Toxicogenomic Analysis Of Hepatotoxicity Induced By Tributyltin

Objective: Tributyltin(TBT)is one of the most widely used organotin biocides,which has severe effects on marine species and mammals.It mainly includes reproductive toxicity,neurotoxicity,immune toxicity and hepatotoxicity.Given that liver plays an important role in the metabolism and detoxification,TBT shows obvious hepatotoxicity.However,most of the available studies on TBT have focused on observations at the cellular level,while studies at the level of genes and proteins are limited;therefore,the molecular mechanisms of TBT-induced hepatotoxicity remains largely unclear.In the present study,we applied a toxicogenomic approach to investigate the effects of TBT on gene expression in the human normal liver cell line HL7702.Method: 1.Cell proliferation was determined by MTT assays after cells were exposed to 0,2,4,6,8,or 10 μM TBT for 2 h.2.Total RNA was extracted after HL7702 cells treated with TBT.The qualities and quantities of extracted RNAs were verified by spectrophotometer and 1 % formaldehyde denaturing gel electrophoresis.3.Gene expression profiling analysis was used to identify the transcriptional levels of whole genes.4.SAM software was used to identify differentially expressed genes(DEGs)between TBT exposure groups and the control group,and the criteria for DEGs were FDR < 0.05 and fold change > 2.0 or < 0.5.5.Annotation and biological interpretation of the identified DEGs were conducted on the basis of the Gene Ontology(GO)database.Functional classification and biological pathway analysis were conducted using DAVID.6.Thirteen representative genes selected from differentially expressed genes in the microarray analysis whose functions were found,in a biological function analysis,to be closely related to apoptosis were verified by quantitative real-time PCR(qRT-PCR).7.The HL7702 cells were exposed to different concentrations of TBT for 2 h,and the apoptotic rate was analyzed with an Annexin V-FITC Apoptosis Detection Kit by flow cytometry analysis.8.IPA includes upstream signal analysis and downstream signal analysis,carrying out a schematic diagram of TBT induced apoptosis.Result: 1.Based on the result of MTT assay,0,2,6,and 10 μM TBT were used in the following experiments.And we defined 2,6,and 10 μM as low-dose,medium-dose,and high-dose respectively.2.According to gene expression profiling analysis,a total of 770,1028,and 1221 genes were identified to be DEGs for the low-dose,medium-dose,and high-dose TBT-treated groups,respectively.3.The signal pathway analysis of the differentially expressed genes showed that apoptosis was the main cause of TBT hepatotoxicity.4.Thirteen DEGs selected from the pathway analysis were verified by qRT-PCR.The results of qRT-PCR showed similar trends to those revealed in the microarray data.5.Flow cytometry indicated that low-dose TBT treatment did not change the apoptosis rate.However,medium-and high-dose TBT treatment significantly increased the number of apoptotic cells.They were appeared a obvious dose-dependent relationship.6.IPA suggested that the apoptotic pathways relevant to the DEGs were mediated by HSPs,kinases,and TNF receptors.7.Other signaling pathways such as p53 signaling pathway,integrin signaling pathway,regulation of actin cytoskeleton were enriched after exposed to TBT.These signaling pathways may also be involved in the hepatotoxicity of TBT.Conclusion: Gene expression profiling analysis has revealed that apoptosis plays a key role in the hepatotoxicity induced by TBT.Three apoptotic pathways including death receptor pathway,mitochondrial pathway and endoplasmic reticulum pathway are involved in TBT induced apoptosis.