Toxic Effects And Mechanism Of Chlorinated Paraffins On Zebrafish

Chlorinated paraffins(CPs)are widely used in plasticizers,flame retardants and metal processing fluids because their excellent properties of chemical stability,flame retardant and low volatility.On the one hand,the application of CPs in food processing equipment and packaging may lead to the migration of CPs into the food matrix.On the other hand,CPs can persist in the environment for a long time and accumulate in organisms through the food chain.The content of CPs in food can reach 500 μg/kg,and dietary intake is the main route of human exposure to CPs.As an important digestive organ and the largest immune organ,gut can enriched with most pollutants.More and more studies have shown that the disorder of gut microbiota can induce host diseases,including neurodegenerative diseases,intestinal diseases,immune-related diseases,etc.However,the role of gut microbiota in CPs-induced host toxic effects has not been clarified.In addition,zebrafish(Danio rerio)has been broadly applied in the field of toxicity research on account of its small size,good optical clarity of the embryo,rapid development,and high genetic and tissue homology with humans.Therefore,this study took zebrafish as the research object to explore the embryonal developmental toxicity,neurotoxicity,immunotoxicity and intestinal toxicity induced by CPs,and analyzed the mechanism of CPs-induced host toxicity by non-targeted metabolomics,16SrRNA sequencing and targeted metabolomics of short-chain fatty acids(SCFAs).Specific research contents are as follows:(1)Short-chain chlorinated paraffins(SCCPs),Medium-chain chlorinated paraffins(MCCPs),and CPs were used to expose zebrafish embryos to research the developmental toxicity induced by them.After fertilization for 6 hpf,embryos were exposed to 0 mg/L,0.01 mg/L,0.1 mg/L and 1 mg/L SCCP,MCCP,and CPs solutions for 120 hpf.After exposure to the three drugs,the 48 hpf rupture rate of oolemma increased,the hatching rate decreased,the heart rate and locomotor behavior of 120 hpf larvae were disturbed.The three drugs exhibited certain oxidative damage,and had weak teratogenic and lethal effects,among which SCCP had the strongest toxic effect.(2)Zebrafish were exposed to different concentrations of CPs to analyze its neurotoxic effects and mechanisms.Zebrafish were exposed to 0 mg/L,1 mg/L and 10 mg/L CPs solutions for 14 days,and it was found that CPs resulted in increased mortality and decreased body weight,body length and brain index of zebrafish.At 10 mg/L CPs exposure,zebrafish showed obvious bottom-dwelling behavior with slow movement,reduced motility,and decreased food intake.Untargeted metabolomics techniques based on HPLC-MS/MS were used to further reveal the mechanism of its neurotoxicity.The results demonstrated that CPs affected the metabolic pathways of arginine and proline,lipids,and D-amino acids.Specifically,differential metabolites phosphatidylcholine affects the generation of Acetyl choline(ACH),Sphingomyelin(SM)affects the production of sphingomyelin,ACH and sphingomyelin are related to the conduction of neuronal excitability.D-proline and N-acetyl-aspartyl-glutamic acid are associated with the production of the inhibitory neurotransmitter glutamic acid.Additionally,CPs causes increased levels of dopamine,glycine,and glutamate,and decreased levels of ACH,leading to neuroexcitatory toxicity and motor behavior changes.(3)The oxidative damage and immunotoxicity of CPs exposure in zebrafish were studied.After 14 days of zebrafish exposure to CPs at 0 mg/L,1 mg/L and 10 mg/L,as a key first-line immune tissue,histological section of liver suggested significant CPs accumulation and nucleus loss.The detection results of malondialdehyde(MDA),superoxide dismutase(SOD)and other substances showed that oxidative damage occurred in liver tissue.Real-time quantitative PCR analysis of immune-related genes implied that CPs caused immune system disorders and liver damage.(4)16SrRNA sequencing and SCFAs targeted metabolomics were used to analyze the imbalance in gut microbiota and SCFAs after CPs exposure,and the mechanism by which CPs induced toxic effects through intestinal microecosystems was explored.After CPs exposure,the intestinal tissue of zebrafish was collected,the intestinal index were significantly reduced,and the histopathological results exhibited that CPs caused damage to intestinal tube wall and intestinal villi.Compared with the control group,the 16SrRNA sequencing analysis was found that the abundance of Proteobacteria and Firmicutes were increased and decreased at the phylum level.At the genus level,the abundance of Aeromonas,Cetobacterium and Shewanella decreased,the abundance of pathogenic bacteria increased,which resulted in abnormal brain function,intestinal barrier function and immune function.In addition,the reduced abundance of Enterobacteriaceae in gut after CPs exposure would reduce the production of SCFAs.Furthermore,targeted metabolomics analysis of SCFAs using GC-MSrepresented that the relative contents of acetic acid,butyric acid,isovaleric acid,valeric acid,isobutyric acid and propionic acid in zebrafish gut were decreased after CPs treatment.This affects macromolecular digestion and absorption,fatty acid metabolism,and glycolysis pathways,which mediate neurodegenerative diseases and immune system disorders.Besides,the level of Nacetyl-aspartyl-glutamic acid in brain tissue and the food intake can regulate intestinal barrier function by affecting the production of glutamine,the occurrence of inflammatory reaction in liver tissue may lead to a series of complications,and the decrease of LZM activity is associated with the increase content of intestinal mucopolysaccharide.