Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

Abstract

It has long been concerned that some secondary air pollutants such as smog components, ozone (O3) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O3, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H2O2 and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca2+ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca2+ concentration responsive to the pulse of PAN, but sensitive to Ca2+ channel blockers, was recorded, indicating that Ca2+ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O3 and PAN.

Figure 1. Yield of PAN signals in n-tridecane as major bands in FTIR analysis. The signals for PAN were extracted from the n-tridecane background signals. The bands at 1830 cm⁻¹, 1727 cm⁻¹, 1295 cm⁻¹, and 1154 cm⁻¹ reflect the C = O stretching, NO₂ asymmetrical stretching, NO₂ symmetrical stretching, and C-O stretching, respectively.

Figure 2. Effect of PAN exposure on induced cell death in two cell lines of tobacco differed in sensitivity to O₃. (A) Cell death after exposure to PAN (1–100 sec), cells were incubated for 1 h to allow cell death development. Then cells stained with Evans blue were counted under microscope. Each data point represents the mean and SD from four replicates with each replicate covering 50–100 cells. (B) Typical images of PAN-induced cell death in Bel-W3 cells stained with Evans blue. The plasmolysis was detected in PAN-treated Bel-W3 cells

Figure 3. Effect of ROS scavengers on PAN-induced cell death in tobacco cells. (A) Effect of DMTU (50 mM). (B) Effects of AsA(1 mM, 5 mM) and cysteine (10 mM). (C) Effect of α-tocopherol. The cells of Bel-B (left) and Bel-W3 (right) were pretreated with above chemicals, 5 min prior to the exposure to PAN. Each bar with error bar represents the mean with SD determined from four replicates as described in Figure 2.

Figure 4. Effect of metal chelating agents on PAN-induced cell death in tobacco cells. The cells of Bel-B (left) and Bel-W3 (right) were pretreated with 2 mM o-Phe or Bipy, 5 min prior to the exposure to PAN. Each bar with error bar represents the mean with SD determined from four replicates as described in Figure 2.

Figure 5. Effect of SHAM against the PAN-induced cell death in tobacco cells. The cells of Bel-B (left) and Bel-W3 (right) were pretreated with 0.5 mM SHAM, 5 min prior to the exposure to PAN. Each bar with error bar represents the mean with SD determined from four replicates as described in Figure 2.

Figure 6. Detection of PAN-induced intracellular hydrogen peroxide accumulation in Bel-W3 cells visualized by DCFH-DA. The typical data from three replicates are shown.

Figure 7. Effect of calcium ion chelator against the PAN-induced cell death in tobacco cells. The cells of Bel-B (left) and Bel-W3 (right) were pretreated with 5 mM BAPTA, 5 min prior to the exposure to PAN. Each bar with error bar represents the mean with SD determined from four replicates as described in Figure 2.

Figure 8. Effect of PAN on cytosolic calcium ion level in aequorin-expressing Bel-W3 cells. (A) Monitoring of PAN-induced increases in [Ca²⁺]_c. (B) Inhibition of PAN-dependent increase in [Ca²⁺]_c by calcium channel inhibitors and redox-related inhibitors. Bel-W3 cells were pretreated with 5 mM LaCl₃, 5 mM AlCl₃, 50 mM DMTU, 2 mM o-Phe, or 2 mM Bipy, 5 min prior to the exposure to PAN. Each bar and error bar represent the mean and S.D., respectively, from four independent replicates.