Single-Walled Carbon Nanotubes Attenuate Cytotoxic and Oxidative Stress Response of Pb in Human Lung Epithelial (A549) Cells

Combined exposure of single-walled carbon nanotubes (SWCNTs) and trace metal lead (Pb) in ambient air is unavoidable. Most of the previous studies on the toxicity of SWCNTs and Pb have been conducted individually. There is a scarcity of information on the combined toxicity of SWCNTs and Pb in human cells. This work was designed to explore the combined effects of SWCNTs and Pb in human lung epithelial (A549) cells. SWCNTs were prepared through the plasma-enhanced vapor deposition technique. Prepared SWCNTs were characterized by x-ray diffraction, x-ray photoelectron spectroscopy, scanning electron microscopy, and dynamic light scattering. We observed that SWCNTs up to a concentration of 100 µg/mL was safe, while Pb induced dose-dependent (5–100 µg/mL) cytotoxicity in A549 cells. Importantly, cytotoxicity, cell cycle arrest, mitochondrial membrane potential depletion, lipid peroxidation, and induction of caspase-3 and -9 enzymes following Pb exposure (50 µg/mL for 24 h) were efficiently attenuated by the co-exposure of SWCNTs (10 µg/mL for 24 h). Furthermore, generation of Pb-induced pro-oxidants (reactive oxygen species and hydrogen peroxide) and the reduction of antioxidants (antioxidant enzymes and glutathione) were also mitigated by the co-exposure of SWCNTs. Inductively coupled plasma-mass spectrometry results suggest that the adsorption of Pb on the surface of SWCNTs could attenuate the bioavailability and toxicity of Pb in A549 cells. Our data warrant further research on the combined effects of SWCNTs and Pb in animal models.


S1.2. MTT assay
Cell viability was measured by MTT assay [2] with some specific modifications [3]. In brief, 20,000 cells/well seeded in a 96-well plate and allowed for 24 h to attach on the surface of plate. Then, cells were treated for 24 h to different concentrations of SWCNTs (0-200 µg/ml) and Pb (0-100 µg/ml). For combined cytotoxicity study, cells were exposed for 24 to either SWCNTs (10 µg/ml) or Pb (50 µg/ml) or combination of both (10 µg/ml SWCNTs and 50 µg/ml Pb). At the end of exposure time, culture medium was removed from each well to avoid interference of SWCNTs and replaced with new medium containing MTT solution in an amount equal to 10% of culture volume. The 96-well plate is now incubated for 3 h at 37 °C until a purple colour formazan product was developed. The resulting formazan product was dissolved in acidified isopropanol. Further, 96-well plate was centrifuged at 2500×g for 5 min to settle down the SWCNTs, if present in the solution. Then, a 100 µl supernatant was transferred to other fresh wells of 96-well plate and absorbance was measured at 570 using a microplate reader (Synergy-HT, BioTek, Vinnoski, VT, USA).

S1.3. Cell cycle analysis
Cell were exposed for 24 h either SWCNTs (10 µg/ml) or Pb (50 µg/ml) or mixture of both (SWCNTs+Pb). After the completion of exposure time, control and treated cells were harvested and centrifuged at 2500×g for 5 min to get cell pellets. Further, cell pellets were resuspended in 500 µl of phosphate buffer saline (PBS) and fixed with equal volume of chilled 70% ice-cold ethanol, and incubated at 4 °C for 1 h. After two successive washes with PBS cell pellets were re-suspended in PBS and stained with 50 µg propidium iodide (PI)/ml containing 0.1% Triton X-100 and 0.5 mg/ml RNAase A for 1 h at 30 °C in the dark.
Fluorescence of the PI was measured by flow cytometry (Beckman Coulter, Coulter Epics XL/Xl-MCL, Miami, USA) through a FL4 filter (585 nm) and 10,000 events were acquired.
The data were analyzed by Coulter Epics XL/XL-MCL, System II Software, Version 3.0. Cell debris was characterized by a low FSC/SSC was excluded from the analysis [4].

S1.4. Assay of caspase-3 and caspase-9 enzymes activity
Activity of caspase-3 and -9 enzymes was assayed using commercial kits (BioVision, Milpitas, CA, USA). This assay is based on the principle that activated caspases in apoptotic cells cleave the synthetic substrates to release free chromophore p-nitroanilide (pNA), which was recorded at 405 nm. The pNA produced after specific action of caspase-3 and caspase-9 on tertrapeptide substrates were DEVD-pNA and LEHD-pNA, respectively. In brief, reaction mixture consisted of 50 µl of control and treated cell extract protein (50 µg), 50 µl of 2X reaction buffer (containing 10 mM dithiothreitol) and 5 µl of 4 mM DEVD-pNA (for caspase-3) or LEHD-pNA (for caspase-9) substrate in a total volume of 105 µl. The reaction mixture was incubated at 37 °C for1 h and absorbance of the product was measured using a microplate reader (Synergy-HT, BioTek) at 405 nm according to manufacturer's instruction.

S1.5. Mitochondrial membrane potential assay
Mitochondrial membrane potential (MMP) was quantified using rhodamine-123 probe (Rh-123, Sigma-Aldrich) [5]. Cell were exposed either SWCNT (10 µg/ml) or Pb (50 µg/ml) or mixture of both (SWCNTs+Pb) for 24 h. Briefly, 20,000 cells/well seeded in a 96-well plate and allowed for 24 h to attach on the surface. After the completion of exposure time, cells were harvested and washed twice with PBS. Cells were further exposed with 10 µg/ml of Rh-123 probe for 1 h at 37 °C in the dark. Again, cells were washed with PBS and fluorescent intensity of Rh-123 was measured at a microplate reader (Synergy-HT, BioTek).

S1.6. Reactive oxygen species generation assay
Intracellular ROS generation was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) as reported earlier [5]. ROS level was estimated through two procedures; quantitative analysis and microscopic fluorescence imaging. For quantitative assay, cells (20000cells/well) were seeded in 96-well black-bottomed culture plates and allowed to adhere for 24 h in a CO2 incubator at 37 °C. Further, cells were exposed to either SWCNT (10 µg/ml)

S1.7. Preparation of crude cell extract
For the assay of Malondialdehyde (MDA) level, glutathione (GSH) level, and activity of several antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) cell extract was prepared. In brief, cells were cultured in 75-cm 2 culture flask and exposed either SWCNT (10 µg/ml) or Pb (50 µg/ml) or mixture of both

S1.8. Malondialdehyde estimation
Malondialdehyde (MDA), an end product of lipid peroxidation was estimated using procedures of Ohkawa et al [6]. In brief, a mixture of 0.1 ml cell extract and 1.9 ml of 0.1 M sodium phosphate buffer (pH 7.4) was incubated at 37 °C for 1 h. Then, mixture was precipitated with 5% trichloroacetic acid (TCA) and centrifuged (2500×g) for 15 min to collect supernatant. Furthermore, 1.0 ml of 1% thiobarbituric acid (TBA) was added to the supernatant and placed in the boiling water for 15 min. After cooling to room temperature absorbance of the mixture was taken at 532 nm and was converted to MDA and expressed in nmole MDA/mg protein using molar extinction coefficient of 1.56 ×10 5 M -1 cm -1.

S1.9. Glutathione assay
The glutathione (GSH) level was quantified using Ellman's method [7]. In brief, a mixture of 0.1 ml of cell extract and 0.9 ml of 5% TCA was centrifuged (2500×g) for 15 min at 4 °C. Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01% 5,5'-dithiobis-(2nitrobenzoic acid (DTNB) and the reaction was monitored at 412 nm. The amount of GSH was expressed in terms of nanomole/mg protein.

S1.11. Catalase enzyme assay
Enzymatic activity of catalase was assayed according to the protocol of Sinha et al [9].
In this method, dichromate in acetic acid is reduced to chromic acetate in the presence of H2O2.
Briefly, reaction mixture (150 µl) contained 100 µl of 0.01M phosphate buffer, 10 µl cell extract, and 40 µl of 2M H2O2 was prepared. The reaction was stopped by mixing of 200 µl of dichromoacetic acid reagent (5% of potassium dichromate and glacial acetic acid in 1:3 ratio) and absorbance was recorded at 530 nm.

S1.11. Interaction of Pb and SWCNTs in culture medium
Adsorption of Pb on the surface of SWCNTs in complete cell culture medium (DMEM+10%FBS) was determined by inductively coupled plasma mass spectrometry (ICP-MS) [10]. Briefly, samples were categorized into three groups (n=3); Pb group (50 μg/ml Pb in culture medium), co-exposure group (10 μg/ml SWCNTs and 50 μg/ml Pb in culture medium) and control group (only culture media). All three samples were incubated for 0 and 24 h with gentle shaking. Then, supernatant was collected after high speed centrifugation.
Supernatants were further digested with nitric acid. The digested solution was further dissolved in 4% nitric acid and Pb content was measured by ICP-MS. The adsorbed amount of Pb on the surface of SWCNTs was equal to decreased level of Pb in supernatant over 24 h.

S1.12. Effect SWCNTs on cellular uptake of Pb
Effect of SWCNTs on cellular uptake of Pb was also measured by ICP-MS [10].
Briefly, 20000 cells/well cultured in 96-well plate and allowed 24 h to attach on the surface.
Then, cells were exposed for 24 to 50 μg/ml of Pb with or without SWCNTs (10 μg/ml). After the completion of exposure time, cells were washed several times with PBS and harvested.
Then, harvested cells were digested in nitric acid. The digested solution was further dissolved in 4% nitric acid and Pb content was measured by ICP-MS. The intracellular level of Pb was presented in the unit of picogram (pg) per cell. Figure S1 showed the attenuating effects of different concentrations of SWCNTs (1-200 µg/ml) against Pb-induced cytotoxicity (50 µg/ml) in A549 cells. We can see that SWCNTs at a concentration of 10 µg/ml achieved maximum attenuation effect against 50 µg/ml Pb induced cytotoxicity in A549 cells. Above the concentration of 10 µg/ml SWCNTs attenuating effects against Pb induced toxicity was not much different. This indicated that 10 µg/ml SWCNTs were enough to adsorbed most of Pb (50 µg/ml) present in the culture media.