Ozone-induced cell death mediated with oxidative and calcium signaling pathways in tobacco bel-w3 and bel-B cell suspension cultures

Ozone (O(3))-induced cell death in two suspension-cultured cell lines of tobacco (Nicotiana tabacum L.) derived from Bel-W3 (hyper-sensitive to O(3)) and Bel-B (highly tolerant to O(3)) varieties were studied. By exposing the newly prepared cell lines to the pulse of ozonized air, we could reproduce the conditions demonstrating the difference in O(3) sensitivity as observed in their original plants, depending on the exposure time. Since O(3)-induced acute cell death was observed in the dark, the requirement for photochemical reactions could be eliminated. Addition of several ROS scavengers and chelators inhibited the cell death induced by O(3), indicating that singlet oxygen ((1)O(2)), hydrogen peroxide (H(2)O(2)), hydroxyl radical and redox-active metals such as Fe(2+) play central roles in O(3)-induced acute damages to the cells. As expected, we observed the generation of (1)O(2) and H(2)O(2) in the O(3)-treated cells using chemiluminescent probes. On the other hand, an NADPH oxidase inhibitor, superoxide dismutase (SOD), and some SOD mimics showed no inhibitory effect. Thiols added as antioxidants unexpectedly behaved as prooxidants drastically enhancing the O(3)-induced cell death. It is noteworthy that some ROS scavengers effectively rescued the cells from dying even treated after the pulse of O(3) exposure, confirming the post-ozone progress of ROS-dependent cell death mechanism. Since one of the key differences between Bel-B and Bel-W3 was suggested to be the capacity for ROS detoxification by catalase, the endogenous catalase activities were compared in vivo in two cell lines. As expected, catalase activity in Bel-B cells was ca. 7-fold greater than that in Bel-W3 cells. Interestingly, Ca(2+) chelators added prior to (not after) the pulse of O(3) effectively inhibited the induction of cell death. In addition, increases in cytosolic Ca(2+) concentration sensitive to Ca(2+) chelators, ion channel blockers, and ROS scavengers were observed in the transgenic Bel-W3 cells expressing aequorin, suggesting the action of Ca(2+) as a secondary messenger initiating the oxidative cell death. The O(3)-induced calcium response in Bel-W3 cells was much greater than Bel-B cells. Based on the results, possible pathways for O(3)-dependent generation of the lethal level of ROS and corresponding signaling mechanism for induction of cell death were discussed.