Mechanisms Of Endoplasmic Reticulum Stress In Hepatic Cells Damage Induced By Tributyltin

Objective: Tributyltin(TBT)is a common organotin compound that is widely used as wood preservative,hull antifouling agent.As a common environmental endocrine disruptor,TBT causes serious harm to marine organisms and mammals,mainly related to reproductive toxicity,neurotoxicity,immunotoxicity and hepatotoxicity.As the body's main detoxification organ,liver is also the target organ of TBT.Our previous microarray study identified that endoplasmic reticulum stress may play a key role in TBT-induced hepatotoxicity.The present study further studied the mechanism of TBT-induced endoplasmic reticulum stress using high throughput proteomics technology.Materials and methods: 1.The HL7702 cells were exposed to different concentrations of TBT(0,2,4,6,8,10 ?M)for 2 h,and the cell proliferation was detected by an MTT assay.2.Total protein was extracted and the expression levels of all proteins were detected using labeled quantitative proteome iTRAQ ?.3.Proteinpilot software was used to identify the differentially expressed proteins after TBT expose.4.Five endoplasmic reticulum stress-related genes found in the proteomic analysis were verified by real-time PCR.5.Eight endoplasmic reticulum stress-associated proteins were verified by western blotting.6.The ultrastructural changes of HL7702 cells treated with TBT were observed under transmission electron microscope.Results: 1.TBT exposure affected the proliferation of HL7702 cells and showed the tendency of low concentration to promote cell proliferation and high concentration to inhibit cell proliferation.2.According to proteomic experiments,349,598,801 differentially expressed proteins appeared after exposure to low,medium and high concentrations of TBT.3.KEGG pathway analysis of proteomic results showed that endoplasmic reticulum stress was the major cause of TBT hepatotoxicity.4.Five endoplasmic reticulum stress-related genes selected from the pathway analysis were verified by qRT-PCR,and the trends of change were consistent with those revealed in the proteomic data.5.Eight endoplasmic reticulum stress-related proteins selected from the pathway analysis were validated by Western Blot,and the trends of change were consistent with those revealed in the proteomic data.6.Under the electron microscope,nuclear condensation,endoplasmic reticulum cavity expansion and ribosome shedding were observed in the experimental group treated with low concentration of TBT,indicating that endoplasmic reticulum stress occurred.With the further increase of the concentration,the cell died and disintegrated.Conclusion: The results of proteomics indicate that endoplasmic reticulum stress is the major cause of TBT hepatotoxicity.ATF6,PERK,IRE1 three receptors protein-mediated unfolded protein pathway all involved in TBT-induced endoplasmic reticulum stress.