Response of Acanthamoeba castellanii mitochondria to oxidative stress.

Abstract

The purpose of this study was to examine the effects of oxidative stress caused by hydroperoxide (H(2)O(2)) in the presence of iron ions (Fe(2+)) on mitochondria of the amoeba Acanthamoeba castellanii. We used isolated mitochondria of A. castellanii and exposed them to four levels of H(2)O(2) 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 (DeltaPsi(m)), ability to accumulate Ca(2+) , and cytochrome c release. Our results show that the increasing concentrations of H(2)O(2) stimulates respiration in states 3 and 4. The highest concentration of H(2)O(2) 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 Ca(2+) also decreased with the increasing stress treatment. The lowest stress treatment (0.5 mM H(2)O(2)) significantly decreased oxygen consumption in state 3 and 4, RC, and membrane potential. The ADP/O ratio decreased significantly under 5 mM H(2)O(2) treatment, while Ca(2+) accumulation rate decreased significantly at 15 mM H(2)O(2). We also observed cytochrome c release under increasing stress conditions. However, this release was not linear. These results indicate that as low as 0.5 mM H(2)O(2) with Fe(2+) damage the basic energetics of mitochondria of the unicellular eukaryotic organism Acanthamoeba castellanii.

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress.

“…It is revealed by no changes in respiratory rates, ∆Ψ values and coupling parameters found in the presence of the alternative oxidase inhibitor BHAM. In contrast, using similar conditions (1 min incubation with 0.5-25 mM H 2 O 2 , in the presence of Fe 2+ ) Trocha and Stobienia (2007) observed a significant increase in both state 3 and state 4 respiration accompanied by a considerable decrease in ∆Ψ values and coupling parameters. The results described by those authors seem to be inconsistent, as the increase in phosphorylating respiration found for H 2 O 2treated mitochondria (e.g., a 3-fold increase for 25 mM H 2 O 2 ) was accompanied by an enormous cytochrome c release.…”

“…The aim of the present work was to establish the basic energetic parameters of amoeba A. castellanii mitochondria respiring with malate and their response to oxidative stress caused by H 2 O 2 in the presence of Fe 2+ . Our aim was to verify the results obtained recently by Trocha and Stobienia (2007) with isolated A. castellanii mitochondria treated for a short time (1 min) with increasing concentrations of H 2 O 2 (up to 25 mM) in the presence of Fe 2+ . The puzzling results reported by those authors included unexpectedly quite high values of coupling parameters obtained with malate under control conditions (ADP/O ratio of 2.9 and RCR of 11) and inconsistent changes observed after 1 min incubation of mitochondria with H 2 O 2 , i.e., up to a 3-fold increase in phosphorylating respiration accompanied by an enormous cytochrome c release, as well as a decrease in the coupling parameters.…”

“…Trophozoites of amoeba were collected 60-72 h following inoculation at the early stationary phase (at a density of about 5-6 × 10 6 cells/ml). Mitochondria were isolated as described by Trocha and Stobienia (2007). Mitochondrial protein concentration was determined by the biuret method using BSA as a standard.…”

“…The oxidative stress model. Oxidative stress model conditions (concentration of H 2 O 2 and incubation time) were those applied by Trocha and Stobienia (2007). To trigger the Fenton reaction, samples containing 3 mg of mitochondrial protein (180 µl) were treated with H 2 O 2 (0.5, 5, 15, or 25 mM) in the presence of 0.2 mM FeCl 2 .…”

“…The puzzling results reported by those authors included unexpectedly quite high values of coupling parameters obtained with malate under control conditions (ADP/O ratio of 2.9 and RCR of 11) and inconsistent changes observed after 1 min incubation of mitochondria with H 2 O 2 , i.e., up to a 3-fold increase in phosphorylating respiration accompanied by an enormous cytochrome c release, as well as a decrease in the coupling parameters. Therefore, we repeated all the experiments performed by Trocha and Stobienia (2007), measuring changes in respiratory rates, coupling parameters, membrane potential, Ca 2+ up-take and cytochrome c release/retention in isolated A. castellanii mitochondria. Additionally, we checked cytochrome c oxidase activity and outer mitochondrial membrane integrity.…”