Toxicity Of Multi-walled Carbon Nanotubes Research Articles

Instillation versus inhalation of multiwalled carbon nanotubes: exposure-related health effects, clearance, and the role of particle characteristics.

Inhaled multiwalled carbon nanotubes (MWCNTs) may cause adverse pulmonary responses due to their nanoscale, fibrous morphology and/or biopersistance. This study tested multiple factors (dose, time, physicochemical characteristics, and administration method) shown to affect MWCNT toxicity with the hypothesis that these factors will influence significantly different responses upon MWCNT exposure. The study is unique in that (1) multiple administration methods were tested using particles from the same stock; (2) bulk MWCNT formulations had few differences (metal content, surface area/functionalization); and (3) MWCNT retention was quantified using a specialized approach for measuring unlabeled MWCNTs in rodent lungs. Male Sprague–Dawley rats were exposed to original (O), purified (P), and carboxylic acid functionalized (F) MWCNTs via intratracheal instillation and inhalation. Blood, bronchoalveolar lavage fluid (BALF), and lung tissues were collected at postexposure days 1 and 21 for quantifying biological responses and MWCNTs in lung tissues by programmed thermal analysis. At day 1, MWCNT instillation produced significant BALF neutrophilia and MWCNT-positive macrophages. Instilled O- and P-MWCNTs produced significant inflammation in lung tissues, which resolved by day 21 despite MWCNT retention. MWCNT inhalation produced no BALF neutrophilia and no significant histopathology past day 1. However, on days 1 and 21 postinhalation of nebulized MWCNTs, significantly increased numbers of MWCNT-positive macrophages were observed in BALF. Results suggest (1) MWCNTs produce transient inflammation if any despite persistence in the lungs; (2) instilled O-MWCNTs cause more inflammation than P- or F-MWCNTs; and (3) MWCNT suspension media produce strikingly different effects on physicochemical particle characteristics and pulmonary responses.

Acute toxicity of multi-walled carbon nanotubes, sodium pentachlorophenate, and their complex on earthworm Eisenia fetida

Laboratory experiments were undertaken to relate biomarker responses to the toxicities of multi-walled carbon nanotubes (MWCNTs) and sodium pentachlorophenate (PCP-Na), both individually and combined. The acute toxicities of MWCNTs and PCP-Na on earthworm Eisenia fetida were studied through different exposure methods (filter paper contact test, immersion contact test, and artificial soil contact test). Enzyme activity and malondialdehyde (MDA) content in the earthworm E. fetida exposed to MWCNTs and PCP-Na in filter paper contact test, both individually and under combined exposure, were determined. After exposure, PCP-Na induced observable acute toxicity while the MWCNTs induced slight toxicity. Interestingly the earthworms exposed to the mixture of MWCNTs and PCP-Na demonstrated different expression of enzymatic biomarkers from those exposed to MWCNTs or PCP-Na alone. Our results indicated that the toxicity of PCP-Na on E. fetida may be alleviated by the appearance of MWCNTs for all exposure methods except for immersion contact test.

Drosophila Embryos as Model to Assess Cellular and Developmental Toxicity of Multi-Walled Carbon Nanotubes (MWCNT) in Living Organisms

Different toxicity tests for carbon nanotubes (CNT) have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs) become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

Physicochemical Determinants of Multiwalled Carbon Nanotubes on Cellular Toxicity: Influence of a Synthetic Method and Post-treatment

Since the discovery of carbon nanotubes (CNTs), scientists have performed extensive studies on nanotubes in the fields of materials science, physics, and electronic engineering. Because multiwalled CNTs (MWCNTs) are not homogeneous materials, and because it is not feasible to test every newly synthesized MWCNT, this study was aimed at investigating the physicochemical properties that primarily determine the cellular toxicity of MWCNTs. This study analyzed the relationship between cell viability and physicochemical characteristics following exposure to eight different MWCNTs. We generated eight different MWCNTs using various synthetic methods and post-treatments. From this analysis, we sought to identify the major physicochemical determinants that could predict the cellular toxicity of MWCNTs, regardless of the synthetic method and post-treatment conditions. Creation of binding sites on the tube walls by breaking C-C bonds played a pivotal role in increasing toxicity and was most clearly demonstrated by a Raman G peak shift and the ID/IG ratio. In addition, several factors were found to be strongly related to cellular toxicity: surface charge in the case of MWCNTs created by the chemical vapor deposition method and surface area and EPR intensity in the case of MWCNTs created by the arc discharge based method. The methods developed in this study could be applied to the prediction of the toxicity of newly synthesized MWCNTs.

Evaluation of the combined toxicity of multi-walled carbon nanotubes and sodium pentachlorophenate on the earthworm Eisenia fetida using avoidance bioassay and comet assay

In this study, the adsorption kinetics of sodium pentachlorophenate (PCP-Na) onto multi-walled carbon nanotubes (MWCNTs) was determined and acute toxicity tests were performed to evaluate the toxicity of MWCNTs and PCP-Na on the earthworm Eisenia fetida in artificial soil samples, and then the toxicities of their mixture were investigated using soil avoidance test and comet assay. The results of the acute toxicity test showed that no mortality was observed for MWCNTs concentration up to 1000 mg kg−1. The earthworms suffered DNA damage in soils amended with MWCNTs at 50 mg kg−1 in a natural soil, despite of the fact that no significant (p > 0.05) avoidance had occurred at this level of MWCNTs in artificial soil. The LC50 values of PCP-Na for the 7-d and 14-d were 176.0 and 71.1 mg kg−1, respectively. PCP-Na at the concentration of 35.6 mg kg−1 in soil induced behavior toxicity in avoidance test, and genetic toxicity could be determined at a relatively low concentration (1.4 mg kg−1). The avoidance assay revealed a synergetic effect of the two chemicals on E. fetida, regardless of whether these compounds were both added separately or after the adsorption of PCP-Na to the MWCNTs. The two exposure methods showed no differences on the combined toxicity of MWCNTs and PCP-Na when low concentrations of the chemicals were used. Comet assay is not a suitable method for evaluation of combined toxicity of the two chemicals in this study, because exposure to PCP-Na at high dosages could induce lower values of the parameters compared with the lower dosages.

In vivo translocation and toxicity of multi-walled carbon nanotubes are regulated by microRNAs

We employed an in vivo Caenorhabditis elegans assay system to perform SOLiD sequencing analysis to identify the possible microRNA (miRNA) targets of multi-walled carbon nanotubes (MWCNTs). Bioinformatics analysis on targeted genes for the identified dysregulated miRNAs in MWCNT exposed nematodes demonstrates their involvement in many aspects of biological processes. We used loss-of-function mutants for the identified dysregulated miRNAs to perform toxicity assessment by evaluating functions of primary and secondary targeted organs, and found the miRNA mutants with susceptible or resistant property towards MWCNT toxicity. Both the physiological state of the intestine and defecation behavior were involved in the control of the susceptible or resistant property occurrence for specific miRNA mutants towards MWCNT toxicity. This work provides the molecular basis at the miRNA level for future chemical design to reduce the nanotoxicity of MWCNTs and further elucidation of the related toxicological mechanism.

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice

Aqueous stabilization of carbon nanotubes: effects of surface oxidization and solution chemistry

Surface oxygen functional groups can affect the morphological characteristics, aggregation kinetics, and adsorption capacity of multi-walled carbon nanotubes (MWCNTs). However, little is known about the quantitative relationship between oxygen content and the dispersion stability of MWCNTs. To investigate the effects of surface oxidization, MWCNTs were oxidized using concentrated H2SO4/HNO3 acids for 0, 1, 2, 4, and 8 h, respectively. Experimental results showed that the oxygen content of MWCNTs increased with surface oxidization time. Linear correlations were found to exist between the oxygen content, critical coagulation concentration (CCC) for NaCl, and critical coagulation pH values of MWCNTs detected by optical density at 800 nm. The CCC values for CaCl2 increased with oxygen contents for unmodified MWCNTs and lowly oxidized MWCNTs, while which decreased after further increasing the surface oxidization. CCC ratios in the presence of Ca(2+) to Na(+) were consistent with the empirical Schulze-Hardy rule for unmodified MWCNTs and lowly oxidized MWCNTs; however, which were much lower than the expected values for highly oxidized MWCNTs. Fulvic acid can clearly increase the stability of MWCNT suspension with unmodified MWCNTs and lowly oxidized MWCNTs, while it cannot affect the dispersion of highly oxidized MWCNTs. This study implied that the oxidation and presence of fulvic acid will possibly increase the mobility, exposure, bioavailability, and toxicity of MWCNTs.

Toxicity Study of Multiwalled Carbon Nanotubes on Freshwater Aquatic Algae

Nanotechnology based applications had been extended to almost all aspects of daily life, however, the ecotoxicity of nanomaterials especially multiwalled carbon nanotubes is not well studied. The present study, therefore, dealt with the evaluation of ecotoxicity of multiwalled carbon nanotubes (MWCNTs) against fresh water algae i.e., Chlorella which was collected from the authorised collection centre and maintained in the laboratory giving appropriate growth conditions. MWCNTs suspensions were prepared in various concentrations from 0.06 mg/ml to 0.24 mg/ml using algal test medium and further quantified by UV/Vis spectroscopy. It has been observed that with the increase of the concentrations of MWCNTs, the growth of algae and the lipid production decreases simultaneously. It shows 24.13% less growth and 5.06% less lipid production compare to control at maximum concentration of MWCNTs i.e., 0.24 mg/ml. Toxicity of MWCNTs was further confirmed through transmission and scanning electron microscopic images of algae.

Inhibition of catecholamine secretion by iron-rich and iron-deprived multiwalled carbon nanotubes in chromaffin cells

The assay of the toxic effects of carbon nanotubes (CNTs) on human health is a stringent need in view of their expected increasing exploitation in industrial and biomedical applications. Most studies so far have been focused on lung toxicity, as the respiratory tract is the main entry of airborne particulate, but there is also recent evidence on the existence of toxic effects of multiwalled carbon nanotubes (MWCNTs) on neuronal and neuroendocrine cells (Belyanskaya et al., 2009; Xu et al., 2009; Gavello et al., 2012). Commercial MWCNTs often contain large amounts of metals deriving from the catalyst used during their synthesis. Since metals, particularly iron, may contribute to the toxicity of MWCNTs, we compared here the effects of two short MWCNTs samples (<5μm length), differing only in their iron content (0.5 versus 0.05% w/w) on the secretory responses of neurotransmitters in mouse chromaffin cells.We found that both iron-rich (MWCNT+Fe) and iron-deprived (MWCNT−Fe) samples enter chromaffin cells after 24h exposure, even though incorporation was attenuated in the latter case (40% versus 78% of cells). As a consequence of MWCNT+Fe or MWCNT−Fe exposure (50–263μg/ml, 24h), catecholamine secretion of chromaffin cells is drastically impaired because of the decreased Ca2+-dependence of exocytosis, reduced size of ready-releasable pool and lowered rate of vesicle release. On the contrary, both MWCNTs were ineffective in changing the kinetics of neurotransmitter release of single chromaffin granules and their quantal content. Overall, our data indicate that both MWCNT samples dramatically impair secretion in chromaffin cells, thus uncovering a true depressive action of CNTs mainly associated to their structure and degree of aggregation. This cellular “loss-of-function” is only partially attenuated in iron-deprived samples, suggesting a minor role of iron impurities on MWCNTs toxicity in chromaffin cells exocytosis.

Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells

BackgroundNanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT.MethodsA co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined.ResultsExposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure.ConclusionThe co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitro method to study the pulmonary toxicity of MWCNT.

Purification and sidewall functionalization of multiwalled carbon nanotubes and resulting bioactivity in two macrophage models

This study examined the consequences of surface carboxylation of multiwalled carbon nanotubes (MWCNT) on bioactivity. Since commercial raw MWCNT contain impurities that may affect their bioactivity, HCl refluxing was exploited to purify raw “as-received” MWCNT by removing the amorphous carbon layer on the MWCNT surface and reducing the metal impurities (e.g. Ni). The removal of amorphous carbon layer was confirmed by Raman spectroscopy and thermogravimetric analysis. Furthermore, the HCl-purified MWCNT provided more available reaction sites, leading to enhanced sidewall functionalization. The sidewall of HCl-purified MWCNT was further functionalized with the –COOH moiety by HNO3 oxidation. This process resulted in four distinct MWCNT: raw, purified, –COOH-terminated raw MWCNT, and –COOH-terminated purified MWCNT. Freshly isolated alveolar macrophages from C57Bl/6 mice were exposed to these nanomaterials to determine the effects of the surface chemistry on the bioactivity in terms of cell viability and inflammasome activation. Inflammasome activation was confirmed using inhibitors of cathepsin B and Caspase-1. Purification reduced the cell toxicity and inflammasome activation slightly compared to raw MWCNT. In contrast, functionalization of MWCNT with the –COOH group dramatically reduced the cytotoxicity and inflammasome activation. Similar results were seen using THP-1 cells supporting their potential use for high-throughput screening. This study demonstrated that the toxicity and bioactivity of MWCNT were diminished by removal of the Ni contamination and/or addition of –COOH groups to the sidewalls.

Two-week Toxicity of Multi-walled Carbon Nanotubes by Whole-body Inhalation Exposure in Rats

To evaluate pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs), F344 rats of both sexes were exposed by inhalation to 0.2, 1 or 5 mg/m3 MWCNT aerosol for 6 h/day, 5 days/week for 2 weeks using a whole-body exposure system. At the end of the 2-week exposure period, one-half of the rats were necropsied, and at the end of an additional 4-week postexposure period, the remaining rats were necropsied. MWCNTs were deposited in the lungs of all MWCNT-exposed groups and mostly remained in the lungs throughout the 4-week postexposure period. Granulomatous changes in the lung were found in the rats exposed to 5 mg/m3 MWCNTs, and these changes were slightly aggravated at the end of the 4-week postexposure period. In the bronchoalveolar lavage fluid (BALF), the numbers of neutrophils, percentages of bi- and multinucleated alveolar macrophages, levels of ALP activity and concentrations of total protein and albumin were elevated in the rats exposed to 1 and 5 mg/m3 MWCNTs. At the end of the 4-week postexposure period, the values of the BALF parameters tended to remain elevated. In addition, goblet cell hyperplasias in the nasal cavity and nasopharynx were observed in the rats exposed to 1 and 5 mg/m3 MWCNTs, but these lesions had largely regressed by the end of the postexposure period. Based on the histopathological and inflammatory changes, the no-observed-adverse-effect level (NOAEL) for inhalation of MWCNTs for 2 weeks was 0.2 mg/m3.

Toxicological effects of multi-walled carbon nanotubes adsorbed with nonylphenol on earthworm Eisenia fetida

The high surface area of multi-walled carbon nanotubes (MWCNTs) tends to adsorb a large variety of toxic chemicals, which may enhance the toxicity of both MWCNTs and chemicals to organisms. In order to evaluate the combined toxicity of nonylphenol (NP) and MWCNTs to the earthworm Eisenia fetida in soil, artificial soil systems containing distilled water, 0.1 g kg(-1) MWCNTs, 1 g kg(-1) MWCNTs, 1 g kg(-1) MWCNTs absorbed 5 mg kg(-1) NP, and 10 mg kg(-1) NP alone were prepared and exposed to earthworms for 7 days. Antioxidative responses, and activities of cellulase, Na(+), K(+)-ATPase and acetylcholinesterase (TChE) as well as DNA damage were chosen as toxicological endpoints. The results showed that 1 g kg(-1) MWCNTs adsorbed 5 mg kg(-1) NP from the soil which caused much more adverse effects on the earthworms than each chemical alone, evident from the responses of cellulase, Na(+), K(+)-ATPase and comet assay. This study indicated that MWCNTs facilitated the bioavailability of NP to the earthworm and increased the harmful effects of NP.

Crucial role of the biological barrier at the primary targeted organs in controlling the translocation and toxicity of multi-walled carbon nanotubes in the nematode Caenorhabditis elegans

Multi-walled carbon nanotubes (MWCNTs) can be translocated into the targeted organs of organisms. We employed a model organism of the nematode Caenorhabditis elegans to investigate the role of a biological barrier at the primary targeted organs in regulating the translocation and toxicity formation of MWCNTs. A prolonged exposure to MWCNTs at predicted environmental relevant concentrations caused adverse effects associated with both the primary and secondary targeted organs on nematodes. The function of PEGylated modification in reducing MWCNTs toxicity might be mainly due to the suppression of their translocation into secondary targeted organs through the primary targeted organs. A biological barrier at the primary targeted organs contributed greatly to the control of MWCNTs translocation into secondary targeted organs, as indicated by functions of Mn-SODs required for prevention of oxidative stress in the primary targeted organs. Over-expression of Mn-SODs in primary targeted organs effectively suppressed the translocation and toxicity of MWCNTs. Our work highlights the crucial role of the biological barrier at the primary targeted organs in regulating the translocation and toxicity formation of MWCNTs. Our data also shed light on the future development of engineered nanomaterials (ENMs) with improved biocompatibility and design of prevention strategies against ENMs toxicity.

Pulmonary Inflammation of Well-Dispersed Multi-Wall Carbon Nanotubes Following Intratracheal Instillation: Toxicity by Fiber of 1–5 µm in Length

The pulmonary toxicity of multi-wall carbon nanotubes (MWCNT) were examined by intratracheal instillation. We prepared a well-dispersed MWCNT dispersion including MWCNTs of 3.71 µm geometric average length. The fiber length of most of the MWCNTs in the dispersion was 10 µm or less. The MWCNT dispersion was administered to rat lung by single intratracheal instillation at doses of 0.2 mg and 0.6 mg/rat. Bronchoalveolar lavage fluid (BALF) was collected at 3 days, 1 week, 1 month, 3 months, and 6 months after instillation. The influences of the longer MWCNTs on the induction of inflammation and oxidative stress were examined by the number of neutrophils, cytokine induced neutrophil chemoattractant-1 (CINC-1), CINC-2, CINC-3 and HO-1 in the BALF. Additionally, ho-1 gene expression in the lung was examined. The intratracheal instillation of MWCNT induced transient inflammation dose dependently in the lung. The number of neutrophils was highest at 3 days after instillation and then decreased. However, the neutrophils in the MWCNT administered animals tended to be higher than in the control group until 3 months after instillation. The CINC-1 and CINC-2 concentrations in the BALF increased at 1 month after instillation. There were no significant differences in CINC-3 and HO-1 between the MWCNT administered animals and the control animals. These results revealed that the MWCNTs of 1–10 µm in length induced persistent inflammation in rat lung. There were no remarkable differences between the MWCNTs in the present study and previously reported, shorter MWCNTs prepared from “the same” raw MWCNT material.

Evaluation of reproductive and developmental toxicity of multi-wall carbon nanotubes in pregnant mice after tail vein administration

Evaluation of reproductive and developmental toxicity of multi-wall carbon nanotubes in pregnant mice after tail vein administration

Water-Soluble Taurine-Functionalized Multi-Walled Carbon Nanotubes Induce Less Damage to Mitochondria of RAW 264.7 Cells

Multi-walled carbon nanotubes (MWCNTs) are increasingly used and many studies have already showed their cytotoxicity in different kinds of cell lines, while their mechanism is not fully understood. Mitochondria are the relatively sensitive organelle in response to the stress challenged by the exogenous chemicals. The study aimed to evaluate mitochondrial injury and cytotoxicity in mouse peritoneal macrophage cell line (RAW 264.7) induced by the water-soluble taurine functionalized MWCNTs (tau-MWCNTs) and pristine raw MWCNTs (raw-MWCNTs). The different concentrations including 0, 5, 20, 40 and 80 microg/ml for both kinds of MWCNTs were adopted. The cytotoxicity and the mitochondrial function were assessed after 12 h and 24 h treatment. The results revealed that both MWCNTs could produce significantly dose-dependent cytotoxicity and mitochondrial injury including the decrease of mitochondrial membrane potential, the release of cytochrome c from mitochondria and the increase of cytoplasm Ca2+ content. The activities of adenosine triphosphatases (ATPase) and succinodehydrogenase (SDH) of mitochondria were all inhibited after MWCNTs treatment. In conclusion, MWCNTs could lead to mitochondrial injury, which might be one possible pathway of apoptotic cell induction. The findings that tau-MWCNTs exhibited less toxicity compared with raw-MWCNTs support a useful approach to reduce the toxicity of MWCNTs by surface-functionization with taurine.

Systematic and Quantitative Investigation of the Mechanism of Carbon Nanotubes’ Toxicity toward Algae

Concurrent with the increasing production and application of carbon nanotubes (CNTs) comes an increasing likelihood of CNTs presenting in the aquatic environment, and thereby potentially threatening aquatic organisms via toxic mechanisms that are, at present, poorly understood. This study systematically investigated the toxicity of three multiwalled CNT (MWCNT) samples toward a green alga (Chlorella sp.), focusing on examining and quantifying the contributions of five possible mechanisms to the algal growth inhibition. The results showed that the MWCNTs significantly inhibited the algal growth. The contribution of metal catalyst residues in the MWCNTs to the algal growth inhibition was negligible, as was the contribution from the MWCNTs' adsorption of nutrient elements. The algal toxicity of MWCNTs could mainly be explained by the combined effects of oxidative stress, agglomeration and physical interactions, and shading effects, with the quantitative contributions from these mechanisms depending on the MWCNT size and concentration. At MWCNT concentrations around 96 h IC(50), the oxidative stress accounted for approximately 50% of the algal growth inhibition, whereas the agglomeration and physical interactions, and the shading effects each took approximately 25% of the responsibility.

In vitro toxicity of multi-walled carbon nanotubes in C6 rat glioma cells

The present study aimed to evaluate the potential toxicity and the general mechanism involved in multi-walled carbon nanotubes (MWCNT)-induced cytotoxicity in C6 rat glioma cell line. Two kinds of MWCNT, which were coded as MWCNT1 (measured 10–20nm in diameter and 2μm in average length) and MWCNT2 (measured 40–100nm in diameter and 10μm in average length), were used in this study. To elucidate the possible mechanisms of cytotoxicity induced by MWCNT, MTT assay and flow cytometry analysis for apoptosis and cell cycle, MDA and SOD assays for oxidative stress were quantitatively assessed. The exposure of C6 rat glioma cells to different sizes of two kinds of carbon nanotubes at concentrations between 25 and 400μg/ml decreased the cell viability in a concentration- and time-dependent manner. The exposure of C6 rat glioma cells to MWCNT (200–400μg/ml) resulted in a concentration dependent cell apoptosis and G1 cell cycle arrest, and increased the level of oxidative stress. Results demonstrate that smaller size of MWCNT seems to be more toxic than that of larger one. MWCNT-induced cytotoxicity in C6 cells is probably due to the increased oxidative stress.