Vol. 55 No. 2/2008, 349–355 Regular paper on-line at: www.actabp.pl Basic energetic parameters of Acanthamoeba castellanii mitochondria

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.


INTRODUCTION
Mitochondria are the key cellular source of superoxide (O 2 •-) and hydrogen peroxide (H 2 O 2 ).There are many different exogenous inducers of reactive oxygen species (ROS) formation, such as UV irradiance and relatively high concentrations of iron (Fe 2+ ) ions in the cell.Fe 2+ reacts with H 2 O 2 in the Fenton reaction, producing the highly reactive hydroxyl radical ( • OH) (Minotti & Aust, 1987;Fleury et al., 2002).The oxidative stress resulting from an increase in ROS generation leads to a damage of mitochondrial DNA, proteins, membranes, ageing ac-celeration, and in many cases to cell death (Papa & Skulachev, 1997).Hydrogen peroxide is damaging to mitochondria and other cell components because it can oxidize biomolecules directly or through the formation of hydroxyl radical.The consequences of elevated mitochondrial H 2 O 2 concentrations could be lipid peroxidation, disruption of calcium homeostasis, cytochrome c release, inactivation of respiratory chain carriers and other mitochondrial enzymes, and uncoupling or decrease in respiration (Vladimirov et al., 1980;Malis & Bonventre, 1988;Zhang et al., 1990;Radi et al., 1993;Slyshenkov et al., 1996;Sherer et al., 2002;Winger et al., 2007).

W. Jarmuszkiewicz and others
Acanthamoeba castellanii is a small non-photosynthesizing free-living amoeba found in soil and in marine and freshwater environments.A. castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct human diseases.In molecular phylogenesis, A. castellanii appears on a branch basal to the divergence points of plants, animals and fungi (Wainright et al., 1993).Under axenic non-pathogenic conditions, A. castellanii has been used frequently as a model organism to study mitochondrial energy-dissipating systems such as a cyanide-resistant alternative oxidase (Jarmuszkiewicz et al., 1997;1998;2001;2005a), an ATP-sensitive potassium channel (Kicinska et al., 2007), and an uncoupling protein (Jarmuszkiewicz et al., 1999;2004a;2004b;2005b;Czarna et al., 2007;Swida et al., 2007).Mitochondria of A. castellanii contain a plant-type respiratory chain with additional (in addition to the four classical) electron carriers: external and internal NADH dehydrogenases and an alternative cyanideresistant quinol oxidase that consumes mitochondrial reducing power without energy conservation in the proton electrochemical gradient (ΔµH + ) (Jarmuszkiewicz et al., 1997;2005a).We have shown that in A. castellanii, like in plant mitochondria (Popov, 2003;Vercesi et al., 2006), the two mitochondrial energydissipating systems, the alternative oxidase and the uncoupling protein, may play a role in the energetic status of the cell (decreasing the yield of ATP synthesis) (Jarmuszkiewicz et al., 1998;1999;2004b;2005b) and in attenuating ROS production (Czarna & Jarmuszkiewicz, 2005).Moreover, the contribution of both energy-dissipating systems in the prevention of mitochondrial ROS generation in vivo could ensure their constant level throughout the growth cycle of A. castellanii batch culture (Czarna et al., 2007).
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.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.

MATERIALS AND METHODS
Cell culture and mitochondria isolation.The soil amoeba Acanthamoeba castellanii, avirulent strain Neff, was cultured as described previously (Jarmuszkiewicz et al., 1997).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.Mitochondria preparations were diluted to a concentration of 50 mg protein/ ml.All measurements were carried out within 5-6 h after mitochondria isolation, as their bioenergetic features were found to weaken after this time.
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 .Hydrogen peroxide (Sigma, INFARM) concentrations were determined using a molar absorbance coefficient of 43.6 M -1 cm -1 at 240 nm.Stock solution of 10 mM FeCl 2 was prepared daily by adding the solid iron salt to 2.5 M NaCl (plus 5 mM Tris/HCl), pH 7.0, exhaustively bubbled with argon.The solution was capped, protected from light, and used within 4 h (Minotti & Aust, 1987).A small volume (3.6 µl) of Fe 2+ solution was added to the mitochondria incubation reaction to reach a final concentration of 0.2 mM FeCl 2 and 50 mM NaCl.Incubations were performed for 1 or 5 min at 0-4 o C. Control (H 2 O 2untreated) mitochondria were incubated for 1 or 5 min in the absence of H 2 O 2 but in the presence of 0.2 mM FeCl 2 and 50 mM NaCl (pH = 7.0).The reactions were stopped by a 100-fold dilution with the incubation medium (0.25 M sucrose, 10 mM Tris/HCl, pH 7.4).The samples were centrifuged at 10 000 × g at 0°C for 7 min to remove remaining H 2 O 2 .The pellets containing stressed (H 2 O 2 -treated) or control (H 2 O 2 -untreated) mitochondria were suspended gently in 180 µl of the incubation medium (to obtain a concentration of 50 mg of mitochondrial protein per ml) and used immediately for the measurements of oxygen consumption, membrane potential and calcium ion uptake.Supernatants were collected and used to estimate cytochrome c release.Only results obtained after 5 min incuba-Parameters of Acanthamoeba castellanii mitochondria tion of mitochondria with H 2 O 2 in the presence of Fe 2+ are shown, since no significant changes were found after 1 min incubation.
Oxygen uptake and membrane potential.Oxygen uptake was measured polarographically using a Rank Bros. (Cambridge, UK) oxygen electrode or a Hansatech oxygen electrode in 3 ml or 1.5 ml (respectively) of the standard reaction medium (25°C) containing 120 mM KCl, 20 mM Tris/HCl, pH 7.4, 3 mM KH 2 PO 4 , 8 mM MgCl 2 and 0.2% BSA, with 3 or 1.5 mg of mitochondrial protein (to keep the concentration of 1 mg × ml -1 ).Membrane potential (∆Ψ) of mitochondria was measured simultaneously with the measurements of oxygen uptake using a tetraphenylphosphonium-specific electrode according to Kamo et al. (1979).For calculation of the ∆Ψ value the matrix volume of amoeba mitochondria was assumed as 2.0 µl/mg protein.Values of O 2 uptake are presented in nAt O × min -1 × (mg protein) -1 .∆Ψ values are presented in mV.
All measurements were made in the presence of 3 mM malate (as a respiratory substrate) and 100 nmol ADP (equivalent to 30-60 µM, prepulse).The ADP/O ratio was determined by the ADP pulse method with 450-500 nmol ADP.The total amount of oxygen consumed during state 3 respiration was used for calculation of the ratio.A prepulse of ADP was always applied before the main pulse to ensure that a true state 4 had been achieved and to activate malate dehydrogenase by the produced ATP.Measurements were made in the absence or presence of 2 mM benzohydroxamate (BHAM), an inhibitor of the alternative oxidase.At the end of each measurement, 1 µM FCCP was added to collapse ∆Ψ and to assess the rate of the uncoupled state, i.e., a maximal cytochrome pathway activity.Measurements of ∆Ψ allowed fine determination of the duration of state 3 respiration.
Cytochrome c oxidase activity and outer mitochondrial membrane integrity.Cytochrome c oxidase (COX) activity was measured with an oxygen electrode using 0.5 mg of mitochondrial protein (in 1.5 ml of the standard reaction medium), without exogenously added respiratory substrate and in the presence of 2 mM BHAM.Respiratory rates were measured during sequential additions of antimycin A (4 µg/mg mitochondrial protein), 8 mM ascorbate, 0.06% cytochrome c and up to 2.5 mM TMPD.The rate of oxygen consumption following the addition of TMPD reflected the maximal O 2 consumption by COX (complex IV).Outer mitochondrial membrane integrity was assayed as the latency of COX activity during the same measurements (acceleration of respiration by addition of cytochrome c prior to addition of TMPD).
Calcium ion uptake.The measurements of Ca 2+ uptake and accumulation by mitochondria were carried out using a tetraphenylphosphonium-specific electrode at 25 o C with 3 mg mitochondrial protein per individual measurement.Standard reaction medium (3 ml) was used with 3 mM malate as a respiratory substrate.After addition of malate (state 4 conditions), known aliquots of calcium ions (about 200 nmol/mg of mitochondrial protein) were sequentially added to the medium until the membrane potential collapsed.The total amount of accumulated calcium ions and the total time of calcium ion accumulation were measured.Total loading of mitochondria with Ca 2+ was assumed when no rebuilding of ΔΨ was observed.
Cytochrome c assay.Cytochrome c release was assessed by three independent spectrophotometric approaches, i.e., at 550 nm, at 550 minus 540 nm, and by spectrum measurements (420-620 nm).We used supernatants obtained after mitochondria (H 2 O 2 -treated or -untreated) suspension and centrifugation in a 100-fold volume of the incubation medium.Three milligrams of mitochondrial protein per reaction was used.The measurements were monitored in quartz cuvettes containing 1 ml of the supernatant solution in the absence or presence of 0.1 mM potassium ferricyanide (to oxidize the reduced cytochrome c) and in the absence or presence of a few grains of sodium dithionite (to reduce cytochrome c).Cytochrome c concentrations were determined using a molar absorbance coefficient of 21 mM -1 cm -1 at 550 minus 540 nm and 27.8 mM -1 cm -1 at 550 nm.

Respiration rates, coupling parameters and membrane potential
The capacity of cytochrome pathway-dependent respiration of isolated A. castellanii mitochondria was measured in the presence of BHAM, an inhibitor of the alternative oxidase, and BSA that binds free fatty acids thereby excluding uncoupling protein activity.Malate was used as a respiratory substrate.In the presence of BHAM, in control, H 2 O 2 -untreated mitochondria the ADP/O ratio was 2.3 ± 0.2 and respiratory control ratio (RCR) was 3.3 ± 0.3 (S.D., for five different mitochondria preparations).Table 1 shows that the incubation of mitochondria with 0.5-25 mM H 2 O 2 in the presence of Fe 2+ results in no marked changes in the cytochrome pathway activity.In particular, in the presence of BHAM, there were no differences in the rates of uncoupled (FCCP-stimulated) respiration, state 3 (ADP-stimulated) respiration, RCR and ADP/O ratio in the absence and in the presence of up to 25 mM W. Jarmuszkiewicz and others H 2 O 2 .However, in the absence of BHAM, non-phosphorylating state 4 respiration (but not state 3 and uncoupled state) progressively decreased with the increasing concentrations (0.5-25 mM) of H 2 O 2 (by up to 31% with 25 mM H 2 O 2 , P = 0.0086) while the coupling parameters, RCR and ADP/O ratio slightly improved (respectively by up to 20 and 10% with 25 mM H 2 O 2 , P = 0.0073 and 0.0325).This indicates that short (5 min) incubation of mitochondria with H 2 O 2 in the presence of Fe 2+ could decrease the alternative oxidase activity.In addition, a slight (by up to 10% with 25 mM H 2 O 2 , P = 0.0175) decrease in the activity of COX (complex IV) was observed.
The values of mitochondrial ∆Ψ generated during malate oxidation both in nonphosphorylating (state 4) and phosphorylating (state 3) conditions are presented in Table 2.As expected, in the absence and presence of BHAM, no significant changes in the ∆Ψ4 and ∆Ψ3 values for H 2 O 2 -treated compared to control (H 2 O 2 -untreated) mitochondria were ob-served.This confirms our before-mentioned observations that a 5-min incubation of isolated A. castellanii mitochondria with H 2 O 2 (up to 25 mM) does not decrease the cytochrome pathway-mediated respiration.

Accumulation of Ca 2+
Calcium ion accumulation by A. castellanii mitochondria leads to an increase in oxygen consumption in the resting state (state 4) and simultaneously decreases ΔΨ.In A. castellanii mitochondria, ∆Ψ-driven ruthenium red-sensitive Ca 2+ uptake is mediated through a Ca 2+ uniporter (Domka-Popek & Michejda, 1986).In the present work, active Ca 2+ accumulation was measured when portions of 200 nmol Ca 2+ were sequentially added to the reaction medium containing H 2 O 2 -treated or control (H 2 O 2 -untreated) mitochondria respiring under state 4 conditions (Table 3).The measurements were performed as long as  the mitochondrial ∆Ψ was no longer restored after the uptake of a subsequent portion of Ca 2+ , indicating total loading of mitochondria with these ions.These measurements allowed us to calculate Ca 2+ accumulation rates expressed as the amount of Ca 2+ (µmol) accumulated during one minute per one milligram of mitochondrial protein.

Mitochondrial outer membrane integrity and cytochrome c retention
The influence of external cytochrome c on the respiratory rate during measurements of COX maximal activity was used to assess the outer mitochondrial membrane integrity of A. castellanii mitochondria.No significant difference in this feature was found between control (H 2 O 2 -untreated) and stressed (0.5-25 mM H 2 O 2 -treated) mitochondria.Namely, the outer mitochondrial membrane integrity averaged 98 ± 4% and 95 ± 5% for H 2 O 2 -untreated and 25 mM H 2 O 2 -treated mitochondria, respectively (for five different mitochondria preparations).
To measure cytochrome c release from the mitochondrial intermembrane space as a result of a possible outer membrane damage after 5 min incubation of mitochondria with H 2 O 2 in the presence of Fe 2+ , we carried out spectrophotometric measurements at 550 nm, at 550 minus 540 nm, and continuous at 420-620 nm.The absorbance values obtained at 550 minus 540 nm and at continuous spectrum measurements show no cytochrome c release from mitochondria of A. castellanii subjected to increasing H 2 O 2 concentrations under 1 or 5 min incubation (not shown).The absorbance values recorded only at 550 nm were discarded as they were apparently due to some light-absorbing contaminations that was clearly indicated by the continuous spectral measurements and those at 550 minus 540 nm.In intact isolated A. castellanii mitochondria, total c-type cytochromes (c + c 1 ) content estimated from the difference spectra obtained at room temperature (substrate-reduced minus oxidized) of the α peaks at 550 minus 540 nm (using absorbance coefficient 19 mM -1 cm -1 ) was 1.8 ± 0.4 nmol/mg mitochondrial protein (S.D., n = 5).The estimated content of c-type cytochromes is close to that found previously for A. castellanii mitochondria with the difference spectrum obtained at low temperature (Edwards et al., 1977).

DISCUSSION
Our results show that a 5-min incubation of A. castellanii mitochondria with H 2 O 2 at a concentration up to 25 mM (in the presence of 0.2 mM FeCl 2 ) is not associated with marked changes in cytochrome pathway activity.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.Moreover, the coupling parameters obtained for control conditions (H 2 O 2untreated mitochondria) with malate as the respiratory substrate are quite different in the present work (ADP/O = 2.3 and RCR = 3.3) from those of Trocha and Stobienia (2007) (ADP/O of 2.9 and RCR of 11.4).According to our results, cancellation of ∆Ψ4 after Ca 2+ accumulation indicates mitochondria saturation by these positively charged ions but not mitochondria disruption (Trocha & Stobienia, 2007).Contrary to Trocha and Stobienia (2007), in our experiments incubation of mitochondria with H 2 O 2 had no effect on Ca 2+ uptake, in agreement with unchanged ∆Ψ4 values.Moreover, according to those authors, the total amount of cytochrome c released from A. castellanii mitochondria treated with 15 mM H 2 O 2 would be (after calculation of the obtained absorption values) around 1.4 µmol/mg mitochondrial protein, thus three orders of magnitude higher than the amount found presently and by Edwards et al. (1977) with the difference spectrum obtained at room temperature and at low temperature, respectively.
Although a slight decrease in the COX activity was observed in the present study, it seems that it should not influence the overall cytochrome pathway activity, as the terminal oxidase of the respiratory chain of A. castellanii mitochondria is not ratelimiting for the cytochrome pathway-sustained respiration (Czarna et al., 2007).However, our results point to a partial inactivation of two Fe-containing proteins, the alternative oxidase and COX (the nonheme and heme-bound proteins, respectively) in H 2 O 2 -treated mitochondria.This may mean that these proteins could be highly sensitive to oxidation under treatment of isolated A. castellanii mitochondria with H 2 O 2 in the presence of FeCl 2 .

W. Jarmuszkiewicz and others
It can be summarized that 0.5-25 mM H 2 O 2treated mitochondria of A. castellanii did not show substantial impairment.No marked changes in cytochrome pathway activity were found since in the presence of an alternative oxidase inhibitor no effects were observed on either the rates of uncoupled and phosphorylating respiration or on the coupling parameters.Moreover, our results indicate no H 2 O 2 -induced change in ∆Ψ values, Ca 2+ uptake and accumulation ability, and the maintenance of mitochondrial integrity and cytochrome c content.Only in the absence of BHAM, non-phosphorylating respiration progressively decreased with increasing concentration of H 2 O 2 , while the coupling parameters slightly improved, which may indicate inactivation of alternative oxidase activity.Thus it can be concluded that short (5 min) incubation of isolated mitochondria with H 2 O 2 (up to 25 mM) does not significantly damage the basic energetics of A. castellanii mitochondria.To observe an impact of oxidative stress, such as elevated H 2 O 2 concentrations, on the function of A. castellanii mitochondria, cell culture should perhaps be treated with H 2 O 2 for a couple of days.Such a treatment may allow to observe a response to stress at the level of mitochondrial protein composition and activity.Sweetlove et al. (2002) demonstrated for example that treatment of Arabidopsis cell cultures with 88 mM H 2 O 2 for 7 days resulted in degradation of the tricarboxylic acid cycle and some respiratory chain proteins and led to a decrease in respiration, while mitochondrial integrity was maintained.The consequences of elevated H 2 O 2 concentrations could also be lipid peroxidation, disruption of calcium homeostasis and cytochrome c release (Vladimirov et al., 1980;Malis & Bonventre, 1988;Zhang et al., 1990;Radi et al., 1993;Sherer et al., 2002;Sweetlove et al. 2002;Winger et al., 2007).In our opinion, to observe such a response of A. castellanii mitochondria, exposure of isolated mitochondria to a stronger oxidative stress (longer exposure and/or higher H 2 O 2 concentration) would be required.In this study, under the stress conditions applied (5-min incubation at 4 o C with up to 25 mM H 2 O 2 in the presence of 0.2 mM FeCl 2 ), A. castellanii mitochondria displayed remarkable resistance to H 2 O 2 treatment.

Table 1 . Respiratory rates, coupling parameters and COX activity of A. castellanii mitochondria oxidizing malate.
Rates of state 3 (phosphorylating state), state 4 (nonphosphorylating state) and state U (uncoupled state) are presented.For control (no H 2 O 2 ) and 25 mM H 2 O 2 -treated mitochondria measurements were additionally performed in the presence of 2 mM BHAM (values in parentheses).Values of O 2 uptake and COX activity are expressed in nAt O × min -1 × (mg protein) -1 .Mean values for five different mitochondria preparations ± S.D. are shown.Values marked with * are significantly different from those from control (no H 2 O 2 ) mitochondria at the level of P < 0.05 (paired Student's t-test).