Unique physicochemical properties of carbon nanomaterials (CNMs) have opened a new era for therapeutics and diagnosis (known as theranostics) of various diseases. This exponential increase in application makes them important for toxicology studies. The present study was aimed at exploring the toxic potential of one of the CNMs, that is, bucky tubes (BTs), in human lung adenocarcinoma (A549) cell line. BTs were characterised by electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform spectroscopy (FTIR), and X-ray diffraction (XRD). Flow cytometric study showed a concentration and time dependent increase in intracellular internalization as well as reduction in cell viability upon exposure to BTs. However, a significant increase in intracellular reactive oxygen species (ROS) production was observed as evident by increased fluorescence intensity of 2′,7′-dichlorofluorescein (DCF). BTs induced oxidative stress in cells as evident by depletion in glutathione with concomitant increase in lipid peroxidation with increasing concentrations. A significant increase in micronucleus formation and apoptotic cell population and loss of mitochondrial membrane potential (MMP) as compared to control were observed. Moreover, in the present study, BTs were found to be mild toxic and it is encouraging to conclude that BTs having outer diameter in the range of 7–12 nm and length 0.5–10
Nanotechnology has developed tremendously in past few decades which lead to the discovery and production of various nanomaterials (NMs), that is, metallic (silver and gold) and metallic oxide nanoparticles (ZnO and TiO2), quantum dots and carbon nanomaterials. These NMs find potential applications in diverse areas including physical, chemical, and biological sciences such as cosmetics, food packaging industries, electronics, medicines, and biomedical engineering [
Carbon nanotubes (CNTs) are the distinct form of carbon based nanomaterials (CNMs) comprised of single or concentrically stacked graphene sheets rolled seamlessly, showing astonishing structural and physicochemical properties. These have been explored for diverse applications ranging from electronics to biomedical applications in applied sciences such as nanoinjectors, tissue engineering, drug delivery, gene therapy, and biosensor technology [
An occupational survey on exposure of engineered nanoparticles suggested that the workers got chronic obstruction and pulmonary and cardiac diseases due to exposure of nanoparticles [
According to previous reports, MWCNTs were found to be immunotoxic when exposed to murine macrophages for 16 h, 24 h, and 32 h (0–100
Several groups suggested that inhalation is the primary route of exposure of MWCNTs and hence induces pulmonary toxicity [
Therefore, keeping in view the above mentioned reports regarding pulmonary toxicity, present study was designed to examine the toxic effect of bucky tubes [BTs, a type of multiwall carbon nanotubes] on human lung alveolar cell lines (A549).
Bucky tubes (CAS number 308068-56-6), propidium iodide (PI), 2,7-dichlorofluorescein diacetate (DCFDA) dye, 5,5′,6,6′-tetrachloro-1,1′3′3′-tetraethylbenzimidazolecarbocyanine iodide (JC-1) dye, ethidium bromide (EtBr), Triton X-100, 5,5′-dithiobisnitrobenzoic acid (DTNB), and glutathione (GSH) were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). Phosphate buffered saline (Ca+2, Mg+2 free; PBS), Dulbecco’s modified eagle medium : nutrient mixture F-12 (Ham) (1 : 1) powder (DMEM F-12), trypsin-EDTA, fetal bovine serum (FBS), trypan blue, antibiotic, and antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin, and 25
The particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform spectroscopy (FTIR), and X-ray diffraction (XRD).
Electron microscopy was carried out for the assessment of morphology and size of BTs. Samples were prepared by suspending BTs in Milli-Q water at a concentration of 25
Bucky tubes were suspended in complete growth medium, that is, DMEMF-12 supplemented with 10% fetal bovine serum (FBS) at a concentration of 150
FTIR analysis of BTs has been performed with the scan range 400–4000 cm−1 at the resolution of 8 cm−1 using ATR accessory on Agilent Cary 630 FTIR spectrometer.
Powder X-ray diffraction of BTs has been done using Rigaku Miniflex-II bench top X-ray diffractometer with tube voltage of 30 KV.
The human lung epithelial cells (A549) were purchased from the National Centre for Cell Sciences (NCCS), Pune, India, and maintained in DMEMF-12 (1 : 1) medium supplemented with 10% heat inactivated FBS, 0.2% sodium bicarbonate, and 1% antibiotic and antimycotic solution at 37°C under a humidified atmosphere of 5% CO2.
A549 cells were cultured in complete medium having all supplements and were harvested at 80–85% confluency using 0.25% trypsin-EDTA solution and were seeded at a density of 1 × 104 cells/mL/well in a flat bottom 96-well plate, 1 × 105 cells/mL/well in a 12-well plate, and 2 × 105 cells/mL in a 6-well plate and culture flasks according to the need of the experiment. After 22 h of seeding, cells were incubated with varying concentrations of BTs (1, 10, 25, 50, and 100
The uptake of BTs using flow cytometry was measured according to the method of Suzuki et al. [
Cytotoxicity of BTs was determined by trypan blue dye exclusion assay and propidium iodide (PI) staining assay.
MMP was determined by using fluorescent, lipophilic cationic carbocyanine 5,5′,6,6′-tetrachloro-1,1′3′3′-tetraethylbenzimidazolecarbocyanine iodide (JC-1) dye which exhibits dual fluorescence emission depending upon the membrane potential state of mitochondria. Exposed cells were harvested and washed with 1x PBS and then incubated with 10
Apoptosis on exposure of BTs was measured by using Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) as per the manufacturer’s protocol. Briefly, treated cells were harvested; cell pellet was washed with 1x PBS and resuspended in 100
Flow cytometric analysis of micronucleus formation (MN) was done according to the method of Pandey et al. [
Effect of BTs on cell cycle progression was determined by using flow cytometer. Briefly, the treated cells were harvested and fixed in 70% ethanol for 30 min at 4°C and then lysed with 0.2% triton X-100 for 30 min at 4°C. After lysis, cells were incubated for 30 min in dark at 37°C in RNaseA (10 mg/mL) and finally stained with PI (1 mg/mL) for 60 min at 4°C. Then, the flow cytometric analysis was performed using BD FACS Canto II flow cytometer equipped with FACS Diva software (version 6.1.2, BD Biosciences) and results were expressed in percentage cell population in different phases of cell cycle.
All assays were done in three independent sets of experiments and results were expressed as mean ± SEM. Data of treated cells were compared with their respective controls and were analysed using one way analysis of variance (ANOVA) with Dunnett post hoc test to determine significance. In all cases
BTs were first analysed by TEM to assess the particle morphology and size. TEM analysis shows that the particles were tubular in shape with an average (bundle diameter) size of ~37.2 nm at scale bar of 100 nm (Figure
Characterization of BTs: (a) TEM photomicrograph: TEM analysis revealed that BTs were tubular/rod shaped having ~37.2 nm average (bundle) size at scale bar of 100 nm. (b) FTIR analysis revealed the presence of characteristic peaks corresponding to two-dimensional carbon nanostructures. (c) XRD pattern of BTs.
Further, the mean hydrodynamic diameter and zeta potential of BTs in cell culture medium DMEM F-12 supplemented with 10% FBS were estimated using DLS and found to be in range of 179.7 nm and 12.9 mV, respectively, with polydispersity index (PdI) 0.354 (Table
Characterisation of BTs by DLS.
S. Number | Medium | Hydrodynamic diameter | PdI | Zeta potential |
---|---|---|---|---|
1 | Culture medium (DMEM F-12 supplemented with 10% FBS) | 179.7 nm | 0.354 | 12.9 mV |
FT-IR spectra of BTs exhibited the presence of characteristic peaks at ~2600 cm−1, 2325 cm−1, 1111 cm−1, and 465 cm−1 corresponding to the two-dimensional carbon nanostructures (Figure
X-ray diffraction pattern of BTs taken in the 2
The cellular uptake of BTs was assessed by flow cytometry. The change in side scattering (SSC) mean and forward scattering (FSC) mean represents the relative change in granularity and size of the cell, respectively. There was an increase in SSC mean with the increase in concentration at 1, 10, 25, 50, and 100
Internalization of BTs in A549 cells after 6 h and 24 h exposure. Increase in SSC mean correlates with the increased granularity of cells which was used as a marker for internalization of NPs. Values represent mean ± SEM of three independent experiments. (
For determining cytotoxicity by trypan blue dye exclusion assay and PI staining assay, cells were exposed to varying concentration (1
Cytotoxic effect of BTs NPs in A549 cells by (a) trypan blue dye exclusion assay using automatic cell counter and (b) propidium iodide (PI) uptake method in which cells were stained with PI and analysed by flow cytometer after 6 h and 24 h exposure. Results were expressed as the percentage cell death after exposure of BT relative to control cells and were represented as mean ± SEM of three independent experiments. (
A549 cells were loaded with 2′,7′-dichlorodihydrofluorescein diacetate and it was observed that BTs induced ROS generation in both time and concentration dependent manner which was revealed from increase in DCF fluorescence. The fluorescence intensity increased from 101.71%, 106.37%, 119.21%, and 142.49% after 1 h and 109.77%, 130.12%, 144.55%, and 202.43% after 3 h exposure of BTs as compared to control. The fluorescence intensity was decreased from 95.7%, 120.98%, 137.11%, and 195.23% after 6 h and 89.58%, 109.57%, 100.29%, and 126.32% after 24 h exposure of BTs at concentrations (10–100
Effect of BTs NPs on (a) induction of intracellular reactive oxygen species (ROS), (b) cellular level of glutathione (GSH), and (c) lipid peroxidation (LPO) in A549 cells. The % ROS generation of the control cells was considered 100% and fluorescence intensity was measured by microplate reader while glutathione level and lipid peroxidation were measured spectrophotometrically. Data represents mean ± SEM of three independent experiments. (
Cells treated with BTs showed a statistically significant (
Flow cytometric detection of change in mitochondrial membrane potential (MMP) in A549 cells exposed to BTs using JC-1 dye. (a) Representative bar graph of three independent experiments analysed. (b) Representative dot plots. Data represent mean ± SEM of three experiments. (
The cell population of interest was gated on the basis of untreated control stained cells. These were divided into four quadrants and were analysed as FITC negative and PI negative (viable normal cells), FITC positive and PI negative (early apoptotic), FITC positive and PI positive (late apoptotic), and FITC negative and PI positive (necrotic). It was observed that there was increase in percentage of FITC− and PI+ population and decrease in FITC+ PI+ and FITC+ PI- population with increasing concentration which suggest that at lower concentration BTs cause apoptosis in A549 cells and at higher concentration cause necrosis (Figures
BTs induced cell death in A549 cells analysed by flow cytometry. Data represent mean ± SEM of three experiments. (a) Representative bar graph. (b) Dot plots. (
A statistically significant (
Flow cytometric detection of chromosomal damage by micronucleus formation after 3 h and 6 h of exposure. Data represent mean ± SEM of three experiments. (
Flow cytometric analysis of cell cycle of A549 cells exposed to BTs for 24 h revealed that there was a significant concentration dependent increase in cells present in sub-G1 phase (4.4%, 2.8%, 3.7%, 5.3%, and 8.3%) in comparison to control (2.6%) while there is no significant change in percentage of cells in G1, S, and G2/M phase of cell cycle which revealed that there was increase in apoptotic cell population (Figures
Flow cytometric analysis of cell cycle of A549 cells exposed to BTs for 24 h. (a) Bar graph representing cell population in different phases of cell cycle and (b) representing % cell population in sub-G1 phase which ultimately represents apoptotic population and (c) representative histograms. (
MWCNTs are produced in huge amount and exponentially applied in almost all sectors. These increasing applications necessitate the evaluation of risk of MWCNTs exposure and their adverse health effects.
Therefore, present study was designed to evaluate the toxicity of BTs (type of MWCNTs) and add to the present knowledge by concluding that BTs get internalized and cause cellular toxicity. It causes oxidative stress by generation of intracellular ROS which induce lipid peroxidation that leads to imbalance in level of antioxidants. It has been also observed that BTs exposure causes mitochondrial dysfunctioning, chromosomal damage, and cell cycle arrest for which oxidative stress may be one of the possible reasons.
Prior to investigating the
Another method used was DLS which characterize the particle in the cell culture medium which was used for treatment of BTs to A549 so as to characterise the particle by simulating the culture conditions. The mean hydrodynamic diameter and zeta potential of BTs in culture medium obtained from DLS were in the range of 179.7 nm and 12.9 mV, respectively, at physiological pH which was more than the size reported (7–12 nm) by its commercial supplier (Sigma-Aldrich). The reason behind the vast difference in particle size may be that DLS characterise the particle considering it spherical in shape which is not in the case of BTs as these are tubular in structure. Moreover, difference in particle size may be due to agglomeration which is influenced by intrinsic and extrinsic factors [
A FT-IR spectrum of BTs illustrates the presence of characteristic peaks related to 2-dimensional carbon nanostructures. Stretching at around ~2600 cm−1 has been assigned to C–O stretching that may correspond to the presence of –COOH groups. Several minor peaks appeared due to amorphous content, C–H stretching vibrations (~2325 cm−1, ~1111 cm−1, and ~465 cm−1), and so forth; stretching at ~2100 cm−1 is due to the interference of CO2 during the course of the spectral measurements.
X-ray diffraction pattern of BTs showed the graphitic structure with interlayer spacing of 0.33 nm corresponding to the d002 reflection at 25.6°. Peaks at 42.4°, 53.5°, and 77.2° were attributed to the diffraction of (1 0 0), (0 0 4), and (1 1 0) planes, respectively, which suggested the BTs structural resemblance to the multiwalled carbon nanotubes.
To explore the toxicity potential of BTs, it is necessary to evaluate the internalization of BTs which can be correlated with varying biological responses. The cellular uptake of nanoparticles was considered as a primary method of screening of nanotoxicity by flow cytometer [
After internalization studies, effect of BTs on cellular viability was examined by trypan blue dye exclusion assay and PI assay. It was found that BTs exposure causes a statistically significant decrease in cell viability in lung alveolar cells in both assays at higher concentration at different time points. Several groups have reported that, after internalization, NPs interact with cellular components that induce alteration in cellular/functional responses which ultimately leads to cell death [
Further, to investigate the cause of cytotoxicity, we tried to identify the generation of free radicals which are considered to be the primary cause of toxicity due to exposure of NPs [
It is documented that oxidative stress occurs in a cell or tissue when the concentration of ROS generated exceeds the antioxidant capability of that cell [
In addition, overproduction of ROS may result in damage to critical biomolecules including cellular fatty acids which are readily oxidized to produce lipid peroxyl radicals and lipid hydroperoxides. Lipid peroxyl radicals are subsequently propagated into malondialdehyde (MDA) which is the major carbonyl produced during LPO and potent mutagen and carcinogenic compound. We explored the level of cellular fatty acids and found that there was increase in level of LPO with increasing concentration which represents the increased toxicity due to free radical generation.
Based on a study on wide body of literature it is seen that severe oxidative stress can cause instability in mitochondrial membrane potential, DNA damage, and/or cell death and even moderate oxidation can trigger apoptosis, while more intense stresses may cause necrosis [
Moreover, it can be said that disruption of mitochondrial activity is the distinctive feature in cell death and in accordance with this we assessed cell fate by Annexin V/PI double staining and found that at lower concentration BTs cause apoptosis in A549 cells and at higher concentration causes necrosis. It can be concluded that BTs were mild toxic towards A549 and lead to cell death. Some of the previous reports have shown that mitochondrial dysfunction is activated with accidental cell death (necrosis) or programmed cell death [
Another important outcome of these deleterious reactive radicals is that they can diffuse through membranes and may also interact with cellular DNA and nitrogen bases by forming adducts resulting in a loss of cellular homeostasis [
Further, in response to DNA damage, progression of cell cycle was assessed by PI staining flow cytometrically and it was observed that there was statistically significant increase in apoptotic cell population in exposed cells at 100
Present study demonstrated that BTs get internalized intracellularly to human lung alveolar cells and cause cell death with increasing concentrations and time points which was observed by flow cytometer. Exposure of these nanoparticles causes increased ROS production and lipid peroxidation with concomitant depletion in glutathione level which confirms the induction of oxidative stress which ultimately triggers loss in MMP at 100
The authors declare that there is no conflict of interests.
The authors gratefully acknowledge the funding from CSIR, New Delhi, for EMPOWER scheme (OLP-06) and Network project NanoSHE (BSC0112). Authors are thankful to Dr. L. K. S. Chauhan for his help in TEM imaging.