Research On The Mechanism Of Hepatic Ferroptosis Induced By Subacute Oral Exposure To Polypropylene Microplastics

Due to the massive production of plastics,coupled with poor biodegradability,easy migration,and imperfect recycling management measures,microplastics(microplastics,MPs)have caused widespread pollution to the environment.Currently,evidence of MPs has been found in oceans,rivers,drinking water and food.With the passing of the food chain,people can easily take up MPs from the environment through ingestion and respiration.Therefore,it is necessary to study the biological safety of MPs.However,the existing studies on the toxicity of MPs in mammals are mainly focused on commercial polystyrene particles,and there are few studies on other types of MPs,especially polypropylene(PP)particles which are widely distributed in water environment.In addition,the biosafety of PP particles less than 10 μm in the environment has not been reported.Thus,the health effects of polypropylene microplastics(PP-MPs)need to be further studied.In this study,male C57BL/6N mice were used as experimental animals,and PP particles(8 μm and 70 μm)were prepared by ball milling.Nile red fluorescence labeled PP particles were used as experimental materials to investigate the accumulation of PP-MPs particles in mice after subacute oral exposure.Non-fluorescent PP particles were used as experimental materials to investigate the effects of PP-MPs particles on body metabolism and the mechanism of toxicity to liver after subacute oral exposure.The exposure doses of both PP-MPs were 0.1,1.0 and 10 mg/m L and the exposure time was 28 days.The main findings are as follows:(1)Fluorescent PP-MPs was detected to have significant accumulation in stomach,intestine,liver and testis in a concentration-dependent manner.It was also observed that at the same exposure concentration,the smaller the particle size of PP-MPs,the greater the accumulation.These suggested the possibility of liver and testis as distal target organs of PP-MPs.(2)After PP-MPs exposure,the intestinal barrier of mice was damaged,suggesting that PP-MPs enter the blood through the damaged intestinal barrier and then reach other organs.After PP-MPs exposure,compared with the solvent control group,the levels of occludin(occludin)and Na-K-2Cl cotransporter 1(NKCC1)in the colon of mice were significantly decreased(p < 0.05)in a concentration-dependent manner.At the same time,with the increase of PP-MPs concentration,the levels of zonula occludens 1(ZO-1),claudin-1(claudin-1),solute carrier family 26 member 6(SLC26A6)and cystic fibrosis transmembrane conductance regulator(CFTR)were significantly lower than those in solvent control group(p < 0.05).In addition,the levels of ZO-1,claudin-1,occludin and NKCC1 were significantly lower in the 8 μm PP-MPs treatment group than those in the 70 μm PP-MPs treatment group(p < 0.05)at the exposure concentration of 10 mg/m L.(3)After PP-MPs exposure,the liver function of mice was damaged,suggesting that liver is one of the distal target organs of PP-MPs.Compared with the solvent control group,the expressions of total protein(TP),albumin(ALB)and alanine aminotransferase(ALT)were not significantly changed in all PP-MPs treated groups;However,serum aspartate aminotransferase(AST)and alkaline phosphatase(ALP)levels of mice were significantly increased(p < 0.05)after exposure to PP-MPs(8 and70 μm)at concentration of 10 mg/m L,while serum globulin(GLB)levels was significantly decreased(p < 0.05)after exposure to PP-MPs(8 μm)at the same concentration.(4)After PP-MPs exposure,lipid metabolism and amino acid metabolism were abnormal in mice.The results of serum untargeted metabolomics showed that,in POS mode,the significantly changed metabolites represented by 3-hydroxyanthranilic acid,L-Glutamine,Betaine,2,3-dinor-8-iso-prostaglandin-f2.Alpha,Leukotriene f4 and PC(16:0/16:0)were screened out;In NEG mode,the significantly changed metabolites represented by L-Pyroglutamic acid,Dihydroxyacetone phosphate,Glycine and Glutamic acid were screened out.KEGG pathway analysis revealed that subacute exposure to PP-MPs significantly altered the processes of arachidonic acid metabolism and glutathione metabolism in mice.(5)After PP-MPs exposure,pathological damage and ultrastructural changes,as well as oxidative stress,were observed in the liver of mice.Different degrees of pathological changes in the liver of mice exposed to PP-MPs were found,including congestion,inflammatory cell infiltration and necrosis.The results of electron microscope showed that there were different degrees of mitochondrial damage in the liver of mice,mainly manifested as disordered arrangement of ridges,dissolution and fracture,and partial swelling and vacuolation.In addition,the levels of reduced glutathione(GSH),superoxide dismutase(SOD)and catalase(CAT)in the liver tissue of mice were significantly decreased compared with the solvent control group,while the levels of oxidized glutathione(GSSG)and malondialdehyde(MDA)were significantly increased(p < 0.05),and the changes in the levels of GSSG,MDA,SOD and CAT were concentration-dependent;It was also observed that there were significant differences in the levels of GSSG,MDA,SOD and CAT between 8 μm and70 μm PP-MPs groups at 1 and 10 mg/m L exposure concentrations(p < 0.05).(6)After PP-MPs exposure,arachidonic acid(AA)metabolism in mice was significantly altered,suggesting lipid peroxidation.Compared with the solvent control group,the levels of AA and its metabolites leukotriene B4(LTB4),prostaglandin E2(PGE2)and thromboxane A2(TXA2)in the liver of mice were significantly increased(p < 0.05)in a concentration-dependent manner.In addition,the levels of AA,LTB4,TXA2 and PGE2 were significantly higher in the 8 μm PP-MP treatment group than those in the 70 μm PP-MP treatment group(p < 0.05)at the exposure concentration of 1and 10 mg/m L.(7)After PP-MPs exposure,ferroptosis occurred in the liver of mice in a concentration-dependent manner.In addition,8 μm PP-MPs made the liver more sensitive to ferroptosis.Compared with the solvent control group,the levels of tissue iron,transferrin receptor(Tf R),Acyl-Co A synthetase long-chain family member 4(ACSL4),Lysophosphatidylcholine acyltransferase 3(LPCAT3)and phospho-lipoxygenase 5(p-ALOX5)in liver increased significantly,while the levels of ferritin heavy chain 1(FTH1),ferritin light chain(FTL),solute carrier family 7member 11(SLC7A11)and glutathione peroxidase 4(GPX4)decreased significantly(p< 0.05).The differences were all concentration-dependent.In addition,at the exposure concentration of 1 mg/m L and 10 mg/m L,the level of ACSL4 was significantly higher in the 8 μm PP-MPs treatment group than that in the 70 μm PP-MPs treatment,while the levels of FTH1,SLC7A11 and GPX4 were significantly lower than those in the 70μm PP-MP treatment group(p < 0.05);At the exposure concentration of 10 mg/m L,the levels of tissue iron,Tf R,LPCAT3 and p-ALOX5 were significantly higher in the 8 μm PP-MPs treatment group than those in the 70 μm PP-MPs treatment group,while the level of FTL was significantly lower than that in the 70 μm PP-MPs treatment group(p< 0.05).In summary,after subacute oral exposure,PP-MPs can enter the blood circulation through the intestinal barrier and deposit in the liver.This causes liver function damage and metabolic abnormalities,especially glutathione metabolism and arachidonic acid metabolism;induces pathological damage and mitochondrial structure damage of liver;leads to oxidative stress and lipid peroxidation,and further induces ferroptosis of liver.