Figure 2: Iron toxicity impairs mitochondrial function and increases ROS production.
Synchronized worms (0.25 million/plate) were transferred to plate with or without iron (0 and 35 μM). Mitochondria were isolated after 3 days and quantified for: A). State 3 respiration is the maximum respiratory rates following addition of ATP. Data are mean ±SEM N=3 independent replicates. *p<0.05, Unpaired t test. B). State 4 respiration is the minimum respiratory rates upon depletion of ATP. Data are mean ±SEM N=3 independent replicates. ns not significant, Unpaired t test. C). Respiratory control ratio (RCR) which is the ratio of maximum respiration state 3 over that of minimum respiration state 4. Data are mean ±SEM N=3 independent replicates. **p<0.001, Unpaired t test. D). Iron toxicity reduced mitochondrial complex I enzyme activity. Data are mean ±SEM N=3 independent replicates. *p<0.05, Unpaired t test. E). Iron toxicity reduced citrate synthase activity. Data are mean ±SEM N=3 independent replicates. *p<0.0001, Unpaired t test. F). Iron toxicity increased mitochondrial Superoxide (O2•−) production. Data are mean ±SEM N=7 independent replicates. ***p=0.0001, Unpaired t test. G). DMT1 (smf-3) knock out reduced mitochondrial iron uptake. Mitochondrial iron was measured using ICP-MS in isolated mitochondrial from WT, WT + 35 μM iron, smf-3 KO, and smf-3 + 35 μM iron. Data are mean ±SEM, N=3 independent biological replicates. ns not significant, ***p=0.0001, ***p<0.0001, one-way ANOVA, Tukey post hoc test. H). DMT1 (smf-3) knock out abolished iron induced increase in mitochondrial calcium uptake. Mitochondrial Ca was measured using ICP-MS in isolated mitochondrial from WT, WT + 35 μM iron, smf-3 KO, and smf-3 + 35 μM iron. Data are mean ±SEM, N=3 independent biological replicates. ns not significant, *p<0.05, **p<0.001, ***p<0.0001, one-way ANOVA, Tukey post hoc test. I). Mitochondrial Zn is not impacted by iron toxicity in DMT1 knock out. Mitochondrial Zn was measured using ICP-MS in isolated mitochondrial from WT, WT + 35 μM iron, smf-3 KO, and smf-3 + 35 μM iron. Data are mean ±SEM, N=3 independent biological replicates. ns not significant, *p<0.05, **p<0.001, one-way ANOVA, Tukey post hoc test. J). Mitochondrial Cu is not impacted by iron toxicity in DMT1 knock. Mitochondrial Cu was measured using ICP-MS in isolated mitochondrial from WT, WT + 35 μM iron, smf-3 KO, and smf-3 + 35 μM iron. Data are mean ±SEM, N=3 independent biological replicates. ns not significant, ***p<0.0001, one-way ANOVA, Tukey post hoc test. K). No effect of iron on mitochondrial Mn in DMT1 knock. Mitochondrial Mn was measured using ICP-MS in isolated mitochondrial from WT, WT + 35 μM iron, smf-3 KO, and smf-3 + 35 μM iron. Data are mean ±SEM, N=3 independent biological replicates. ns not significant, ***p=0.0001, one-way ANOVA, Tukey post hoc test.