Cytotoxic effects of various stressors on PC12 cells: Involvement of oxidative stress and effect of antioxidants

In order to specifically elucidate the involvement of oxidative stress, the effects of various types of stressors and antioxidants on PC12 cells were examined. In this study, the following four stressors were studied in detail: free radicals generated from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), 4-hydroxynonenal (HNE), 7-ketocholesterol (KC), and arsenic trioxide (As₂O₃). Undifferentiated PC12 cells were treated with 50% lethal concentration (LC₅₀) of these stressors, and subsequently the viability, apoptosis/necrosis ratio, reactive oxygen species (ROS) production, caspase-3 activity, and protection by antioxidants were measured to elucidate the underlying mechanisms that determine the action of these stressors on PC12 cells. The cytotoxicity did not correlate directly with the intracellular formation of ROS. For example, as compared to AAPH, As₂O₃ produced considerably smaller amounts of ROS at LC₅₀. As observed in the cells incubated with As₂O₃, KC and HNE exerted cell toxicity, but with a moderate production of ROS. With the exception of HNE, the apoptosis/necrosis ratio of all the stressors evaluated by annexin V and propidium iodide assays increased with an increase in the incubation time at the LC₅₀ values of these stressors. In accordance with apoptosis ratio, caspase activity was detected in the cells incubated with AAPH, As₂O₃, and KC, but not HNE at LC₅₀ for 24 h. The protective effect of α-tocopherol, 17β-estradiol, 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO653), glutathione, and N-acetylcysteine (NAC) against cytotoxicity depended on the type of stressors. These antioxidants were found to be effective against the abovementioned stressors, except As₂O₃ against which only NAC was effective. These results suggest that the involvement of ROS and the protective effect of antioxidants depend on the type of stressors.