Multi-walled carbon nanotubes induce cytotoxicity, genotoxicity and apoptosis in normal human dermal fibroblast cells

Abstract

Multi-walled carbon nanotubes (MWCNT) have won enormous popularity in nanotechnology. Due to their unusual, one dimensional, hollow nanostructure and unique physicochemical properties they are highly desirable for use within the commercial, environmental and medical sectors. Despite their wide application, little information is known concerning their impact on human health and the environment. While nanotechnology looms large with commercial promise and potential benefit, an equally large issue is the evaluation of potential effects on humans and other biological systems. Our research is focused on cellular response to purified MWCNT in normal human dermal fibroblast cells (NHDF). Three doses (40, 200, 400 microg/mL) of MWCNT and control (tween-80+0.9% saline) were used in this study. Following exposure to MWCNT, cytotoxicity, genotoxicity and apoptosis assays were performed using standard protocols. Our results demonstrated a dose-dependent toxicity with MWCNT. It was found to be toxic and induced massive loss of cell viability through DNA damage and programmed cell-death of all doses compared to control. Our results demonstrate that carbon nanotubes indeed can be very toxic at sufficiently high concentrations and that careful monitoring of toxicity studies is essential for risk assessment.

Figure1

TEM Structures of COOH Functionalized Carbon Nanotubes. (A) low magnification, (B) high magnification, (C) internal diameter and (D) Photograph showing uptake of carbon nanotube by the cells.

Figure 2

(A) Raman Spectrum of COOH Functionalized Carbon Nanotubes. (B) FTIR Spectrum Data of COOH Functionalized Carbon Nanotubes.

Figure 2

(A) Raman Spectrum of COOH Functionalized Carbon Nanotubes. (B) FTIR Spectrum Data of COOH Functionalized Carbon Nanotubes.

Figure 3

(A) Dose and time-dependent increase in normal human fibroblast lactate dehydrogenase (LDH) by MWCNTs. (B) Dose and time-dependent reduction in Normal Human fibroblast cell viability by MWCNTs. The values were calculated as percent of control (untreated cells) and represent the mean + SD for three independent determinations. Statistically significance is indicated in the figure as (*) for p<0.05.

Figure 3

(A) Dose and time-dependent increase in normal human fibroblast lactate dehydrogenase (LDH) by MWCNTs. (B) Dose and time-dependent reduction in Normal Human fibroblast cell viability by MWCNTs. The values were calculated as percent of control (untreated cells) and represent the mean + SD for three independent determinations. Statistically significance is indicated in the figure as (*) for p<0.05.

Figure 4

(A). Comet assay photographs of NHDF cells exposed to MWCNTs. Representative comet images A= control; B= 40 μg/ml; C= 200 μg/ml; 400 μg/ml MWCNT (B) Genotoxicity of MWCNT in NHDF cells.

Figure 4

(A). Comet assay photographs of NHDF cells exposed to MWCNTs. Representative comet images A= control; B= 40 μg/ml; C= 200 μg/ml; 400 μg/ml MWCNT (B) Genotoxicity of MWCNT in NHDF cells.

Figure 5

Agarose gel electrophoresis of DNA extracted from NHDF untreated and treated cells with different concentrations of MWCNT for 48 hours. Lane 1=control; Lane 2=40 μg/ml; Lane 3=200 μg/ml and Lane 4=400 μg/ml, M=Molecular marker.

Figure 6

MWCNTs induction of apoptosis of NHDF cells. (A) Immunofloresence images of NHDF cells treated for 48 hours with MWCNT. Cells treated with oxidized MWCNT stained positive with annexin V-FITC, A= control; B= 40 μg/ml; C= 200μg/ml; and D: 400μg/ml. (B) Graph shows percentage of annexin V positive cells after incubation for 48 hours with 0, 40, 200 and 400 μg/ml of MWCNT. The values represent mean + SD for three independent experiments. Statistical significance is indicated by (*) in the figure for p<0.05.