Study On The Joint Toxic Effect And Mechanism Of Arsenic,Dichloroacetamide And Iron On HepG2 Cells

Drinking water safety is a hot issue of global concern.Arsenic(As)pollution in the water can cause cancer and a variety of non-cancer diseases,posing a threat to human health.Disinfection byproducts(DBPs)and some conventional elements(such as Fe)are also present in drinking water,and it is not clear how they affect the accumulation and metabolism of As in the body and their mechanisms.It is urgent to study the combined toxicity effect of different types of pollutants in drinking water,such as As,DBPs and Fe,so as to provide more reliable toxicity data for the safe toxicity assessment of drinking water.In this paper,using human hepatoma cells(Hep G2)as test cells,the effects of typical nitrogen-containing DBPs,dichloroacetamide(DCAcAm),and Fe on As toxicity were systematically studied by high-throughput PCR and metabonomics techniques through binary and ternary exposure systems.The main research contents are as follows:(1)Effects of DCAcAm and Fe on bioavailability and metabolic transformation of As;(2)Cytotoxic effects and toxic mechanisms of combined exposure to DCAcAm and As;(3)Cytotoxic effects and toxic mechanisms of combined exposure to Fe,DCAcAm and As.The specific results and conclusions are as follows:(1)Combined exposure of As and DCAcAm significantly promoted the accumulation of As in cells,and the higher the exposure concentration of DCAcAm,the higher the intracellular concentration of As.The addition of Fe reduced the total intracellular concentration of As,and the higher the exposure concentration of DCAcAm,the more significant the effect of Fe.Combined exposure to As and DCAcAm promoted the methylation of As.The addition of Fe increased the methylation of low concentration(0.375 mg/L)of As but decreased the methylation of high concentration(0.75 mg/L)of As.Combined exposure of As and DCAcAm increased the content of intracellular methyl donor SAM,and the changing trend was consistent with the methylation change of As.The addition of Fe further increased the content of intracellular SAM.Under the influence of combined exposure of As and DCAcAm,the expression of intracellular DNA methylation regulatory genes was inhibited,and the addition of Fe further inhibited gene expression.(2)Combined exposure of As and DCAcAm significantly inhibited cell growth,and the higher the concentration of DCAcAm,the stronger the inhibitory effect,indicating that these two substances have synergistic toxic effects on cells.The highthroughput PCR study found that 46.3% of the 54 genes detected were differentially expressed due to the single and combined exposure of As,and the number of differentially expressed genes caused by the combined exposure of 0.375 mg/L As and DCAcAm was the same as that of the single 0.375 mg/L As exposure group,while the combined exposure of 0.75 mg/L As and DCAcAm led to more differentially expressed genes.As and DCAcAm combined exposure caused significant differences in gene function and pathway enrichment,including intracellular oxidative stress,inflammatory immune response,apoptosis,lipid metabolism,and DNA repair related genes;Among them,oxidative stress showed the most significant changes,with 6-9more DEGs involved in this pathway than those exposed to As alone,and the expression multiples of EGFR and AKT1 of typical genes increased by 28.1% and 18.6% after combined exposure of 0.375 mg/L As and DCAcAm,respectively.The metabonomic analysis showed that As and DCAcAm combined exposure significantly inhibited the amino acid metabolism of Hep G2 cells,decreased the levels of valine,leucine and isoleucine,and inhibited the process of aminoacyl-t RNA biosynthesis.The above results showed that compared with As exposure alone,low concentration of DCAcAm exposure significantly increased the effect of As on cell growth and metabolism,and a high concentration of DCAcAm exposure even significantly affected cell gene expression.(3)Combined exposure of As and Fe had a synergistic effect on Hep G2 cells,and its effect on the growth of Hep G2 cells was similar to that of the combined effect of As and DCAcAm.On the basis of As and DCAcAm exposure,the addition of Fe significantly reduced the cell survival rate.The results of gene expression analysis showed that the interference of gene expression caused by combined exposure of 0.375mg/L As and DCAcAm was less affected by Fe,and Fe increased the differential expression of genes exposed to 0.75 mg/L As and DCAcAm.Triad exposure led to more DEGs enrichment on oxidative stress,cell detoxification,and apoptosis pathways.The number of DEGs in the 0.375 mg /L As,DCAcAm and Fe combined exposure group participating in the GO:0006979,GO:0098754 and GO:0097190 pathways was 6-13 more than that of As exposure alone and As combined with Fe exposure.Similarly,the results of the 0.75 mg/L As,DCAcAm and Fe combined exposure group compared with the corresponding As exposure group and the As and Fe combined exposure group showed that 1-19 more DEGs were enriched in these pathways in the former group than in the latter group.The metabonomic analysis showed that the addition of Fe increased the number of differential metabolites and their metabolic pathways exposed to As and DCAcAm,including Beta-alanine metabolism,arginine and proline metabolism,glyoxylic acid and dicarboxylic acid metabolism,biosynthesis and degradation of valine,leucine and isoleucine,and metabolism of alanine,aspartic acid and glutamic acid.The above results showed that compared with As alone and As combined with the DCAcAm exposure group,Fe significantly increased their cell proliferation toxicity and enhanced the disturbance of cell gene expression and metabolism.To sum up,DCAcAm can promote the absorption and methylation of As,increase the toxic effect of As on Hep G2,and the addition of Fe further increases the combined toxicity of DCAcAm and As.The results provide a new idea for the assessment of the combined pollution of drinking water.While paying attention to the inorganic As pollution of drinking water,the effects of typical organic pollutants,DBPs and major element Fe in drinking water on the toxicity of As can not be ignored.