Study On Pulmonary Toxicity Of Multi-Wall Carbon Nanotubes In Rats

Carbon nanotubes are unique nanostructures with remarkable electronic, thermal and mechanical properties. They are large carbon molecules shaped like tubes, which are about 1-3 nanometers in diameter, and hundreds to thousands of nanometers long. Carbon nanotubes are expected to be one of nanotech's most promising materials because they have a wide range of potential commercial applications, such as tiny sensors, electronic and optical devices, catalysts, batteries, disease diagnostics and drug delivery vehicles. There are two forms of carbon nanotubes: multi-wall carbon nanotubes (MWCNTs) and single-wall carbon nanotubes (SWCNTs).Despite the huge amount of interest and investment in carbon nanotubes, the toxicological impacts of them are still unknown. Considering that the physical and chemical properties of nanosized materials mostly differ from the existing microsized materials, the potential impacts of carbon nanotubes on human health and the environment raised great concern.The aim of the present study is to investigate the potential pulmonary toxicity of MWCNTs in rats and explore for its possible mechanism. Our study includes two parts: the first part was to assess the in vitro cytotoxicity and oxidative injury of MWCNTs in cultured macrophage and cultured alveolar epithelium cell II; the second part was to study the in vivo pulmonary toxicity of MWCNTs in Wistar rats.Cultured macrophage and alveolar epithelium cell II were selected for in vitro study. Because alveolar macrophage is considered to be the first defense line of respiratory tract, which exerts the function of phagocytosis, clearance and protection; and AT II cell are indispensable for the renovation of alveolar structure after lung injury. The cytotoxicity and oxidative stress response of MWCNTs were investigated in cultured macrophage (RAW264.7 cell line) and cultured alveolar epithelium cell II (A549 cell line). Cells were exposed to blank control, solvent control, and different doses of MWCNTs suspension (2.5, 10, 25 and 100μg/ml) for 24h. Cell viability was assessed by MTT assays. Indicators of cytotoxicity and oxidative stress, including total protein (TP), lactate dehydrogenase(LDH), nitrogen monoxidum(NO), superoxide dismutase (SOD), glutathion(GSH) and malondialdehyde (MDA) in supernate as well as in cultured cells, were quantitatively assessed by EIA methods. Oxygen free radical was detected by luminol chemiluminescence method. Phagocytosis in RAW264.7 was tested by Wright's staining. Reactive oxygen species (ROS) production in A549 cell was measured by chemiluminescence method. The morphology change of RAW 264.7 cell and A549 cell were observed also. Results showed that after 24h exposure to MWCNTs, the viability of A549 cells was decreased significantly in a dose-dependent manner with comparison to control. The levels of TP, LDH, NO and MDA in exposed groups were higher, while GSH and SOD level were lower than control with dose-effect relationship. Injury of cell membrane and rate of oxidative stress were more severe in A549 cell than in RAW264.7 cell. Oxygen free radical assay showed that MWCNTs can induce oxygen free radical activity in both cell lines. Phagocytotic effect of RAW264.7 cells was defected by MWCNTs treatment, and ROS production in A549 was induced. Cells exposed to MWCNTs exhibited increased cell mortality, necrosis, altered morphology of macrophages and alveolar epithelium cells, and cell debris caused by apoptosis. The increased production of LDH and MDA release from the cells indicated membrane damage and lipid peroxidation. In summary, exposure to MWCNTs results in a dose-dependent cytotoxicity in cultured RAW264.7 cells and A549 cells that is closely correlated to increased oxidative stress.In vivo pulmonary toxicity of MWCNTs was investigated in Wistar rats. One hundred and ninety-two Wistar rats were divided into six groups at random and exposed to blank control, solvent control, ultrafine carbon black (UFCB), and different doses of MWCNTs (2.0, 10.0,20.0mg/ml) by intratracheally instillation once a day for 3 continuous days. Rats were sacrificed at 24h, 7d, 28d and 90d after exposure. The bronchoalveolar lavage fluids(BALF) were collected, and lung morphological change was observed under light microscopic. Indicators of cytotoxicity and oxidative stress, including TP, LDH, AKP, GSH, SOD and MDA in BALF, were determined. Results showed that the level of TP, LDH, NO and MDA in BALF were higher, while GSH and SOD level were lower than control significantly in a dose-dependent manner. Pathological examination showed that the severity level of inflammation (infiltration by macrophage and lymphocytes, vascular injection, epithelial lesion, etc.), which extended from bronchiole to alveolus and interstitute, rose with the increase of administrative doses. Different to UFCB group, multifocal granulomas and fibroblasts were found in MWCNTs-treated group at 90d. Nucleus membrane damage, ring-shaped mitochondria, and swelling of endoplasmic reticulum were found in MWCNTs-treated group. The oxidative stress response, cytotoxicity and pathologic changes tended to recover with time. Interestingly, MWCNTs was found to translocate to the lymph node from 28d, indicating that lymph circulation helps to eliminate the MWCNTs from lung tissue. The in vivo results suggested that MWCNTs can induce acute inflammation, dust cell granuloma, and oxidative stress injury in lung.In conclusion, MWCNTs may induce pulmonary inflammatory response and cytotoxicity under current experimental conditions, which is correlated to increased oxidative stress to macrophage and ATII cells.MWCNTs was provided by Nagoya Institute of Technology. The characterization showed it an average diameter of 10nm, average length below 1μm, and carbon purity exceed 95%. In order to improve the dispersity of MWCNTs, DNA sodium salt was added into the suspensions at different doses and at 1:1 proportion to MWCNT. The composition analysis of DNA sodium salt showed it consists phosphorus (<9 %) and protein (<5 %). It is suggested that small size, large surface area and high concentration are important factors for MWCNTs-induced lung injury and inflammatory response.