Relation Between Reactive Oxygen Species, Calcium Mobilization And Apoptosis In Cadmium-induced Stress

The roles of reactive oxygen species (ROS), intracellular calcium ([Ca²⁺]_i) and apoptosis induced by cadmium were compared using normal cells (Human liver cell, HL-7702) and tumor cells (Raji Cell, CL86) in vitro. Apoptosis was assessed by using Hoechst33258 staining. Changes in intracellular calcium concentration and ROS production were assessed by using fluorescent dyes (Flu-3/AM, 6h; DCFH-DA, 24h respectively) under a flow cytometry. To investigate the intracellular calcium elevation generated from inside or outside of the cells induced by Cd²⁺, cells were treated with EGTA (20mM) blocking extracellular Ca²⁺ and BAPTA-AM (10μM) blocking intracellular Ca²⁺. To further investigate the role of intracellular Ca²⁺ and ROS in Cd-induced apoptosis, cells were treated with BAPTA-AM (10μM) to inhibit intracellular Ca²⁺ elevation and NAC (15mM) to scavenge ROS in the presence of Cd²⁺ (20μM), respectively. Excessive apoptosis was observed in the HL-7702 and Raji cells treated with Cd²⁺. Significant elevation of intracellular Ca²⁺ and ROS were seen in the cells with higher levels of apoptosis. BAMTA/AM blocked the Cd-elicited intracellular Ca²⁺ inactivation (all,p<0.001), ROS accumulation (all, p<0.001) and apoptosis (all,p<0.001). Furthermore, inhibition of intracellular Ca²⁺ and scavenging of ROS significantly reduced apoptosis in the cells. EGTA decreased Cd-elicited intracellular Ca²⁺ (p<0.01 or p<0.001) and ROS accumulation (all, p<0.001) effectively except apoptosis. These studies identified that the Ca²⁺ in calcium elevation was primarily (about 78% in HL-7702 and 59% in Raji) intracellular-generated and only a small portion of it was extracellular-generated. The apoptosis is positively correlated with ROS content (r=0.8781 in HL-7702 and 0.6919 in Raji) and calcium elevation (r=0.9073 in HL-7702 and 0.8692 in Raji). The ROS content and calcium elevation are positively correlated (r=0.8619 in HL-7702 and 0.9061 in Raji). We conclude that 1) the intracellular calcium elevation generated mostly from inside of the cells induced by Cd²⁺; 2) ROS accumulation and calcium mobilization were concurrent and they were two crucial events interactive in the normal cell and tumor cell apoptosis approach induced by Cd²⁺.