| With the rapid development of social economy,the ecological environment of the Yangtze River has been deteriorating,and shipping accidents have occurred frequently.Water pollution has become the main factor leading to the sharp decrease of aquatic biological resources,such as fish.Among the main components of water pollution,Polycyclic aromatic hydrocarbons(PAHs)play important roles.As a class of global persistent organic pollutants,PAHs are widely found in various water ecological environments such as the Yangtze River.Up to now,lots of explorations have been carried out on the toxicity of PAHs,most of which focus on their parent compounds.However,studies have shown that most of the PAHs in nature have been structurally added with various substituent groups through diverse reactions.As a result,the actual PAHs in the environment become a complex mixture of various compounds,for instance oxygenatedPAHs(oxy-PAHs)and others.Systematical and profound explorations are required on the toxicological response of fish to these forms of PAHs and relative mechanism.Puffer fish Takifugu obscurus is an anadromous species,taxonomically belonging tothe Tetraodontiformes,Tetraodontidae.This species mainly distributes in the middle and lower reaches of the Yangtze River and sea areas in China,as well as the Korean Peninsula and the west coast of Japan.The sexually mature broodstocks in the late spring and early summer migrate upstream to lay eggs to the middle and lower reaches of the Yangtze River from the East China Sea.In the second year of spring,T.obscurus juveniles return to the sea and continuely grow.In recent years,the resources of wild T.obscurus in the Yangtze River have declined sharply.Water pollution of PAHs may be one of the the adverse factors leading to this situation.To this end,this study systematically explored the sublethal toxicity of 9,10phenanthrenequinone(9,10-PQ,a kind of oxy-PAHs)to T.obscurus based on toxicology,tissue immunology,metabonomics and transcriptomics.In addition,the toxicity of 9,10-PQ,its parent compound phenanthrene(Phe)and alkyl compound retene to T.obscurus were further investigated.The main contents and results of this dissertation are as follows:Firstly,in this study,the average body length(10.19±0.45)cm,average body weight(24.96±0.97)g of T.obscurus was utilized,and the experiment was applied in semihydrostatic contact experiment.The pre-test indicated that 9,10-PQ was the most toxic of the three compounds.According to the pre-experimental results and the semi-lethal concentration preparation requirements,different concentration gradients were set according to the logarithmic distance of 0.1 concentration,and the DMSO solution(V(DMSO):V(water)=1‰)was used as the control.The 24 h,48 h and 96 h lethal concentration(LC)experiment of 9,10-PQ on T.obscurus was calculated by linear regression method to be 164.55 μg/L,124.76 μg/L and 88.59μg/L,respectively.Then the sublethal toxicity test of 9,10-PQ was conducted basing on the 50%96 h-LC50(44.30μg/L)with Phe and retene.After 96 hours exposure,all the fish were anesthetized with plasma,gill,liver and other tissues collected.The results of histological studies indicated that the degree of damage in livers and gills was different when exposed to Phe,retene,and 9,10-PQ,respectively.All the three compounds exerted toxicity effect on T.obscurus while 9,10-PQ was the most toxic.The phenomenia showed that the liver and gill were important tissues of the defense system on immune responses to the three PAHs for T.obscurus.Secondly,plasma and hepatic metabolites of T.obscurus were detected by LC-MS after exposure to 9,10-PQ.After identifiing and screening in comparison with the control,the corresponding differential expressed metabolites(DEMs)in the plasma were mainly related to glycerophospholipid metabolism,biosynthesis of phenylalanine,tyrosine and tryptophan,and phenylalanine metabolism in ESI+model,as well as purine metabolism and sulphur metabolism in ESI-model.The DEMs in the liver were mainly related to the pathways on biosynthesis of unsaturated fatty acids,metabolism of phenylalanine and retinol.These DEMs and pathways indicated that 9,10-phenanthraquinone possibly caused cytotoxicity,neurotoxicity and immunotoxicity to Takifugu obscurus.Thirdly,transcriptome sequencing of liver tissues was performed through RNA-Seq technology.Each group received about 5 GB of Clean reads.The total number of unigenes obtained after assembly was 22,414.Based on the comparison with those of the control group,11627 differential expressed genes(DEGs)were screened in the liver samples of the fish after exposure to 9,10-PQ.Among them,767 DEGs were significantly altered with 462 up-regulated and 305 down-regulated.The results showed that 9.10-PQ activated glycolysis/gluconeogenesis,FOXO signaling pathway,phagosome pathway and steroid biosynthesis.These phenomenia indicated that 9,10-PQ could promote tumor growth,produce carcinogenic toxicity,affect immune function,inhibit fat metabolism and fatty acid synthesis,inhibit energy metabolism,and interfere with endocrine system.Fourthly,the DEMs and DEGs were analyzed together to explore the toxicity effect in response to 9,10-PQ.The enrichment of 22 metabolic pathways was found in both metabolic and transcriptome assays,Glycolysis/Gluconeogenesis was analyzed as the most disturbed pathway after screening.The activation of this pathway indicated that 9,10-PQ treatment had exerted impact on energy metabolism and immune stress in T.obscurus.These results indicated the shortcomings of single assay and confirmed that the combination of metabolome and transcriptome of the present study could provide a theoretical basis to T.obscurus for the systematic analysis and mutual validation of the toxic effects of 9,10-PQ on its immune defense mechanism under impact of oxidized PAHs.Fifthly,the alterations of SOD,CAT,MDA and C3 in the liver and AST,ALT,TG,TC,cortisol,T-Ig,LZM and glucose in the plasma were detected so as to verify the toxicity effect of oxidative stress,lipid peroxidation and damage to the immune system in this puffer fish following 9,10-PQ exposure,which was a complement to the result in aforementioned conjoint analysis of metabolome and transcriptome.The results of the above alterations further confirmed a more toxicity of 9,10-PQ than Phe and retene to T.obscurus. |
