Proteomic Study Of MWCNT-induced Cytotoxicity To A549Cells

Since the discovery of multi-walled carbon nanotube (MWCNT) in1991, they have been widely used in numerous applications due to their unique physicochemical properties. However, with their wide application, the chances of exposure to MWCNT are greatly increased for both the human population and environment. Thus, there is growing concern regarding the possible negative impacts of MWCNT on human health and the environment. Many in vitro and in vivo studies have shown that MWCNT and/or associated contaminants or catalytic materials that arise during the production process may induce oxidative stress, prominent pulmonary inflammation and neural injuries. The cytoxicity of MWCNT has been reported in many cell lines, while contradictive results also existed. Results from animal studies suggest that the increased concentration of nanoparticles, higher reactive surface area per unit mass, along with the unique chemistry and functionality, are important for the acute and chronic inflammatory response in lung. Recent studies also suggested certain similarities between the pathogenic properties of MWCNT and those of asbestos fibers, as MWCNT could induce lung cancer or mesothelioma in rats. On the other hand, there were also reports showing no cancer occurred after long-term exposure to MWCNT. Therefore, the toxic effects of MWCNT are not clear and still controversial.To clarify the toxic effects of MWCNT and the underlying mechanisms, in the current study, the cytotoxic and genotoxic effects of MWCNT on A549cells were examined, then a proteomic approach (2-DE coupled with LC-MS/MS) was applied. By screening and identifying the differentially expressed protein spots, we investigated the possible roles of the differentially expressed proteins in MWCNT-induced cytotoxicity to A549cells. As a result, MWCNT induced time-and dose-dependent cytotoxicity in A549cells. Within a relatively low dosage range (30μg/ml) and short time period (24h), MWCNT treatment did not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, MWCNT treatment caused significant protein expression changes. A total of106proteins showed altered expression at different time points and dosages, and among them,52proteins were further identified by MS. Among them,29were up-regulated, and23were down-regulated. The identified proteins were involved in cellular processes such as proliferation, stress response, or cellular skeleton organization, etc. Three identified proteins, namely14-3-3ε, Actin and Hsp27, were verified by Western blot and immunofluorescent microscopy, which were consistent with2-DE results. In particular, MWCNT treatment caused increase in actin expression, leading to increased migration capacity, in which reactive oxygen species (ROS) and Ca2+might be involved.