Preliminary Study On Biological Effects And Mechanisms About Joint Action Of Several Kinds Of Food Additives

Foods contain various additions that affect our daily lives.The food industry could not develop if separated from food additives.As a kind of natural or synthetic chemicals,the safety of food additives has been widely concerned by human beings.The national standards for food additives GB2760-2014 approved the use of food additives have been through systematic toxicological safety evaluation and risk analysis,according to the provisions to add,we can ensure food safety.At present,the food additive safety evaluation standard of state is based on the toxic effect of additive alone.But in real life,foods often contain two or more kinds of food additives,and human daily intake of a variety of foods.So food additives used in combination may produce additive,synergistic or antagonistic joint action.A single additive is safe in the permitted range,but some additives used at the same time,joint action may have different forms,it puts forward a new challenge to the traditional food toxicology theory and the risk assessment of food additives,urging researchers to develop alternative approaches to in vivo studies for predicting joint action of food additives.In this study,we developed a multiparametric protocol performed on cultured HepG2 cells for assessing the potential joint action of food additives.The preservative sodium sulfite,sodium metabisulfite and pigment sunset yellow,lemon yellow,erythrosine,carmine,allura red were used as subjects beccause of usually existing at the same time in the food for the separate action research of cell viability assessment by CCK-8 assay.Selecting the sunset yellow and sodium sulfite which have greater inhibition ability for subsequent joint test.2x2 analysis of variance of factorial design asseses the cell proliferation by CCK-8 assay.Multi-parameter analyses of cytotoxicity(cell number,membrane permeability,ROS release and DNA damage)were detected by High Content Analysis(HCA).To further corroborate the results presented,using preservative Potassium sorbate and antioxidant D-sodium erythorbate as parallel test compounds to repeat and verify test at the same time.Then the effects of sunset yellow and sodium sulfite,potassium sorbate and D-sodium erythorbate alone or combined with HepG2 cells on cell apoptosis(Caspase-3/7,intracellular calcium concentration and mitochondrial membrance potential)and cell cycle were detected by HCA for the research of the apoptosis mechanism preliminarily.At last,the expression of Caspase-3,Bax,Bcl-2,Caspase-12 and p53 were detected by qPCR and Western blotting after sunset yellow and sodium sulfite?potassium sorbate and D-sodium erythorbate alone or combined treatment.The current study was to investigate the joint toxicity and damage mechanism of food additives upon HepG2 cells,main results of our research VI states as follows:All test compounds dose-dependently decreased the growth of HepG2 cells,order of the toxicity of these additives is as follows:sodium sulfite>sodium metabisulfite,sunset yellow>carmine>Lemon yellow>allura red>amaranth.sunset yellow and sodium sulfite with an IC50 of 1.06�0.07,and 0.30�0.02 g/L at 24h,respectively.Potassium sorbate and D-sodium erythorbate dose-and time-dependently decreased the growth of HepG2 cells with an IC50 of 1.35�0.11 g/L and 1.58�0.17 g/L at 24h,respectively.The HCA results showed that both sunset yellow and sodium sulfite could significantly decrease the cell number,accompanied by the increase of membrane permeability,the rate of dead/live and ROS release,their joint approach showed a synergistic effect in these targets.When exposure Potassium sorbate or D-sodium erythorbate combined,their joint approach also showed a synergistic effect.All food additives in the research could induce HepG2 cell cycle arrest.Different concentrations of the individual additive induce different cycle arrest,concentrated in the G0/G1 phase and S phase,and their mixture induced G0/G1 phase arrest.The increase of Caspase-3/7,intracellular calcium level and mitochondrial membrance potential were dose-related,all the mix groups showed a synergistic effect when compared to the separate group.But only the high-dose group had an effect in DNA damage.The results of qPCR and Western blot showed that sunset yellow and sodium sulfite,Potassium sorbate and D-sodium erythorbate alone or combined could inhibit HepG2 cells proliferation by significantly increasing the expression of mRNA and protein p53,activated Caspase-3,Bax,and decreasing the expression of Bcl-2;But Caspase-12 had no obvious change.In this work,The results showed that food additives could induce multi parameter cytotoxicity and cell apptosis,change cell cycle,and high dose groups induced genotoxicity.In conclusion,sunset yellow and sodium sulfite,potassium sorbate and D-sodium erythorbate combined action in HepG2 cells showed a synergistic effect;and mainly through the mitochondrial apoptosis pathway induced cell apoptosis.