Figure 2.

Oxidative stress is involved in nickel-induced apoptosis. (A, B) Nickel mainly induces H₂O₂ generation, not O₂^•−. BEAS-2B (4 × 10⁵) cells were seeded in a 60 mm dish overnight and then treated without or with different concentrations of Ni₃S₂ for 48 h. The cells were labeled with CM-H₂DCFDA and DHE, respectively, followed by flow cytometry as described in the Materials and Methods. The rightward shift of the overlay reflected the ROS generation. Illustrated overlays are representatives of at least three separate experiments. Quantifications of both H₂O₂ and O₂^•− generation are displayed on the right panel. Each bar represents the mean ± SE of the three independent experiments. *P < 0.05 as determined by the treatment vs control. (C, D) NAC and catalase inhibited nickel-induced ROS generation. BEAS-2B cells (4 × 10⁵) were seeded in a 60 mm dish overnight and then pretreated with or without NAC (10 mM) or catalase (2000 units) for 2 h, respectively, and then treated without or with 2 μg/cm² Ni₃S₂ for 48 h. The cells were stained with CM-H₂DCFDA and measured by flow cytometry. Overlays shown here are representatives of at least three separate experiments. Quantifications of H₂O₂ generation are displayed on the right panel. Each bar represents the mean ± SE of the three independent experiments. *P < 0.05 between the indicated two groups. (E) Vitamin E ameliorated nickel-induced ROS generation. BEAS-2B cells (4 × 10⁵) were seeded in a 60 mm dish overnight and then pretreated with or without vitamin E (20 μM) for 2 h and then treated without or with 2 μg/cm² Ni₃S₂ for 48 h. The cells were stained with CM-H₂DCFDA and measured by flow cytometry. The overlay shown here is representative of at least three separate experiments. Quantifications of H₂O₂ generation are displayed on the right panel. Each bar represents the mean ± SE of the three independent experiments. *P < 0.05 between the indicated two groups. (F) NAC attenuated nickel-induced apoptosis. BEAS-2B cells (4 × 10⁵) were seeded in a 60 mm dish overnight, then pretreated with or without NAC (10 mM) for 2 h, and then treated without or with 4 μg/cm² Ni₃S₂ for 48 h. The cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry as described in the Materials and Methods. The results shown are the means of triplicate determinations ± SE. *P < 0.05 as determined by the indicated two groups. (G) Nickel decreased the protein expression of catalase, not Cu/Zn SOD or Mn SOD. BEAS-2B cells were treated without or with various concentrations of Ni₃S₂ for 48 h as indicated and then measured by Western blot assay. Four more additional experiments achieved equivalent results. Band densities of catalase in the Western blots were normalized against β-actin (shown on right panel). Each bar represents the mean ± SE of the three independent experiments. All means marked with * (P < 0.05) are significantly different from the control.