(527f) Micrornas As Biomarkers for Cytotoxicity Evaluation of Multi-Wall Carbon Nanotube

Multi-wall carbon nanotubes (MWCNT) are one of the most promising engineered nanomaterials because of their excellent physical and chemical characteristics. However, the applications of MWCNT also raise health concerns. The small size and light weight of MWCNT allow them to enter the working environment as particulate matter of respirable sizes. Many studies have already reported that inhaled CNTs showed significant cytotoxicity both in vitro and vivo, and the toxicity potential will increase with decreasing particle sizes.

MicroRNAs, a family of short endogenous noncoding RNAs, harbor critical functions in many fundamental biological processes, such as proliferation and apoptosis. Intracellular and circulating microRNAs have been implicated as promising biomarkers in the diagnosis of lung cancer and pulmonary diseases. Here, we seek to identify both intracellular and circulating microRNAs as biomarkers for MWCNT induced pulmonary toxicity.

Total 4 types of MWCNT with different lengths (0.2-2µm and 10-50µm) and surface modifications (with and without carboxyl modification) were selected and their cytotoxicity was evaluated in human bronchial epithelial cells (BEAS-2B). The cytotoxic effects of 4 types of MWCNT were first characterized by the cell growth measurement and the reactive oxygen species (ROS) assay. BEAS-2B cells were treated with MWCNT at concentrations from 0 to 50µg/mL. At 48 h post treatment, all 4 types of MWCNT inhibited the growth of BEAS-2B cells and generated significant ROS stress. The MWCNT toxicity was dose and tube type dependent. The shorter length and carboxyl modification enhanced the MWCNT toxicity because of better dispersion in aqueous solutions and higher cellular uptake.

Next, a microRNA toxicology panel was used to identify the dysregulated microRNAs post MWCNT exposure. Profiling of 87 miRNAs related with toxicology showed that the expression of total 7 miRNAs was changed at least two folds compared with untreated controls. Among 87 miRNAs, miRNA-29b, miRNA-21, and miRNA-486 were selected for further investigation because they are closely related with lung diseases including pulmonary fibrosis and lung cancer. At 48 h post treatment, the expression of intracellular miRNA-29b was up-regulated up to ~10 folds, the expression of intracellular miR-486 was down-regulated up to ~55% and the expression of intracellular miR-21 remained unchanged compared with untreated controls. However, the expression of circulating miRNA-29b was up-regulated up to ~4 folds, the expression of circulating miR-486 was up-regulated up to ~10 folds and the expression of circulating miR-21 was up-regulated up to ~50 folds compared with untreated controls. The expression of intracellular and circulating miRNAs was also dose and tube type dependent. Among 4 types of MWCNT, short MWCNT with carboxyl modification showed the most significant impact on the expression of both intracellular and circulating miRNAs. And the expression of intracellular and circulating miRNAs were able to distinguish the toxicity effects induced by different types of MWCNT.

     Our results demonstrated that intracellular and circulating microRNAs are sensitive biomarkers for MWCNT induced pulmonary cytotoxicity, and they hold great potential in the early detection of adverse health effects associated with exposure to MWCNT.