The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H2O2) in the presence of iron ions (Fe2+) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. Castellanii and exposed them to four levels of H2O2 concentration: 0.5, 5, 15, and 25 mM. We measured basic energetics of mitochondria: oxygen consumption in phosphorylation state (state 3) and resting state (state 4), respiratory coefficient rates (RC), ADP/O ratios, membrane potential (ΔΨm), ability to accumulate Ca2+ , and cytochrome crelease. Our results show that the increasing concentrations of H2O2 stimulates respiration in states 3 and 4. The highest concentration of H2O2 caused a 3-fold increase in respiration in state 3 compared to the control. Respiratory coefficients and ADP/O ratios decreased with increasing stress conditions. Membrane potential significantly collapsed with increasing hydroperoxide concentration. The ability to accumulate Ca2+ also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H2O2) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H2O2 treatment, while Ca2+ accumulation rate decreased significantly at 15 mM H2O2. We also observed cytochrome crelease under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H2O2 with Fe2+ damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii
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