| Takifugu obscurus,commonly known as pufferfish,is classified under the order Tetraodontidae which is nested within the series Tetraodontiformes.It is a traditional rare and anadromous fish in the middle and lower reaches of theChangjiangRiver and mainly distributed in the Bohai Sea,the Yellow Sea,the East China Sea and the middle and lower reaches of the ChangjiangRiver in China.Tributyltin chloride(TBT-Cl)is one of the most toxic and widely used organotin compounds.At present,TBT-Cl still has a high residue in marine and freshwater environment,and has a high re-exposure risk under the change of natural environment or human disturbance.Previous studies have shown that TBT-Cl exposure can produce immunotoxicity,reproductive toxicity,neurotoxicity,growth and development obstacles and other toxic effects on aquatic animals,which poses a great threat to the life history of aquatic animals.However,the toxicological effects of TBT-Cl on fish are relatively weak;especially the toxic effects and mechanisms of TBT-Cl on T.obscurus distributed in seawater,fresh water and estuarine area have not been reported.In this study,the acute toxicity of TBT-C1 exposure to juvenile.obscurus was studied.The toxicological response mechanism of T.obscurus to TBT-Cl exposure was studied by transcriptomics and metabolomics from gene level and metabolite level,in order to provide support for the evaluation of water ecological safety of TBT-Cl,and provide basic data for the establishment of TBT-Cl residue limit system(or amendment)in key water areas such as important migration channels and spawning grounds,as well as provide new ideas and research methods for exploring the toxicity mechanism of TBT-Cl exposure to aquatic organisms.Firstly,TBT-Cl was applied to the juvenile T.obscurus(average body length:10 ±1.5 cm,average body weight:25 ±1g)by semi-hydrostatic contact test.The results showed that the LC50 of TBT-Cl was 61.02,40.66 and 19.62 ?g·L-1 for 24 h,48 h and 96 h,respectively.According to the empirical formula,the safe concentration of TBT-Cl to juvenile T.obscurus was 1.962?g·L-1.The results showed that TBT-Cl was a highly toxic chemical substance.The presence or concentration of TBT-C1 in water environment would pose a serious threat to.obscurus and all fishery resources,which should be strictly controlled.The results provided a scientific basis for setting TBT-C1 concentration in the study of chronic toxicity and recovery effect in the future,and also provided a reference for toxicological study of TBT-C1 in other fishes.Secondly,in order to find out the molecular mechanism of the response of juvenile T obscurus after acute exposure to TBT-Cl,three concentrations of 10%(groupA),20%(groupB)and 50%(groupC)96 h-LC50TBT-Cl as well as a control group were set up in this study,and the livers of the experimental fish were harvested at 96 h for comparative analysis of transcriptomics.The results showed that 4,028 differentially expressed genes(DEGs)were screened from the liver transcriptome of each treatment group and control group.The up-regulated genes were mainly about immune response related genes,and the down-regulated genes were mainly about protein synthesis together with carbohydrate and lipid metabolism related genes.The DEGs were mainly enriched into 109 pathways through KEGG and 1,218 categories in GO database.These genes were mainly involved in signal transduction,immune response,autophagy and adhesion,among which MAPK signaling pathway,JAK/STAT signaling pathway,Wnt and TollI-like receptor signaling pathway were involved.In this study,comparative transcriptomics of response to TBT-C1 exposure in.obscurus was conducted for the first time,which laid a solid foundation for further exploring the toxicological response mechanism of related functional genes and pathways in.obscurus.Four up-regulated genes and two down-regulated genes may play an important role in the TBT-Cl exposure process in the screen and analysis of differential genes.Certain differences were revealed about the temporal and spatial expression patterns of these six genes were through qRT-PCR experiment.UBE3D was significantly induced by TBT-CI within gill in the acute and chronic treatment,and the maximum induction values existed in group B and 20 dand were 6.18 and 3.17 times of those incorresponding control groups,respectively.Meanwhile,this gene was significantly inhibited in the liver,and the minimum inhibitory values appeared in group C and 30 d,respectively,which were 0.10 and 0.03 times of those in corresponding control groups.In both acute and chronic treatments,STARD3 was significantly induced in gills,and the maximum values appeared in group B and 20 d and were 19.38 times and 4.03 times of those in corresponding control groups,respectively.The gene was also significantly induced in the liver of group A,with a maximum value 6.96 times of the control group.RBBP5 was significantly induced in the liver and gill of acute experiment while the maximum values appeared in group A and group Band were 11.60 and 6.18 times of that in the control group,respectively.As for chronic stress,it was also significantly inducedin the gill as the maximum value appeared at 20 d and was 10.50 times much as that of the control group.CCNE1 was significantly induced in the liver and gill of the acute exposure and in the gill of thechronic exposure.The maximum values of CCNE1 appeared in group B,group C and 20 d,and were 8.77,14.64 and 12.87 times of those in corresponding control group,respectively.ANGPTL2b was significantly induced in gill of both acute and chronic experiments,which were 23.55 and 9.36 times higher in group C and 20 d than those in corresponding control group,respectively.Prdx6 was significantly inhibited in the gill of acute experiment as the minimum value was in group Cand0.12 times of that in corresponding control.Contrarily Prdx6 was evidently induced in the liver of chronic experimentas the maximum value was at 20 d and 3.26 times of that in corresponding control.As this aspect of research indicated,these DEGs may be involved in TBT-Cl exposure to T.obscurus by activating the immune system,initiating immune signal response,participating in the repair pathway of immune injury,accelerating adaptive inflammation and maintaining the steady state of tissue environment in vivo.However,the specific biological mechanisms of these genes require further exploration.Thirdly,in order to further study the response mechanism of juvenile.obscurus to TBT-Cl,HSP90?1 gene,which may be involved in immune response and is highly conserved in structure,was identified based on the differential genes of transcriptome library.HSP90?1 gene was cloned successfully by RACE technology with a total length of 2,775 bp,including 2,412 bp of open reading frame,encoding 803 amino acids.The phylogenetic tree of NJ system was constructed by Mega 5 and the phylogenetic relationship was analyzed.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of HSP90?1 mRNA in various tissues.Then,acute and chronic TBT-C1 experiments were designed to detect mRNA levels of HSP90?1 and damages of.obscurus by qRT-PCR,histochemistry and immunohistochemistry.The results showed that HSP90?1 mRNA transcripts were widely expressed in brain,liver,gill,heart,muscle,kidney,spleen,blood cells,stomach and intestine,and the expression level was higher in liver and gill.HSP90?1 expressions in gill were 5.67,4.97 and 8.77 times higher than that in control group,and 0.70,1.01 and 2.55 times higher in liver,respectively.In chronic experiment,HSP90?1 expression was 6.11,18.21 and 2.63 times higher in gill and 0.13,0.60 and 0.12 times higher in liver than those in control group at 10 d,20 d and 30 d,respectively.At 15 d and 30 d of the recovery experiment,it was 1.52 and 1.11 times higher in gill and 0.70 and 1.00 times higher in liver compared with the control group,respectively.The injury degree of liver and gill under different concentrations of TBT-Cl was different from each other.The expression of HSP90?1 increased significantly after TBT-Cl stress,and was involved in the cytoplasmic reorganization of immune stress process.The expression level of HSP90?1 mRNA in gill was higher than that in liver.The expression pattern of HSP90?1 mRNA in gill from different groups was different from with each other as the experiments of chronic toxicological experiment and recovery experiment proceeding.The liver and gill,as the important tissues of defense system of T.obscurus,are immune to the toxic effects of TBT-C1.HSP90?1 may be involved in the defense of the invasion of TBT-Cl within T.obscurus to maintain its immune homeostasis.The results will be helpful to understand the specific effects of HSP90?1 on TBT-C1 toxicity and the possible mechanisms of HSP90?1 on immunotoxicity.Finally,in order to study the response of juvenile.obscurus to TBT-C1 acute exposure at metabolite level,serum and liver samples of three acute treatment groups and control group were tested by LC-MC technique,and metabolomics was compared and analyzed.The results showed that 87 and 34 different metabolites were screened from serum metabonomics under positive and negative ion modes,which were classified as phosphoglycerides,unsaturated fatty acids,and amino acids and so on.These metabolites were mainly related to sphingolipid metabolism,phosphoglyceride metabolism and biosynthesis of unsaturated fatty acids.There were 92 and 71 different metabolites in the liver metabolism group,which were classified as phosphoglycerides,unsaturated fatty acids,and amino acids and so on.These metabolites were mainly related to the metabolic pathways of phosphoglycerides,arachidonic acid and unsaturated fatty acid biosynthesis.Further analysis showed that the relative contents of phospholipids(16 PC,13 PE,1 PS,etc.)involved in phosphoglyceride metabolism from the liver of.obscurus of the three treatment groups were significantly higher than those of the control group(P<0.01),and the highest increase in PC(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))was 4,263.90 times higher than that of the control group.LysoPC(18:2(9Z,12Z))in the 20%96h-LC50 treatment group was up-regulated by 1.58 times compared with that in the control group.In 10%,20%and 50%96h-LC50 treatment groups,prostaglandin E2 was up-regulated by 6.67,6.93 and 7.86 times respectively while arachidonic acid was down-regulated by 15.93,22.50 and 16.07 times respectively.Acetylcholine in serum decreased by 2.84,2.30 and 2.63 times,sphingosine decreased by 1.39,1.89 and 2.07 times and sphingomyelin increased by 2.69,2.42 and 2.49 times,respectively.After TBT-Cl exposure,the pathways of PC to acetylcholine and sphingosine synthesis in.obscurus were inhibited,ARA metabolism and other unsaturated fatty acid synthesis in vivo were activated after liver injury,and the process of phosphoglycerides in vivo was accelerated.With the increase of lysophosphatidylcholine and prostaglandin E2 concentration in metabolites,detoxification function increased and inflammatory reaction decreased.The metabolic response mechanism may be one of the important reasons for the higher tolerance of juvenile T.obscurus to TBT-Cl than other fishes.The study of metabolomics provided the possibility to understand the specific situation of the disturbance of metabolic pathway in.obscurus under TBT-Cl stress and to analyze the relationship among the changes of metabolite content,related gene changes and biological phenotype changes,and provided a new theoretical guidance for the toxicity mechanism of TBT-Cl exposure to T.obscurus. |
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