Study On The Toxic Effects Of Citrate Plasticizers In Food Packaging Materials On Cells

In the production of food packaging materials,a variety of plasticizers are usually used in combination to meet the requirements of material flexibility and ductility.The safety of plasticizers in packaging materials,which are prone to food contamination through migration and leaching,has caused widespread concern worldwide.Due to toxic effects such as endocrine interference,the most commonly used phthalate plasticizers have been gradually banned in food packaging materials in many countries around the world.Citrate ester plasticizers,as a new type of environmentally friendly plasticizers,have become the main alternatives of traditional plasticizers such as phthalates.In recent years,the use of citrate ester plasticizers have shown a rapid growth.However,citrate ester plasticizers also suffer from migration during use and their safety issue is still poorly studied.Therefore,studying the single and binary toxic effects of citrate plasticizers at human daily exposure levels is of great significance for assessing their safety,formulating limit standards,and ensuring food safety.In this paper,based on the cell alternative toxicology technology,the cell viability of Human Hepatoma cells(Hep G2)which were induced by single and binary exposure of 10-1000 mg/kg of Tributyl Citrate(TBC),Tributyl O-acetylcitrate(ATBC)and Triethyl O-acetylcitrate(ATEC)for 48 h were analyzed.The results of the CCK-8(Cell Counting Kit-8)cell activity assay showed that TBC at 10-200 mg/kg showed toxic effects on Hep G2 cells;ATBC at 50-1000 mg/kg showed toxic effects;and ATEC at 200-1000 mg/kg showed toxic effects.TBC,ATBC and ATEC showed an overall dose-dependent toxic effect,which increased with increasing exposure concentrations.Sorting by toxic effect:TBC>ATEC>ATBC.The binary mixture of TBC and ATEC was further investigated for cytotoxicity based on the iso-effect concentration,and the combined action mode of the binary exposure was analyzed using the Concentration Addition(CA),Independent Addition(IA),and Combination Index(CI)models.The results showed that the CI model was more accurate in predicting the effects of the binary mixture.The CI indices were calculated by CompuSyn software,and TBC and ATEC showed antagonistic effects at concentrations of 371.86-867.66 mg/kg with Log CI>0.An untargeted analytical method for small molecule metabolites was established based on the Ultra High Performance Liquid Chromatography Q-Exactive mass spectrometer(UHPLC-QE-MS),515 small molecule metabolites were screened after Hep G2 cells exposed to 125 mg/kg ATEC for 48 h.31 differential metabolites such as valyl-aspartate,2-propylpiperidine,phenylalanyl-methionine were obtained based on the P-value<0.05,VIP value>1,FoldChange>2 or<0.5,16 metabolic pathways related to differential metabolites were identified by the enrichment analysis and topological analysis,among which glycerophospholipid metabolism,arginine-proline metabolism,alanine-aspartate-glutamate metabolism correlation highest.Transcriptomics-based techniques were used to obtain differentially expressed genes in Hep G2 cells after ATEC exposure.After normalization,p-test,and multiple correction test,4733 differentially expressed genes were obtained according to the condition of padj?0.05 and log2FoldChange>0.0,among them 3065 up-regulated genes and 1668 down-regulated genes.GO enrichment analysis mainly involves genes such as anchoring junction,adherens junction,etc.KEGG enrichment analysis mainly involves endocytosis,PI3K-Akt signaling pathway,etc.Multi-omics association analysis was conducted based on the metabolomics and transcriptomics analysis,metabolic networks of ATEC treatment group in the positive and negative ion mode were constructed according to the condition of padj<0.001 and the absolute value of log2FoldChange>1.A multi-omics correlation analysis verified that ATEC exposure significantly affects glycerophospholipid metabolism,arginine-proline metabolism,and alanine-aspartate-glutamate metabolism,and identified the metabolites phosphatidylcholine and phosphatidyl ethanol amine,and the genes LIPG and GCLM as biomarkers of ATEC contamination.This paper investigated the individual and combined toxic effects of TBC,ATBC and ATEC,typical citrate plasticizers in food packaging materials,after exposure of Hep G2 cells,and obtained the combined action mode of binary mixed exposure.The mechanisms of toxic effects were investigated by metabolomics and transcriptomics analysis,and the differential metabolites and differential genes of ATEC exposure were obtained.Through multi-omics correlation analysis,it was clarified that the regulatory network of glycerophospholipid metabolism,urea cycle and arginine,proline,glutamate,aspartate and asparagine metabolism were the most important networks producing the toxic effects.This paper investigated the toxic effects of citrate plasticizers in Hep G2 cells and the toxicity mechanism of ATEC at the metabolic level and transcription level.Based on the results of this study,when using TBC,ATBC,ATEC as plasticizers in food packaging materials,attention should be paid to the concentration of addition,with no increased safety risk when the two plasticizers are used in combination,and migration levels of less than 10,50 and 200 mg/kg respectively when used alone.The results of this study are of great importance for assessing the migration risk of the three citrate plasticizers and for ensuring food safety.