| Drinking water disinfection by-products(DBPs)are a kind of chemicals that are formed by the reaction of disinfectants with natural organic matter and halides in the process of drinking water disinfection and harmful to human health.Many studies have shown that DBPs have cytotoxicity,genotoxicity,potential carcinogenicity,and reproductive and developmental toxicity.Iodoacetic acid(IAA)is an unregulated DBP.The concentration of IAA in drinking water is lower than that of regulated DBPs,but its toxicity is higher than those of regulated chloro-and bromo-acetic acid.Although our previous studies have shown that IAA may have thyroid disrupting effects,the mechanism is still unclear.Therefore,this study aims to use transcriptome sequencing technology to screen the differentially expressed genes related to thyroid function after IAA treatment of human normal thyroid Nthy-ori 3-1 cells,then verify the differentially expressed genes and their proteins related to thyroid hormone synthesis,and explore the effects of IAA on the iodine uptake function and oxidative stress of cells.The results of this study will provide references for elucidating the mechanism of thyroid interference effect of IAA.Part Ⅰ The effects of iodoacetic acid on the gene expression profile of human normal thyroid Nthy-ori 3-1 cellsObjective: To determine the effect of IAA on the gene expression profile of Nthy-ori 3-1 cells by using transcriptome sequencing technology,and provide a reference for exploring the interference mechanism of IAA on TH synthesis.Methods: Human normal thyroid Nthy-ori 3-1 cells were treated with IAA.The negative control group was ultrapure water.According to the cytotoxicity experiment,the experimental group was determined to be 2 μM IAA.There are3 parallels in each group.After 24 hours,the cells were sequenced with transcriptome based on the Illumina sequencing platform to obtain gene expression profiles and screen differentially expressed genes.Then these genes were analyzed by GO function enrichment and KEGG pathway enrichment.Results: Transcriptome sequencing results showed thata total of 1218 differentially expressed genes were screened in the IAA group compared with the negative control group,including 695 up-regulated genes and 523down-regulated genes.The GO function enrichment analysis of differentially expressed genes revealed that the biological process(BP),cellular component(CC)and molecular function(MF)were significantly enriched in 313,60,and23 terms,respectively.The most significant term of BP was the regulation of DNA biosynthetic process.CC was cell-substrate junction,and MF was cell adhesion molecule binding.KEGG enrichment analysis of differentially expressed genes obtained a total of 292 pathways,and 7 pathways were significantly enriched pathways.IAA could affect the gene expression in pathway related to TH synthesis.The three up-regulated genes were heat shock protein 90 beta family member 1(HSP90B1,also known as GRP94),phospholipase C beta 4(PLCB4)and G protein subunit alpha q(GNAQ),respectively.And the four down-regulated genes were glutathione peroxidase 1(GPx1),adenylate cyclase 6(AC6),phospholipase C beta 3(PLCB3)and inositol 1,4,5-trisphosphate receptor type 3(ITPR3),respectively.Among them,the expression of HSP90B1 gene was significantly up-regulated,and the expression of GPx1 and AC6 genes were significantly down-regulated.Conclusion: IAA can affect the gene expression of Nthy-ori 3-1 cells,including genes of related to thyroid hormone synthesis pathways.The m RNA expression of HSP90B1,PLCB4 and GNAQ genes are up-regulated,and the m RNA expression of GPx1,AC6,PLCB3 and ITPR3 genes are down-regulated.Part Ⅱ The effects of iodoacetic acid on thyroid hormone synthesis related genes,iodine uptake and oxidative stressObjective: To determine the expression of the related genes and their proteins,iodine uptake function and oxidative stress in TH synthesis on Nthy-ori 3-1 cells after IAA treatment,and explore the possible mechanism of TH synthesis.Methods: Human normal thyroid Nthy-ori 3-1 cells were treated with IAA.The negative control group was ultrapure water,and the experimental groups were 0.5,1 and 2 μM IAA,respectively.There are 3 parallels in each group.After 24 hours,real-time PCR(RT-PCR)was used to detect the m RNA expression of TH synthesis-related genes in cells,including thyroid stimulating hormone receptor(TSHR),guanine nucleotide binding regulatory protein(Gs),AC6,cyclic adenosine monophosphate(c AMP),protein kinase A(PKA),thyroid transcription factor-1(TTF-1),forkhead box protein E1(FOXE1),c AMP-response element binding protein(CREB),sodium iodide cotransporter(NIS),thyroid peroxidase(TPO),thyroglobulin(TG),HSP90B1 and GPx1.Western blot(WB)was used to detect the expression of TH synthesis related proteins of cells,including TSHR,PKA,TTF-1,FOXE1,PAX-8,NIS and TPO.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the level of TG secreted by cells.The ATP and Na+-K+-ATPase activities of cells were measured with related kits,and the non-radioactive Sandell-Kolthoff reaction was used to detect the iodine uptake of cells.The changes of reactive oxygen species(ROS),reduced glutathione(GSH),glutathione peroxidase(GPx),and malondialdehyde(MDA)in cells were determined after IAA treatment.Results: Compared with the negative control group,IAA could down-regulate the m RNA expression of the TH synthesis-related genes in the Nthy-ori 3-1 cells,including TSHR,AC6,c AMP,PKA,PAX-8,CREB,TG,TPO and GPx1(P<0.05).However,the m RNA expression of Gs,TTF-1,FOXE1,NIS and HSP90B1 m RNA had not significant change(P>0.05).Compared with the negative control group,IAA could not change the expression of TSHR,PKA,TTF-1,PAX-8,FOXE1,NIS and TPO proteins in the Nthy-ori 3-1 cells.No significant changes were observed in the levels of secreted TG and ATP(P>0.05).Compared with the negative control group,IAA could significantly reduce the Na+-K+-ATPase activity and iodine uptake in cells(P<0.05).IAA had no effects on GSH,GPx and MDA of Nthy-ori 3-1 cells(P>0.05),but it can significantly increase the level of ROS in cells(P<0.05).Conclusion: IAA may disrupt the synthesis of TH,and its mechanism may be related to reducing the expression of TH synthesis-related genes,inhibiting the iodine uptake function of cells,and inducing oxidative stress. |
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