There have been some reports on the toxicity of CNTs using different cell types25
where biological responses, such as cell viability, morphology change and DNA content, were reported to be different according to the types of cells. Recently, we found that the differences in the sensitivity to MWCNTs were caused by the volume ratio between cell and MWCNTs. However, our previous study revealed that U937 human monoblastic leukemia cells do not show a significant cytotoxic response to purified MWCNTs.27
Therefore, in the present study, we examined four different cell lines in order to investigate the difference in biological responses to MWCNTs. The neuroblast was added in order to evaluate neurotoxicity because carbon particles able to cross the blood–brain barrier are known to affect the central nervous system28
and MWCNTs are known to influence a protein associated with neurons.21
We also examined the effect of MWCNTs on cells at different states of differentiation, and the relationship between cytotoxicity and the cellular uptake of MWCNTs.
Our results indicate that MWCNTs uptake is involved in cytotoxicity. We used two colorimetric assays to evaluate the cytotoxicity. Casey et al reported that CNTs could absorb the dye, interfering in cytotoxicity measurements.30
We also confirmed that the cell viability was observed to be about 30% at 100 μg/mL of MWCNTs using the alamar blue assay. However, there was no large difference in LDH assay and alamar blue assay at levels under 50 μg/mL of MWCNTs. The internalization of MWCNTs to MESO-1, BEAS-2B and differentiated THP-1 cells was marked by less than 50% viability and more than 50% plasma membrane permeability after exposure to less than 50 μg/mL of MWCNTs. At that time, these cells internalized large amounts of MWCNTs and peeled off from the dish. We found that the amount of MWCNTs in the cytoplasm was closely related to the decrease in cell viability. However, the cells that endocytosed MWCNTs could divide when the amount of MWCNTs in the cell was small. These cells also internalized SB but did not show any cytotoxicity. It is reported that the internalized carbon nanomaterials aggregate in the lysosomes and vacuoles.11
SB also aggregates in the lysosomes. However, MWCNTs damaged the lysosomes, but SB did not. Disruption of the lysosome may influence these cytotoxic difference between VGCF and SB and the volume pressure of VGCF which were not enclosed in the lysosome, might cause cell death.
According to a review by Raffa et al, the internalization of mainly single-walled CNTs can occur by phagocytosis, endocytosis or diffusion.32
There are three crucial parameters influencing the CNTs interaction with cells: the degree of dispersion, the formation of supramolecular complexes and the nanotube length. Time-lapse microscopy in the present study showed that the degree of MWCNT dispersion did not seem to influence the internalization of MWCNTs by macrophage- like differentiated THP-1 cells by phagocytosis or by mesothelioma origin MESO-1 cells or pulmonary epithelium origin BEAS-2B cells by endocytosis. We used CytoD, an actin barbed-end capping molecule, to inhibit phagocytosis and endocytosis.33
CytoD inhibited the internalization of not only MWCNTs but also SB, which has a particle size of less than 1 μm. Thus, the results of the present study do not support the theory of MWCNT internalization by diffusion. Because the definition of endocytosis generally includes phagocytosis and we could not differentiate between endocytosis and phagocytosis, we assume that the cells internalized MWCNTs via endocytosis in the present study.
The IMR-32 cells and undifferentiated THP-1 cells maintained more than 50% cell viability and they were not injured at 100 μg/mL of MWCNTs. At this concentration, we observed many MWCNT agglomerates on the bottom of the wells, and the IMR-32 and undifferentiated THP-1 cells were adhered to the aggregated MWCNTs rather than to single MWCNT molecules or thin bundled MWCNTs. In particular, the undifferentiated THP-1 cells adhere to VGCF agglomerates, although they are naturally non-adhesive cells, and secreted IL–8. Rosales and Juliano reported that integrin-mediated adhesion induces a set of immediate early genes that are characteristic of monocytic differentiation containing IL-8.34
In fact, THP-1 cells differentiated by PMA secreted IL-8 in our study. Also, considering the fact that the doubling time of IMR-32 cells and undifferentiated THP-1 cells is about 40 hours and 18 hours, respectively, the biological responses of these cells exposed to MWCNTs might not be cytotoxicity, but active responses including cell differentiation and enhancement of the plasma membrane permeability. Recently, we reported that MWCNTs exhibited differential inducibility on C2C12 cells.35
On the other hand, the reasons why these cells adhered in the aggregated SB were an observed slight decrease in cell permeability and increased IL-8 secretion. It may be that the agglomeration of SB was entirely small and had little effect. However, SB slightly increased the permeability of differentiated THP-1 cells. This biological response may be the same mechanism as in the case of the increased plasma membrane permeability of undifferentiated THP-1 cells exposed to MWCNTs. Walker et al reported that carbon black increased the release of LDH by human aortic endothelial cells without a loss of cell viability.36
It appears that the assessment of cytotoxicity by measurement of cell permeability is not suitable when using carbon materials.
We examined whether intracellular ROS production is involved in biological responses (including cytotoxicity) to MWCNTs. Some studies have reported that the production of ROS is critical in the hazardous effects of CNTs,25
while other studies have not supported this relationship.7
Our results were different for each type of cell, but the production of intracellular ROS did not increase; instead, a scavenger effect for intracellular ROS was observed. Iron is used during the catalytic production of CNTs. This iron remains in CNTs as a residue, and the Fenton reaction is elicited, which is said to cause oxidative stress. However, because the iron content in the VGCF that we used in this study was very low, only a small amount of ROS was produced, and the scavenger effect of the CNTs themselves may have appeared. We also assayed the LPS-induced cytokine production by THP-1 cells. LPS induces nuclear factor-κB, which is induced by ROS,40
and it represents a cytokine secretion pattern unlike the VGCF-induced effects on differentiated THP-1 cells. Hence, the MWCNTs-induced secretion of cytokines is not caused by ROS.
We investigated the cytotoxic effects of MWCNTs by examining the differences in the biological responses of different cell lines to MWCNTs. We found that the biological responses to MWCNTs were different in different cell lines, and even the same cell line exhibited different responses according to its differentiation state. Our results indicate that other biological responses (ie, enhancement of cell membrane permeability and increased cytokine production) may be due to direct contact with MWCNTs, and that endocytosis of MWCNTs is the main reason (or onset mechanism) for MWCNTs cytotoxicity. Since the biological responses apart from the cytotoxicity of MWCNTs are active responses, MWCNTs may possibly be used as biomaterials if used appropriately and with caution. In addition, we confirmed that SB as a negative control in the present study did not cause cytotoxicity. These observations suggest that the key to the safe use of CNTs is avoiding endocytosis and subsequent lysosomal and vacuolar injury.