Mechanism Of Florfenicol-induced Nonalcoholic Fatty Liver Disease In Zebrafish Via Mitochondrial Pathway

As the third-generation phenicols broad-spectrum antibiotic,florfenicol(FF)is extensively used in veterinary clinics and aquaculture.However,recently,following the development of detection technologies and the improvement of people’s health awareness,the residues of Pharmaceuticals and Personal Care Products(PPCPs),especially for antibiotics,are widely concerned by researchers.As one of the top five antibiotics used in China,about 70-80% of FF residues enter the aquatic environment(including sea,lakes,rivers,tap water etc.),due to incomplete metabolism of FF in organisms and in feed residues.Consequently,FF is considered “pseudo-persistent”.Studies have shown that FF mainly accumulates in the liver when it enters the organism and causes hepatic steatosis and liver injury by destroying the oxidative-redox metabolic pathway.The specific mechanisms of liver damage caused by long-term and low concentrations of FF on non-targeted organisms and the potential developmental toxic effects on their unexposed offspring are still unclear.Therefore,in this study,zebrafish were used as a model organism.And multi-omics analysis(including metabolomics,lipidomics,and transcriptomics)combined with biological analysis techniques such as mitochondrial structure and function evaluation were performed to evaluate the changes in small molecules and the degree of mitochondrial damage in zebrafish liver after long-term exposure to FF.The main research contents and results are as follows:First of all,adult zebrafish were exposed to FF at environment-related concentrations of 0.05 mg/L and 0.5 mg/L for 28 consecutive days.And the FF residues in zebrafish liver were detected.The results indicated that FF accumulated in the liver was much higher than original concentration.And the bioconcentration factor(BCF)of 0.05 mg/L FF-treated group was 2.59 times of 0.5 mg/L FF-treated group.The histopathological analysis results showed that 0.5 mg/L FF led to a significant accumulation of lipid droplets in the liver,which can be defined as a mild level of non-alcoholic fatty liver disease(NAFLD).Then,non-targeted omics screening tools(lipidomics,metabolomics,and high-throughput RNA sequencing)combined with quantitative omics analysis(lipids,metabolites,and mitochondrial-related gene expression)were operated to analyze the small molecule changes in FF-induced NAFLD.The results showed that FF induced a global disordered metabolism in zebrafish liver.Specifically,the content of free fatty acids increased significantly.Besides,the total concentration of triglyceride,phospholipids,and lysophospholipids was markedly upregulated.Toxic lipid intermediates including diglyceride and ceramide accumulated in FF-treated livers.Consistent with this result,transport protease-encoding genes were significantly altered at the transcriptional level.Moreover,the mitochondria-centered tricarboxylic acid cycle,amino acid,nucleotide,and glucose metabolism pathways were also dysregulated.Furthermore,compared with the control group,the number of damaged mitochondria in the liver of FF-treated group was increased.The main types of damage included mitochondrial membrane structure destruction and cristae density reduction.The GO-term result showed that the inner and outer membrane structure of mitochondria,mitochondrial matrix,and respiratory chain(RC)complex were damaged.Also,destroyed mitochondrial RC function and reduced complex enzyme activity were found accompanied by a significant decrease in ATP production.The negative effect of excessive generation of reactive oxygen species caused by the disruption of RC function was evaluated.The results showed significantly elevated cardiolipin content in mitochondrial membranes and a disturbance of oxidative-redox metabolic pathways.Finally,the potential developmental toxicity effects and mechanisms on offspring embryos and larvae after exposing F0 female zebrafish to FF for 28 days were investigated.The results showed that FF treatment increased the mortality and decreased hatchability of F1 embryos(2-5 h post-fertilization,hpf).A variety of developmental malformations were observed in F1 larvae(120 hpf),including delayed yolk sac absorption,lack of swim bladder,spinal curvature,and shorter body length.Multi-omics integration analysis showed that the tricarboxylic acid cycle,amino acid metabolism,and lipid metabolism were disordered.At the same time,ATP level was decreased and oxidative redox homeostasis was disrupted in zebrafish larvae.These results were consistent with the impaired mitochondrial membrane potential and RC function,indicating that FF-induced developmental toxicity and metabolic dysregulation in F1 zebrafish were associated with mitochondrial damage.