Toxicological Effects And Mechanism Of Cypermethrin Or/and Sulfamethoxazole Expose On Grass Carp (Ctenopharyngodon Idella)

In recent years,the extensive use of pesticides and antibiotics has led to the increasingly serious destruction of aquatic ecosystem.Because different pollution sources can cause compound pollution,the ecological problems caused by the combined exposure of pesticides and antibiotics have attracted extensive attention.Pesticide cypermethrin(CMN)and antibiotic sulfamethoxazole(SMZ)are two common pollutants in aquatic ecosystem.However,their toxicity mechanism on fish has not been fully studied.As the main freshwater economic fish in China,grass carp are more vulnerable to the source of disease than other carps.At present,grass carp has become a sensitive and effective indicator in environmental science research.In this study,grass carp was used as a model organism to explore the toxic damage effects and mechanism of CMN or/and SMZ through animal and cell experiments.1.Animal experiment: grass carp as the experimental object,and selected the environmental related concentration of CMN(0.651μg/L)and SMZ(0.3μg/L)as the target compound to explore the effects of single and combined exposure on the physiological function of grass carp.The animal experiment is divided into two parts: the first part is the subchronic toxicological experiment of single or combined exposure of CMN and SMZ.The neurotoxicity,immunotoxicity and liver metabolic toxicity of grass carp caused by pollutant exposure were studied by pathological observation,biochemical experiment,intestinal microbial sequencing,transcriptomics and metabolomics sequencing.The results showed that in the brain,liver,intestine and spleen of grass carp,H&E staining showed that inflammatory cell infiltration,nucleolysis,pyknosis,heterotopic and vacuolization appeared in CMN,SMZ and MIX groups;The results of transmission electron microscope showed that the cells in the exposed group showed abnormal phenomena such as endoplasmic reticulum swelling,mitochondria swelling,vacuolization and nuclear pyknosis.CMN and SMZ exposure could reduce the levels of antioxidant indexes CAT,SOD and GSH in liver,intestine and spleen,accompanied by the increase of MDA level(p<0.05 or p<0.01).The activity of SOD and the contents of MDA and8-OHd G increased significantly in brain tissue(p<0.05 or p<0.01).TUNEL staining showed that both CMN and SMZ could significantly induce apoptosis in brain,spleen and liver(p<0.01).The serum biochemical indexes of grass carp showed that the contents of complement C3,Ig M and CRP and lysozyme(Lys)activity in each treatment group decreased(p<0.05 or p<0.01),while the liver function indexes(AST and ALT activities)were significantly higher than those in the control group(p<0.01).In addition,the levels of bloodbrain barrier and intestinal barrier related tight junction proteins,such as Occludin,Claudins,ZO-1 and ZO-2,decreased significantly(p<0.05 or p<0.01).The levels of interleukin(IL-1β,IL-6,IL-8)and TNF-α were increased significantly(p<0.05 or p<0.01).More significant changes were observed in the MIX group compared with the brain,liver,intestine and spleen toxicity caused by the above CMN and SMZ exposure alone.Based on intestinal microbiome analysis,we found that Proteobacteria became the dominant flora after CMN and SMZ were exposed alone and in combination.We sought the similarities and differences between CMN and SMZ induced hepatotoxicity by LC/MS non targeted metabonomic sequencing.The results showed that compared with CMN(45)and SMZ(32),mix group(97)showed more differential metabolites,which were mainly concentrated in glycine,serine and threonine metabolic pathways.The second part is the challenge experiment of Aeromonas hydrophila.Aeromonas hydrophila can cause infectious bleeding in the intestinal tract of grass carp,which threatens the development of grass carp breeding industry.After the exposure experiment,we injected Aeromonas hydrophila into the remaining grass carp.The results showed that after bacterial attack,compared with the control group,the mortality of grass carp in CMN,SMZ and mix groups increased significantly(P<0.01),and the expression of Toll like receptor(TLR)pathway related immune factors(such as TLR2,TLR4,My D88,TNF-α,IL-1β,IL-6 and IL-8)decreased significantly(P<0.01),and the change in mix group was the most significant(P<0.05 or P<0.01).The results showed that CMN and SMZ exposure would eventually destroy the immune recognition system and the ability to eliminate pathogens of grass carp,making them more vulnerable to pathogen infection and death.Therefore,reducing the exposure of environmental pollutants is of great significance to the prevention and control of diseases in the process of grass carp breeding.2.Cell experiment: Based on transcriptome sequencing results,KEGG database was used for enrichment analysis.The results showed that under CMN,SMZ and combined exposure conditions,the differential genes were enriched in proteasome and oxidative phosphorylation signal pathway,drug metabolism cytochrome P450 pathway and peroxisome proliferator activated receptor(PPAR)signal pathway,respectively.L8824 cell line(grass carp hepatocyte line)was used as a model to explore the toxic mechanism of CMN and SMZ.The cell experiment is divided into three parts: the first part discussed the role of proteasome pathway in CMN induced L8824 cytotoxicity.The results showed that the level of reactive oxygen species(ROS),apoptosis rate and the accumulation of 8-OHd G and MDA in L8824 cells were significantly increased after CMN exposure(p<0.05 or p<0.01).CMN significantly inhibited the nuclear accumulation of Nrf2 and the transcriptional levels of downstream antioxidant genes HO-1,NQO1,SOD1,SOD2 and CAT(p<0.01),accompanied by the overall inhibition of antioxidant system.CMN significantly promoted NF-κB and downstream proinflammatory gene IL-1β,IL-8,IL-12p40,IFN-γ2 and TNF-α m RNA levels(p<0.01).Compared with CMN group,the co-treatment of proteasome specific inhibitor MG132 increased the nuclear translocation of Nrf2,activated the transcription of downstream antioxidant genes and the levels of antioxidants such as SOD,CAT,GR and GST(p<0.05 or p<0.01);while reduced the nuclear translocation of NF-κB and alleviated the secretion of inflammatory factors.Therefore,the crosstalk between Nrf2 and NF-κB may reveal new mechanism information of exogenous hepatotoxicity.The second part discussed the role of cytochrome P450 metabolic pathway in SMZ induced L8824 cytotoxicity.The results showed that the concentration and time course analysis suggested that when treated with SMZ 10μM for 2h or 30μM for 48 h,CYP1A increased and decreased to the greatest extent(p<0.01),resspectively.So,we used si RNA knockdown and pc DNA3.1 overexpression to restore the level of CYP1 A,alleviated the production of ROS induced by SMZ,the imbalance of antioxidant system and the increase of apoptosis rate(p<0.01).At the same time,Nrf2/endoplasmic reticulum stress pathway was involved in SMZ induced hepatocyte metabolic defense response.This study showed that CYP1 A can be used as a potential target molecule to alleviate SMZ induced hepatotoxicity.The third part discussed the role of lipid metabolism signaling pathway(PPAR)in L8824 cytotoxicity induced by combined exposure of CMN and SMZ.The results showed that CMN and SMZ exposure significantly induced PPAR-α expression and ROS production(p<0.05 or p<0.01),and inhibited the m RNA expression levels of ACSL1 and CPT-1(p<0.01).Compared with CMN and SMZ groups,the above changes were more significant in MIX group(p<0.05 or p<0.01).Therefore,the combined exposure of CMN and SMZ can significantly activate lipid metabolism,mainly involving PPAR signal pathway,fatty acid synthesis,adipofactor signal pathway,etc.,resulting in the disorder of metabolic system,thus affecting the effective intake and utilization of fish nutrition.In conclusion,this study clarified that CMN and SMZ exposure can lead to neurotoxicity,immunotoxicity and liver metabolic toxicity of grass carp,and reduce the ability of grass carp immune system to recognize and eliminate pathogens.We also explored the similarities and differences of L8824 toxicity under CMN and SMZ exposed alone or in combination.This study provides basic data for the safety evaluation and risk assessment of pesticides and antibiotics.Relevant indicators can be used as biomarkers to effectively predict the potential threat of environmental pollutants to organisms and ecosystems.