gene resources and research means for the molecular toxicology study of PFOA on half smooth tongue sole.