A Biological Effects And Mechanisms Preliminary Study About Joint Action Of Aspartame And Sodium Benzoate

Food additives are the soul of the modern food industry. According to the standard of Chinese food additives of GB2760-2014, it is safe to human when they are used alone. However, different components may interact with each other in the multivariate mixture system, and then they show synergistic, antagonistic or additive effects, which will increase the toxicity of the additive to people after ingestion. So, it is a new challenge to assess the risk of using a multi-component of food additives.In this study, five food additives containing benzene ring structure, aspartame, sodium benzoate, sodium methylparaben, lemon, and sunset yellow, were selected as subjects. Firstly, toxicities of these five food additives were assessed by MMT method using BRL cells from mouse liver. Secondly, three kinds of most toxic additives, aspartame, sodium benzoate, and sodium methylparaben, were selected, and their combined effects showed that aspartame and sodium methylparaben were additive effects, and aspartame and sodium benzoate were synergistic joint actions. Thirdly, cell count, cell cycle, micronucleus rate, necrosis, and apoptosis rate of BRL cells were measured by high content screening method after treated with aspartame and sodium benzoate alone or combined. Fourthly, transcriptome of BRL cells was sequenced using RNA Seq, and a series of different expressed genes were screened out and their GO enrichment and KEGG metabolic pathway were also analysis. Lastly, biochemical indicators of alanine aminotransferase, aspartate aminotransferase, total bile acid content, malondialdehyde, total antioxidant capacity, superoxide dismutase and glutathione peroxidase were detected in mice after exposure to aspartame and sodium benzoate alone or combined for about 90 days. Their heart, liver, and kidney were fixed by paraffin, but no pahtological changed were observed after staining by HE.Main results of our research states as follows:1. There is a positive correlation between the cell inhibitory and exposure time and concentration to five kinds of food additives alone. Order of the toxicity of these additives is as follows:Sodium methylparaben> sodium benzoate> aspartame> lemon≈sunset yellow.2. When exposure to aspartame or sodium benzoate alone or combined, cell cycle was stopped at S or GO/G1 phase, and micronucleus rate was increased, and both cell necrosis and apoptosis rate were decreased.3. RNA_Seq results showed that 306,412 and 532 genes were screened out from cells after exposure to aspartame, sodium benzoate, and combined, respectively. Both GO enrichment and KEGG metabolic pathways analysis revealed different molecular mechanisms of cells responding to different exposure conditions.4. When exposure to aspartame or sodium benzoate alone or combined, mouse weight and its total antioxidant capacity were reduced, while alanine aminotransferase, aspartate aminotransferase, and malondialdehyde were increased. In summary, we propose that aspartame and sodium benzoate have a synergistic joint action when used together.