Molecular Toxicology Of Triclocarban Exposure

Triclocarban(3,4,4,9-trichlorocarbanilide,TCC)is a broad spectrum antibacterial agent containing chlorine,widely used in daily chemicals such as toothpastes,washings and personal care products.The extensive use of antimicrobial agents has led to the almost ubiquitous TCC in the ecological environment,even in human tissues and body fluids.The potential toxicity of TCC brings risks to the environmental and life's health,which has aroused people's wide concern.Although the U.S.Food and Drug Administration(FDA)prohibitted TCC as an antibacterial agent used in over-the-counter hand-washing products in September 2016,they are still used in the other personal care products owing to lacking sufficient evidence to prove their dangers for human and in-depth exploration of toxic molecular mechanism.Therefore,the purpose of this study is to investigate the toxic effects and molecular mechanism of long-term exposure to TCC at different doses,to have a deep understanding of the environmental health effects of TCC,and to provide suggestions for the rational application of antimicrobial agents.In this study,considering the environmental and human TCC exposure level in real life,we selected low(50 ?g/kg),medium(20 mg/kg)and high(100 mg/kg)three different doses of TCC long-term exposure to SD rats by gavage,which were used to simulate below,equivalent to,and above environmental exposure levels,respectively.The non-targeted,targeted metabolomics techniques based on NMR,GC/LC-MS and molecular biological methods were used to study the molecular mechanism of the toxic effects of long-term exposure to TCC at the animal level from the perspective of metabolism,and the toxic mechanism of TCC was further explored at the cellular and molecular levels in vitro.In vivo experiments,combined with histopathological assessments,blood biochemical detection and untargeted 1H NMR metabolic profile analysis,it was found that long-term exposure to TCC affected lipid homeostasis in rats,especially low-dose TCC long-term exposure caused a significant increase in lipids,which induced hepatic steatosis.Further,targeted metabolomic analysis(LC/GC-MS)were used for quantitative analysis of ceramides,phospholipids,fatty acids,and triglycerides respectively,these results showed that low dose of TCC exposure caused a general rise in lipids,but only a few lipids especially toxic lipids such as C16:0 fatty acids and ceramides changed in the high dose of TCC group.Consistently,the lipid synthase gene mRN A expression level was also significantly increased.With the increase of TCC dose,ATF6 signaling pathway was activated and XBP1 protein expression was inhibited,which led to the destruction of endoplasmic reticulum homeostasis in rat hepatocytes.The liver AHR nuclear receptor and the mono-oxygenase P450 enzymes Cyplal and Cyplbl were obviously induced,indicating greater toxicity of high doses of TCC,and the level of inflammatory factors in the serum increased significantly with the increase of TCC exposure dose.In addition,TCC exposure also affected the composition of intestinal flora in rats and promoted the fermentation of intestinal microorganisms.In particular,low-dose TCC caused significant upregulation of short-chain fatty acids,which provided energy for the body and contributed to lipid metabolism disorders in the liver.Taken together,low-dose TCC long-term exposure induced lipid metabolism disorders in rats,while the relative long-term exposure of medium and high doses showed a stronger toxic effect and a risk of developing towards metabolic diseases such as hepatitis.To further study the mechanism of TCC-induced lipid metabolism disorder,we investigated the effect of TCC(1-5 ?M)exposure on the metabolism of hepatocytes(L02)and adipocytes(3T3L1)using in vitro cell exposure assay and 1H NMR non-targeted metabolic and LC/GC-MS targeted metabolic assay combined with molecular biology techniques.The results showed that TCC exposure promoted the differentiation of 3T3L1 preadipocytes in a dose-dependent manner,which was consistent with significant up-regulation of mRN A levels in the key adipogenic markers Fasn,Srebp1,Ap2 and triglyceride(TG),fatty acids,were significantly accumulated during the differentiation process.In addition,ATF4 and XBP1 unfolded protein response signaling pathways were significantly activated during TCC-induced cell differentiation,and caused endoplasmic reticulum stress.To human hepatocytes(L02),the results of untargeted 1H NMR metabolic profile analysis showed that after 12 hours of TCC exposure,GSH and choline contents were dose-dependently decreased,leading to oxidative stress in hepatocytes.TCC exposure for 24 hours,the lipid metabolism of hepatocytes increased significantly,resulting in a significant increase in TG,ceramides and saturated fatty acid C16:0,and interferes with ER homeostasis by activating ATF4 and ATF6 and inhibiting the expression of XBP1 signaling pathway.Thses results were consistent with the changes in liver metabolism in vivo.In addition,the metabolic changes of ceramides in this two different cell lines are opposite,suggesting that ceramides may be involved in the process of cell differentiation.Taken together,TCC exposure in vitro promoted the lipogenesis in 3T3L1 and hepatocytes L02.The effects of TCC exposure on hepatocyte L02 metabolism in vitro are consistent with the changes of liver metabolism in vivo.After TCC exposure to hepatocytes(L02),GSH was down regulated,oxidative stress was induced,and then lipids including TG,SFA(C16:0),ceramides increased significantly.In addition,the homeostasis of endoplasmic reticulum was destroyed by interfering with ATF4,ATF6,XBP1 signaling pathway.This study reveals the molecular mechanism of lipid metabolism abnormalities caused by TCC exposure from the perspective of cells,tissues,animal body fluids,and deeply explores the possible causes of metabolic diseases induced by long-term TCC exposure comprehensively.It provides basic data and new ideas for assessing the health hazard effects of environmental pollutants.