SWCNT (Catalogue Number 652512-G), Rotenone, Glutathione, N-Acetyl Cysteine, Ascorbic acid (Vitamin C), 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Glutathione assay kit were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2,7-dichlorofluoroscein diacetate (DCF-DA) was purchased from Molecular probes (Invitrogen Corp., Carlsbad, CA, USA), Rat lung epithelial cells (RL 65, ATCC; CRL-10354) were purchased from American Type Culture Collection (Manassas, VA). Dulbecco’s minimum essential medium (DMEM), Phosphate buffer saline (PBS), Fetal calf serum were obtained from Gibco (Invitrogen Corp., Carlsbad, CA, USA), Penicillin and Streptomycin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Superoxide dismutase-1 (SOD-1; sc 11407) and superoxide dismutase-2 (SOD-2; sc 18503) antibodies were obtained from Santacruz Biotechnology Inc. (Santacruz, CA, USA).
In our studies, SWCNT particles were suspended in Dimethylformamide (DMF) and sonicated for 5 minutes and henceforth in all control experiments the cells were treated with equivalent volume of DMF.
2.1. Cell Culture and Treatments
Rat lung epithelial cell (LE cells) cultures were maintained in DMEM supplemented with 10% fetal calf serum, Penicillin (100 μg/ml) and Streptomycin (100 μg/ml) under the atmosphere of 5% CO2, 95% air in humidified incubator at 37 °C. The cells were incubated with or without SWCNT or substances in 96 well plates or 6 well plates for time intervals as indicated in the figure legends.
2.2. Measurement of Intracellular ROS
Oxygen radicals collectively called as reactive oxygen species plays a key role in cytotoxicity. Increased ROS levels in cells by chemical compound reflect toxicity and cell death. The level of ROS present in living cells were quantified essentially as described earlier.14
Equal number of rat lung epithelial cells (10,000 number/well in 96 well plate in Hanks Balanced Salt Solution) were treated with 10 μ
M DCF-DA (2,7-dichlorofluoroscein diacetate) for 3 hours. Cells were washed with phosphate buffered saline and treated with different concentrations of SWCNT’s. Following incubation at indicated time intervals the intensity of fluorescence is measured at excitation and emission of wavelength at 485/527 nm, respectively and expressed as fluorescence units.
2.3. MTT Assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, was first described by Mosmann in 1983,15
was performed as described earlier with minor modifications.9
Rat LE cells (2000/well) were cultured in 96 wells plate and were incubated with different concentrations of SWCNT’s for 72 hours at 37 °C. The cells were washed with PBS and MTT at a final concentration (125 μ
g) was added and incubated further for 3 hrs at 37 °C. Then absorbance was measured at 570 nm.
2.4. Effect of Rotenone on SWCNT Induced ROS
The major site of ROS production in a cell is mitochondrial electron transport chain, or the group of enzymes mixed function oxidases that uses cytochrome P450. To determine the role of mitochondria in SWCNT induced ROS production, the LE cells were incubated with rotenone (Sigma) the inhibitor of electron transport chain. Cells were incubated with or without 10 μM rotenone for 3 hours and 24 hours. Then the cells are washed and incubated with SWCNT (10 μg) and DCF-DA for 3 h. The fluorescence was determined as described above.
2.5. Glutathione Assay
Reduced glutathione (GSH) is the major free sulfhydryl groups containing molecule, present in cells and is involved in detoxification of xenobiotics, removal of ROS and maintenance of oxidation state of protein sulfhydryl groups. It is the key antioxidant present in most of the cells. Increased ROS may deplete the concentration of GSH in cells and tissues. The concentration of GSH in cells was measured by glutathione assay kit as per the instructions provided by the manufacturer. In brief equal number of cells were grown in 6 well plates and treated in triplicates with or without SWCNT (10 μg/ml) and incubated for 6 hours at 37 °C. The cells were scraped and homogenized in PBS and deproteinized with 5% 5-sulfosalicylic acid and centrifuged to remove protein precipitate. The supernatant was treated with 5. 5′-dithiobis (2-nitrobenzoic acid; DTNB). GSH reduce DTNB to TNB and oxidized to GSSG. Oxidized GSSG present in cells react with added NADPH to give GSH, which later also reacts with DTNB to give TNB. The total TNB formed is measured by absorption at 412 nm in a spectrophotometer.
2.6. Immuno Blot Analysis of SOD-1 and SOD-2
It is a well known fact that ROS plays an important role in pathophysiology of various diseases from neurological disorders to cancer.16
These constantly produced ROS are scavenged by superoxide dismutase (SOD), glutathione peroxidase, and catalase. SOD specifically processes superoxide anion (O2−
) and produces hydrogen peroxide. Three types of SOD’s have been isolated of which the two major ones SOD-1 and SOD-2 are involved in detoxification of ROS.17
Hence immunoblot analysis of SOD-1 and SOD-2 was done in cell lysate from cells treated with SWCNT for 24 h. Control and SWCNT treated cells were washed with chilled PBS in presence of protease inhibitor and cell extracts were made in PBS by homogenization. Protein concentration was measured by Bradford’s method as described earlier.18
Protein (75 μ
g) was mixed with sample buffer and boiled for 5 minutes and resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nylon membranes. The membranes were then probed with SOD-1 or SOD-2 antibodies, followed by washing thrice with PBS and incubated with second antibody coupled to horse radish peroxidase. To visualize the appearance of the protein bands the membrane was probed with chemiluminiscence reagent by standard procedure as described earlier.9
2.7. Effect of Antioxidants on SWCNT Induced ROS Production
The cells have preventive mechanism to reduce or neutralize the ROS constantly formed. While in the presence of toxic compounds this preventive mechanism is reduced which can be restored by the addition of compounds with known antioxidant activity. The effect of antioxidants also leads to understand the counter measure strategy. LE cells were treated with 1 mM concentrations of glutathione (GSH), N-Acetyl cysteine (NAC), and Vitamin C (Ascorbic acid) for 24 h. These conditioned cells were treated with DCF for 3 h and washed and incubated with or with out SWCNT (10 μg/ml) for additional 3 h. ROS formed in cells were measured by change in DCF fluorescence as described above.