An Investigation Of Methyl Tert-butyl Ether Induced Cytotoxicity On Chinese Hamster Ovary Cells By Proteomic Analysis

Methyl tert-butyl ether(MTBE) is a widely used gasoline additive of small organic molecules, on the growing number of occupational exposure and environmental ex posures to MTBE. Studies have demonstrated that MTBE can exert toxic effects on biological organisms at the biomacromolecular, cellular and in vivo levels. The potentially toxicity impact and biological effects include acute toxicity, reproductive toxicity, genotoxicity, carcinogenicity, apoptosis and oxidative stress. However, the MTBE-induced cytotoxicity and the potential mechanismes are unclear. In this study, we combine the toxicology research method and proteomics technology to investigate the oxidative stress effect and differential proteomic expression of MTBE-induced Chinese hamster ovary(CHO) cells, as well as the potential toxic effect of MTBE on molecular level. This studymainly includes two parts of works as follows. 1. The cytotoxic effects and oxidative stress of the CHO cellsinduced by MTBE.The CHO cells were cultured in the presence of MTBE at various concentrations(0.5-100.0 mmol/L) for 6 h, 12 h, 24 h. Anti-proliferative effect of cells was evaluated and the dose-effect relationship of the MTBE-induced CHO cells by methyl thiazolyl tetrazolium(MTT) assay. The blank control group was setted. Five treatment groups of 0.5, 5.0, 25.0, 50.0 and 100.0 mmol/L under a process-time of 12 h were used. The relevant indicators of oxidative stress were measured in commercial kits, such as lactate dehydrogenase leakage(LDH), maleic dialdehyde(MDA) content, reactive oxygen species(ROS) production, as well as the enzymatic activities of superoxide dismutase(SOD or SOD1), catalase(CAT) and glutathione peroxidase(GSH-Px or GPX1). The mRNA levels and protein expressions of SOD1, CAT and GPX1 were analyzed by Q-PCR and Western blot.MTBE exposure significantly inhibited cell proliferation and induced plasma membrane damage in CHO cells. The ROS generation decreases with the increase of MTBE dose. Elevation of MDA content and enzymatic activities of SOD, CAT and GSH-Px were revealed during the MTBE exposure of CHO cells. In addition, MTBE exposure can induce significantly increase in mRNA levels and protein expressions in SOD, CAT and GPX1, respectively. The results from the present study clearly demonstrated that MTBE exposure induce cytotoxicity and oxidative stress in mammalian ovary cells. 2. The proteomic studies on the cytotoxicity caused by MTBE in CHO cellsBased on the dose-response relationship of MTBE in CHO cells, the cells were treated with three concetrations of MTBE(0 mmol/L, 5.0 mmol/L and 50.0 mmol/L) for 12 h, and then were harvested for protein extraction and protein assay. The proteins were separated by the two-dimensional electrophoresis, The well-matched and differentially expressed protein spots were selected and identified by MALDI-TOF-TOF-MS. and the data on differential expression of proteins were analyzed by Bioinformatics software. then use the Western blot of differentially expressed proteins for further verification.The MTBE-induced differentially expressions were analyzed by a two dimensional fluorescence difference gel electrophoresis and matrix assisted laser desorption/ionization tandem time-of-flight spectrometry(MALDI-TOF-MS). 24 differentially expressed protein spots were found in MTBE-treated CHO cells compared with the control. Among them, 16 protein spots were up-regulated and 8 protein spots were down-regulated. We find out four proteins which related to cell toxicity and oxidativestress, including GRP75, HSC70, RPSA and PPIA. The results of Western Blot demonstrated that the alterations of there proteins were consistent with proteomics. Therefore, we conclude that the cytotoxicity caused by MTBE CHO may be related to several key proteins.