Figure 4

Role of ROS production in MWCNT induced toxicity. A) Dose response of ROS production. HBE cells were treated with different doses of MWCNT (1.5-24 μg/mL equivalents 0.37-6.0 μg/cm²) for 24 hours. Hydrogen peroxide (1 mM) was used as positive control. ROS were labeled with HE and analyzed by flow cytometry. At least 10,000 cells/events excluding debris were captured. B) Time course analysis of NF-κB (REL A/P65) phosphorylation in HBE cells after exposure to 24 μg/mL MWCNT for different time points. Cells based ELISA was performed according to manufacturers recommendations. C) Modulation of MWCNT induced cell death with antioxidants (NAC and CAT Peg). Cells were pre-treated with 5 mM NAC or CAT Peg (1000 I.U) (30 minutes) and then exposed to MWCNT (24 μg/mL) for 24 hours in the presence of antioxidants. Cell death analysis was performed using flow cytometry. D) Modulation of MWCNT induced IL-1β production with antioxidants (NAC and CAT Peg) and NF-κB inhibitor (QNZ). Cells were pre-treated with 5 mM NAC, CAT Peg (1000 I.U) (30 minutes) or QNZ (10 nM) and then exposed to MWCNT (24 μg/mL) for 24 hours in the presence of inhibitors. Cytokine production was analyzed by commercially available ELISA. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test. Graphs show average ± SEM of three independent experiments with triplicate of each condition, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (between media-treated control and treatment).