Study On The Neurocytotoxic Effects Of Bap And PFOS In Adhesives For Food Contact Packaging Materials

Food packaging materials are closely related to food safety,and food contact materials(FCMs)adhesives inevitably introduce some harmful additives during processing and production.Migration experiments have also shown that perfluorinated alkylated substances(PFAS)and polycyclic aromatic hydrocarbons(PAHs)had potential neurotoxic effects after exposure.China has not established maximum residue limits for these substances in packaging materials.Therefore,there is an urgent need to conduct research on the toxic effects of harmful additives in FCMs adhesives,especially the combined toxic effects under common exposure and the possible mechanisms of the toxicity,in order to provide a scientific basis for the development of limitation standards for harmful additives in FCMs adhesives.In this study,benzoapyrene(BaP)and perfluorooctane sulfonates(PFOS)were selected as the objects for individual and combined poisoning experiments in the concentration range of 0.5-5 mg/L in rat glioma cells C6.The analysis results of the CCK-8(Cell Counting Kit-8)after 48 h of exposure showed that the toxic effect of BaP on C6 cells was stronger than PFOS,with the median effect concentration(EC50)values of 1.88 mg/L and 3.36 mg/L,respectively.In the experimental concentration range,the inhibitory effects of individual and combined exposures on cell viability showed concentration dependence,i.e.,the toxic effects increased gradually with increasing concentration.Secondly,the type of combined toxic effect was explored after the combined exposure based on the mixture of equal-effect concentrations,and the fitting results showed that the CI model could better fit the combined toxic effect of BaP and PFOS on C6 cells than the concentration addition(CA)and independent action(IA)models.Further calculated by combined index(CI)model,the results showed that the combined exposure of BaP and PFOS expressed synergistic effects in the range of 0.33-1.31 mg/L.The combined mode of action changed from synergistic to antagonistic at 51.7%of the combined effect,and showed a trend that the synergistic effect gradually increased as the combined toxic effect decreased.The binary combined exposure of BaP and PFOS at actual low doses showed a synergistic effect different from that of single exposure.The intracellular reactive oxygen species(ROS)content,new DNA synthesis,and apoptosis were analyzed based on Harmony high connotation and FlowSight flow cytometry under BaP and PFOS exposure alone and in combination.Consistent with the results of cell viability assay,the BaP-PFOS combined exposure group caused a greater increase in intracellular ROS than the BaP and PFOS alone exposure group;the retardation of intracellular new DNA synthesis by combined exposure was significantly stronger than that by exposure alone;the combined exposure induced apoptosis mainly in early apoptosis,and its ability to induce apoptosis was stronger than the alone exposure.The above experimental results indicated that BaP-PFOS combined exposure showed stronger toxic effects in inducing oxidative stress,blocking DNA synthesis,and promoting apoptosis.Based on transcriptomic analysis changes in gene expression following the individual and combined exposure of C6 cells with concentrations of 1/4EC50,differentially expressed genes(DEGs)were screened at p values<0.05 and |log2FC|≥1.GO enrichment analysis revealed that in the BaP-PFOS combined exposure group,the highest enrichment was mainly in sterols,cholesterol,steroids and other biosynthetic processes,and the terms with the highest number of enriched differential genes were associated with membranes.KEGG enrichment revealed that the differential genes after combined and BaP alone exposure were significantly associated with three pathways:steroid biosynthesis,terpenoid backbone biosynthesis,and metabolism of xenobiotics by cytochrome P450.The differential genes after PFOS alone exposure were significantly associated with DNA replication pathway,indicating that BaP and PFOS exerted their toxic effects by different mechanisms of toxic action.The differences between BaP-PFOS mixed exposure and BaP or PFOS alone exposure were mainly reflected in the pathway enriched with the highest number of differential genes was the metabolic pathway,indicating that the combined toxic effects of BaP and PFOS showed synergistic effects closely related to the metabolic pathway,especially the steroid hormone biosynthesis pathway among them.Among which genes such as Cyp1a1 and Rnaseh2a can be used as potential biomarkers for BaP and PFOS exposure alone and in combination.Eventually,the accuracy of the differential genes was verified based on RT-qPCR technique.BaP and PFOS alone and combined exposure produced large amounts of ROS,on the one hand,BaP alone and BaP-PFOS combined poisoning increased cholesterol synthesis and uptake by significantly upregulating genes such as Acat2,Hmgcs1,Mvd,Nsdhl,Dhcr7,thereby altering lipids in cells;promoting the generation of stable DNA adducts through upregulation of genes such as Cyp1a1,which has a toxic effect on cells;and PFOS activated DNA replication by upregulating the Rnaseh2a gene.On the other hand,altered lipids in cells increase oxidative stress and exacerbate DNA damage,resulting in impaired synthesis of new DNA in C6 cells,insufficient action of repair mechanisms,and also triggering the process of apoptosis,leading to reduced cell viability.The increased cholesterol in the cells can promote steroid hormone biosynthesis and increase hormone metabolites,which affect the delivery process of hormones and their metabolites,causing the accumulation of potentially carcinogenic metabolites and exacerbating C6 neuronal cell damage,manifesting as proliferation-inhibiting neurocytotoxicity.In this study,the neurocytotoxic effects of BaP and PFOS in FCMs adhesives were studied based on C6 cells,and the concentration-effect relationship curves of individual and combined poisoning and the types of combined exposure were obtained;the key regulatory genes and pathways were identified;the mechanisms of toxic effects of BaP and PFOS on neuronal cells C6 were elucidated by combining molecular biology and transcriptomics techniques.The results of the above study will help to understand the mechanism of toxicity of BaP and PFOS on C6 in food packaging.The above findings will help to understand the risk of harmful additives in food packaging material adhesives and provide a scientific basis for the development of harmful additives limit standards.