5b and c). Significant 21.6% and 31.8% reductions of internalization were observed in the presence of chlorpromazine in BEAS-2B cells in Ham’s F12 and HBEpCs in SFGM, respectively, and 50.1% and 28.0% reductions were observed in the presence of indomethacin.

Moreover, we assayed cell growth inhibition by using the AB assay to confirm the influence of the endocytosis inhibitors. Both endocytosis inhibitors suppressed the cell growth inhibition mediated by MWNT-7 in BEAS-2B cells in Ham’s F12 and HBEpCs in SFGM (Fig. 5d). Chlorpromazine suppressed MWNT-7 internalization and cell growth inhibition to a higher degree than did indomethacin in BEAS-2B cells in Ham’s F12, and the reverse pattern see more was observed for HBEpC in SFGM. BEAS-2B cells were originally find more established by infection of normal human bronchial epithelial cells with an adenovirus 12-SV40 hybrid virus (Reddel et al.,

1988). Ke et al. reported that in BEAS-2B cells, most cells at clonal density undergo squamous differentiation when incubated in media containing more than 4% serum (Ke et al., 1988). In this study, BEAS-2B cells in Ham’s F12 internalized MWNT-7 and demonstrated a 50% inhibitory concentration that was approximately 10-fold lower than that of BEAS-2B in SFGM, as shown in Fig. 2. This result supports our hypothesis that the culture medium affects cytotoxicity in BEAS-2B cells. Cellular uptake of MWNT-7 by differentiated BEAS-2B cells observed in the presence of fetal bovine serum was lost when the MWNT-7 treatment was performed in SFGM, which indicates that CNT uptake by BEAS-2B

GNA12 cells is not an original property and is induced by FBS (Fig. 2). Moreover, MWNT-7 was again internalized when BEAS-2B cells that had been cultured in SFGM and had thus lost their capacity for MWNT-7 uptake were again cultured in Ham’s F12. Normal HBEpCs in SFGM showed MWNT-7 internalization and growth inhibition identical to the observations in BEAS-2B cells in Ham’s F12 (Fig. 1 and Fig. 3). We also used another line of HBEpCs purchased from a different company and obtained the same result (data not shown). These cells had an ellipsoid phenotype, although the HBEpCs appeared to be cuboidal, and BEAS-2B cells in Ham’s F12 were squamous. In contrast, BEAS-2B cells in SFGM displayed a spindle shape that is typically observed when normal human bronchial epithelial cells differentiate (Zhang et al., 2011). These results cannot be attributed to the increased solubility of CNTs in serum; rather, they are based on functional changes with resulting morphological changes that occur in the presence of serum (Fig. 3). Cytokine secretion also showed a similar pattern in response to CNT internalization. BEAS-2B cells in Ham’s F12 and HBEpC showed increased secretion of IL-6 and IL-8 upon exposure to CNTs, although there was a large difference in IL-6 secretion between cell types. We did not detect secretion of IL-6 in untreated BEAS-2B cells in SFGM (Fig. 4a).