Study On The Toxic Mechanism Of Arsenic To Cyprinidae And The Alleviating Effect Of Zinc

Arsenic（As）is a carcinogenic metal,which can cause irreversible damage to many systems of the body.Environmental toxins have a profound impact on wildlife and fish.In the aquatic environment,arsenic pollution has a significant impact on related industries by affecting the reproductive health and development of aquatic organisms.In this study,a variety of cyprinid fish cell lines were selected to explore the toxicological mechanism of arsenic poisoning.Zinc（Zn）is an essential trace element in the body and plays a key role in cell homeostasis and immune response.It was found that zinc antagonizes organ toxicity induced by heavy metals in mammals and amphibians.However,the mechanism of interaction between arsenic and zinc in cyprinid fish is still unclear.We studied the mechanism of arsenic poisoning and the alleviating effect of zinc in cyprinid fish from the following aspects:1.IRE1 signaling mediates protective autophagy against As-induced apoptosis in L8824KEGG enrichment analysis based on transcriptome sequencing showed that arsenic significantly affected endoplasmic reticulum（ER）stress and autophagy pathways in grass carp liver cell line L8824（p<0.01）.The expression levels of three ER resident transmembrane proteins,p-PERK,p-IRE1 and ATF6,were significantly increased after 24h of arsenic treatment（p<0.01）.In addition,the apoptotic proteins（CHOP,GADD34 and PARP）downstream of ER stress and fluorescence staining results showed that the apoptosis levels were significantly increased after arsenic treatment for 24h（p<0.01）.The ER stress specific inhibitor 4-PBA significantly alleviated the above ER stress and apoptosis indexes,and improved the cell survival rate（p<0.01）.The results showed that autophagy in this model was a protective response of cells against arsenic toxicity.In addition,JNK inhibitor down-regulated autophagy and c-Jun interactions with Beclin1 promoters.In addition,si-IRE1inhibited the expression of IRE1,p-ASK1 and p-JNK in arsenic-treated L8824 cells,accompanied by significant inhibition of autophagy level（p<0.01）.Together,these results suggest that arsenic can induce ER stress-dependent apoptosis in L8824 cells,and IRE1induces protective autophagy by activating JNK signaling.2.Targeting ferroptosis reduce arsenic induced zebrafish cytotoxicityKEGG enrichment analysis based on transcriptomic sequencing showed that arsenic-induced differential genes were significantly enriched in apoptosis,cell cycle arrest and autophagy in zebrafish fibroblast cell line ZF4 cells（p<0.01）.Validation experiments showed that As³⁺could induce and enhance apoptosis,cell cycle arrest and autophagy death of ZF4cells in a time-dependent manner（24h and 48h）,but only induced ferroptosis at 48h.Therefore,we further analyzed whether Alox12（12-lipoxygenase）and NCOA4（autophagic degradation receptor of ferritin heavy chain 1（FTH1））were the check points of ferroptosis during 24h to48h under As³⁺exposure.When ZF4 cells were treated with As³⁺for 24h,pc DNA3.1-NCOA4significantly reduced the enrichment of FTH1 and led to the leakage of Fe²⁺（p<0.01）.Moreover,pc DNA3.1-Al OX12/NCOA4 further increased cell mortality and ROS production,while the increase of MDA,PTGS2a,CHAC1 and overconsumption of GPx4 indicated the initiation of ferroptosis.When ZF4 was exposed to As³⁺for 48h,ferroptosis was significantly activated,while si-Alox12,si-NCOA4 or Fer-1 combined with As³⁺inhibited ferroptosis and improved cell survival rate（p<0.05 or p<0.01）.This part of the study revealed the role of Alox12 and NCOA4 in arsenic induced ferroptosis,providing evidence for the regulation and targeted treatment of arsenism.3.Arsenic induced immunotoxicity of carp neutrophilsDifferent concentrations of As³⁺（10,20 and 40μM）inhibited the activity of neutrophils in a dose-dependent manner,with the increase of ROS and MDA levels and the significant decrease of SOD,CAT and GSH.Meanwhile,neutrophil migration（CXCa and CXCR1）and phagocytosis（TLR4 and CR3）were inhibited by As³⁺,especially in 40μM group（p<0.01）,with significantly increased IL-1βand TNF-α（p<0.01）.In addition,double staining with SYTOX Green and Hoechst,H3 and MPO levels showed that neutrophil extracellular net（NETs）release increased in an arsenic-dependent manner.These NETs production increases were significantly inhibited by the JAK inhibitor Ruxolitinib.These results suggest that As-induced neutrophil NETs formation in carp is a JAK/STAT-dependent cellular process.4.Protective effect of zinc on chronic arsenism of common carpIn this study,the toxicity of As³⁺（2.83mg/L）and the alleviating effect of Zn²⁺（1mg/L）on common carps were evaluated.The results showed that:arsenic could significantly accumulate in the brain,liver and intestine of carp,and lead to significant pathological changes,oxidative damage and apoptosis.At the same time,it was accompanied by regional inflammation of liver-intestinal axis,increased intestinal permeability and liver lipid metabolism disorder.The toxic reaction of arsenic to common carp can be partially or significantly alleviated by zinc administration.In conclusion,arsenic,as an inducer of ROS,can cause oxidative stress and cascaded damage phenotypes such as endoplasmic reticulum stress,apoptosis,ferroptosis and inflammation in different Cyprinidae fish cell models.Zinc is a component element of many important enzymes in the free radical elimination system of the body,and exerts significant anti-inflammatory,anti-apoptotic,immune regulation and lipid regulation functions in the arsenism model of common carp.This study provides scientific basis for further study and field application of zinc in the prevention and treatment of arsenic poisoning.