| Research Backgrounds:Perchlorate could be released into the environment by anthropogenic sources,such as military and industry,or through atmospheric chemical reactions,by which perchlorate would be enriched into the food chain through natural migration,and then exposed to the human,affecting human health.Perchlorate has a similar structure to iodide and can competitively bind to sodium/iodide isotropic transporters(NIS)with iodide,resulting in disturbance of thyrotropin and thyroid hormone levels and affecting thyroid function.Several studies have shown that perchlorate has thyroid toxicity.And the effects of perchlorate on thyroid hormone homeostasis,thyroid hormone-related action targets and health risk assessment were studied based on the hypothalamic-pituitary-thyroid(HPT)axis.Researches on the adverse health effects of perchlorate on other non-thyroid outcome pathways are limited.The true levels of perchlorate exposure in humans,which contributes to the uncertainty in food safety risk assessment of perchlorate were not taken into account.In this study,HepG2 cell of human liver was selected as the research object,based on the dietary exposure level of perchlorate in the population,the toxic effects of perchlorate were studied on the basis of energy-related metabolic pathways and lipid metabolic pathways.Research Method:(1)According to the results of the sixth dietary survey,referring to the 99 th percentile value of perchlorate dietary exposure level(1.48 μg/kg),1.5 μg/kg sodium perchlorate solution which was at the same level was adopted as the low-dose group in this study,and on the basis of a certain safety factor,six sodium perchlorate standard solutions with different dose concentrations(0 μg/kg,1.5 μg/kg,15 μg/kg,150 μg/kg,1500 μg/kg,15000 μg/kg)were selected for 24-hour toxic effects on HepG2.The changes of cell activity were detected by MTT assay.100 times and10000 times of dietary exposure level were established as medium dose(150 μg/kg)and high dose(15000 μg/kg)perchlorate exposure groups,respectively.Oil red O staining experiments were performed on HepG2 cells exposed to 1.5 μg/kg,150μg/kg and 15000 μg/kg sodium perchlorate standard solution for 24 h respectively.The basic biochemical indexes were determinated.The level of intracellular glucose was analyzed and detected.(2)Based on the untargeted metabolomics method,the changes of energy metabolites in HepG2 cells exposed to 1.5 μg/kg,150 μg/kg and 15000 μg/kg sodium perchlorate standard solution for 24 h were analyzed and detected.Multivariate statistical analysis was used to select potential differential metabolites in each sodium perchlorate exposure group.The differential metabolites were identified.And pathway enrichment analysis was performed.(3)Untargeted and targeted detection methods have been established for differential metabolite expression related to lipid metabolism,lipid metabolite changes of HepG2 cells exposed to 1.5 μg/kg,150 μg/kg and 15000 μg/kg sodium perchlorate standard solution for 24 h were detected and analyzed.And pathway enrichment analysis was performed.(4)Based on the results of metabonomics and lipidomics analysis,biomarkers related to energy metabolism and lipid metabolism disorders were screened out for HepG2 cells exposed to 1.5 μg/kg,150 μg/kg and 15000 μg/kg sodium perchlorate standard solution for 24 h.Result:(1)The toxicity of HepG2 cells at perchlorate doses of 1.5 μg/kg,150 μg/kg and15000 μg/kg was tested by MTT assay.The results showed that the cell activity increased under dietary exposure level of perchlorate,and significantly decreased at medium and high perchlorate concentrations,showing a dose-dependent effect.Oil red O staining showed that perchlorate had a significant influence on lipid droplet formation in HepG2 cells.The results of intracellular basic biochemical indexes indicated that the levels of total cholesterol(TC)and triglyceride(TG)were increased compared with the control group,and the change of TG was significant(p< 0.05).Compared with the control group,the content of glutathione(GSH)was reduced,and malondialdehyde(MDA)was significantly increased in the perchlorate treatment group.The results showed that the antioxidant capacity of cells was significantly decreased due to the oxidative damage of lipid.Intracellular glucose(GLU)levels decreased significantly after perchlorate treatment.The results showed that perchlorate could cause the disturbance of lipid metabolism and energy metabolism in HepG2 cells.(2)MS-DIAL 4.70 and SIMCA P14.1 were used for data visualization and multivariate statistical analysis of untargeted metabolomics mass spectrum information.At the study dose,26 different metabolites were preliminarily screened out.The pathway enrichment analysis of 26 potential differential metabolites was conducted by Metaboanalyst online platform.The results showed that perchlorate affected the metabolism of HepG2 cells through alanine,aspartic acid and glutamic acid metabolism,arginine and proline metabolism,arginine biosynthesis,tricarboxylic acid cycle(TCA cycle)and D-glutamine and D-glutamic acid metabolism.And six biomarkers have been confirmed,namely aspartic acid,arginine,citric acid,malic acid,glutamic acid and citrulline.(3)Data visualization and multivariate statistical analysis were performed based on the untargeted lipid monitoring consequences.The results showed that 73 different lipid metabolites were initially screened out and identified under the conditions of VIP value > 1,P value < 0.05,FC value > 1.2 or < 0.8,among which there were 14 lipid metabolites with remarkable changes occurred in all exposure groups as follow:Cer(d18:1/24:1),Cer(d18:1/25:0),PG(16:0/18:1),PC(16:0/16:0),PI(16:1/16:1),PI(18:2/18:2),PC(18:1/22:6),Stearoyl-EA,Lyso PC(20:1/0:0),Lyso PC(22:1/0:0),PE(14:0/18:1),PE(16:1/18:1),PE(16:1/22:6)and PC(16:0/22:5).The results indicated that perchlorate could affect lipid metabolism in HepG2 cells through glycerophospholipid and sphingolipid metabolic pathways.And 42 biomarkers were identified,including phospholipids,lysophospholipids,sphingolipids and ceramides.Quantitative analysis of targeting omics was performed on these 10 biomarkers:Cer(d18:1/24:1),Cer(d18:1/25:0),Lyso PC(20:1/0:0),Lyso PC(22:1/0:0),PE(14:0/18:1),PE(16:1/18:1),PE(16:1/22:6),PC(16:0/22:5),PC(18:1/22:6)and PC(16:0/16:0).The results showed that the trend was consistent with that of untargeted monitoring,indicating that the conclusion of untargeted lipidomics was credible.Conclusion:In this study,perchlorate dietary exposure dose was used as low dose group,and100 times and 10000 times of dietary exposure level were used as medium and high dose groups,respectively.Perchlorate could mainly affect energy metabolism in HepG2 cells through the tricarboxylic acid cycle and other metabolic pathways.Six biomarkers were screened out and identified,which are aspartic acid,arginine,citric acid,malic acid,glutamic acid and citrulline.The effect of perchlorate on lipid metabolism of HepG2 cells was mainly occurred through glycerophospholipid metabolic pathway and sphingolipid metabolic pathway.A total of 42 biomarkers were screened out and identified,which were phospholipids,lysophospholipids,sphingolipids and ceramides. |
