Mitochondrial Oxidative Stress Is the General Reason for Apoptosis Induced by Different-Valence Heavy Metals in Cells and Mitochondria.

This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of different-valence heavy metals (Ag+, Tl+, Hg2+, Cd2+, Pb2+, Al3+, Ga3+, In3+, As3+, Sb3+, Cr6+, and U6+). The problems of the relationship between the integration of these toxic metals into molecular mechanisms with the subsequent development of pathophysiological processes and the appearance of diseases caused by the accumulation of these metals in the body are also addressed in this review. Such apoptosis is characterized by a reduction in cell viability, the activation of caspase-3 and caspase-9, the expression of pro-apoptotic genes (Bax and Bcl-2), and the activation of protein kinases (ERK, JNK, p53, and p38) by mitogens. Moreover, the oxidative stress manifests as the mitochondrial permeability transition pore (MPTP) opening, mitochondrial swelling, an increase in the production of reactive oxygen species (ROS) and H2O2, lipid peroxidation, cytochrome c release, a decline in the inner mitochondrial membrane potential (ΔΨmito), a decrease in ATP synthesis, and reduced glutathione and oxygen consumption as well as cytoplasm and matrix calcium overload due to Ca2+ release from the endoplasmic reticulum (ER). The apoptosis and respiratory dysfunction induced by these metals are discussed regarding their interaction with cellular and mitochondrial thiol groups and Fe2+ metabolism disturbance. Similarities and differences in the toxic effects of Tl+ from those of other heavy metals under review are discussed. Similarities may be due to the increase in the cytoplasmic calcium concentration induced by Tl+ and these metals. One difference discussed is the failure to decrease Tl+ toxicity through metallothionein-dependent mechanisms. Another difference could be the decrease in reduced glutathione in the matrix due to the reversible oxidation of Tl+ to Tl3+ near the centers of ROS generation in the respiratory chain. The latter may explain why thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc.

A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl+-Induced Mitochondrial Permeability Transition Pore.

Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.

The Joint Influence of Tl+ and Thiol-Modifying Agents on Rat Liver Mitochondrial Parameters In Vitro.

Recent data have shown that the mitochondrial permeability transition pore (MPTP) is the complex of the Ca2+-modified adenine nucleotide translocase (ANT) and the Ca2+-modified ATP synthase. We found in a previous study that ANT conformational changes may be involved in Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria. In this study, the effects of thiol-modifying agents (eosin-5-maleimide (EMA), fluorescein isothiocyanate (FITC), Cu(o-phenanthroline)2 (Cu(OP)2), and embelin (Emb)), and MPTP inhibitors (ADP, cyclosporine A (CsA), n-ethylmaleimide (NEM), and trifluoperazine (TFP)) on MPTP opening were tested simultaneously with increases in swelling, membrane potential (ΔΨmito) decline, decreases in state 3, 4, and 3UDNP (2,4-dinitrophenol-uncoupled) respiration, and changes in the inner membrane free thiol group content. The effects of these thiol-modifying agents on the studied mitochondrial characteristics were multidirectional and showed a clear dependence on their concentration. This research suggests that Tl+-induced MPTP opening in the inner membrane of calcium-loaded mitochondria may be caused by the interaction of used reagents (EMA, FITC, Emb, Cu(OP)2) with active groups of ANT, the mitochondrial phosphate carrier (PiC) and the mitochondrial respiratory chain complexes. This study provides further insight into the causes of thallium toxicity and may be useful in the development of new treatments for thallium poisoning.

Effects of Tl+ on the inner membrane thiol groups, respiration, and swelling in succinate-energized rat liver mitochondria were modified by thiol reagents.

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 resulted in the Tl+-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria.

Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

There we made available information about the effects of the adenine nucleotide translocase (ANT) 'c' conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca(2+)-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25-75 mM TlNO3, 125 mM KNO3, and 100 µM Ca(2+). The fixers and DIDS, in comparison to Ca(2+) alone, greatly increased A540 decline and the rate of Ca(2+)-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in 'm' conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript "To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria" (Korotkov et al., 2016 [1]).

To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

Diamide accelerates opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca(2+) overload and matrix glutathione loss. Fixing the 'm' conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP. Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in 'c' conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl(+)-induced MPTP opening, which was accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria.

Tl+ induces the permeability transition pore in Ca2+-loaded rat liver mitochondria energized by glutamate and malate.

It is known that Ca2+ and heavy metals more actively induce MPTP opening in mitochondria, energized by the I complex substrates. Thus, a rise in a Tl+-induced MPTP was proposed in experiments on isolated rat liver mitochondria energized by the complex I substrate (glutamate and malate). Expose of the mitochondria to Ca2+ into a medium containing TlNO3, glutamate, and malate as well as sucrose or KNO3 resulted in a decrease in state 3, state 4, or DNP-stimulated respiration as well as an increase of both mitochondrial swelling and ΔΨmito dissipation. The MPTP inhibitors, CsA and ADP, almost completely eliminated the effect of Ca2+, which was more pronounced in the presence of the complex I substrates than the complex II substrate (succinate) and rotenone (Korotkov and Saris, 2011). The present study concludes that Tl+-induced MPTP opening is more appreciable in mitochondria energized by glutamate and malate but not succinate in the presence of rotenone. We assume that the Tl+-induced MPTP opening along with followed swelling and possible structural deformations of the complex I in Ca2+-loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl+ to Tl3+ by mitochondrial oxygen reactive species is proposed for the mechanism.

Closure of mitochondrial potassium channels favors opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

It is known that a closure of ATP sensitive (mitoKATP) or BK-type Ca(2+) activated (mitoKCa) potassium channels triggers opening of the mitochondrial permeability transition pore (MPTP) in cells and isolated mitochondria. We found earlier that the Tl(+)-induced MPTP opening in Ca(2+)-loaded rat liver mitochondria was accompanied by a decrease of 2,4-dinitrophenol-uncoupled respiration and increase of mitochondrial swelling and ΔΨmito dissipation in the medium containing TlNO3 and KNO3. On the other hand, our study showed that the mitoKATP inhibitor, 5-hydroxydecanoate favored the Tl(+)-induced MPTP opening in the inner membrane of Ca(2+)-loaded rat heart mitochondria (Korotkov et al. 2013). Here we showed that 5-hydroxydecanoate increased the Tl(+)-induced MPTP opening in the membrane of rat liver mitochondria regardless of the presence of mitoKATP modulators (diazoxide and pinacidil). This manifested in more pronounced decrease in the uncoupled respiration and acceleration of both the swelling and the ΔΨmito dissipation in isolated rat liver mitochondria, incubated in the medium containing TlNO3, KNO3, and Ca(2+). A slight delay in Ca(2+)-induced swelling of the mitochondria exposed to diazoxide could be result of an inhibition of succinate oxidation by the mitoKATP modulator. Mitochondrial calcium retention capacity (CRC) was markedly decreased in the presence of the mitoKATP inhibitor (5-hydroxydecanoate) or the mitoKCa inhibitor (paxilline). We suggest that the closure of mitoKATP or mitoKCa in calcium loaded mitochondria favors opening of the Tl(+)-induced MPTP in the inner mitochondrial membrane.

On the mechanism(s) of membrane permeability transition in liver mitochondria of lamprey, Lampetra fluviatilis L.: insights from cadmium.

Previously we have shown that opening of the mitochondrial permeability transition pore in its low conductance state is the case in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) during reversible metabolic depression taking place in the period of its prespawning migration when the exogenous feeding is switched off. The depression is observed in the last year of the lamprey life cycle and is conditioned by reversible mitochondrial dysfunction (mitochondrial uncoupling in winter and coupling in spring). To further elucidate the mechanism(s) of induction of the mitochondrial permeability transition pore in the lamprey liver, we used Cd(2+) and Ca(2+) plus Pi as the pore inducers. We found that Ca(2+) plus Pi induced the high-amplitude swelling of the isolated "winter" mitochondria both in isotonic sucrose and ammonium nitrate medium while both low and high Cd(2+) did not produce the mitochondrial swelling in these media. Low Cd(2+) enhanced the inhibition of basal respiration rate of the "winter" mitochondria energized by NAD-dependent substrates whereas the same concentrations of the heavy metal evoked its partial stimulation on FAD-dependent substrates. The above changes produced by Cd(2+) or Ca(2+) plus Pi in the "winter" mitochondria were only weakly (if so) sensitive to cyclosporine A (a potent pharmacological desensitizer of the nonselective pore) added alone and they were not sensitive to dithiothreitol (a dithiol reducing agent). Under monitoring of the transmembrane potential of the "spring" lamprey liver mitochondria, we revealed that Cd(2+) produced its decrease on both types of the respiratory substrates used that was strongly hampered by cyclosporine A, and the membrane potential was partially restored by dithiothreitol. The effects of different membrane permeability modulators on the lamprey liver mitochondria function and the seasonal changes in their action are discussed.

Tl(+) showed negligible interaction with inner membrane sulfhydryl groups of rat liver mitochondria, but formed complexes with matrix proteins.

The effects of Tl(+) on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellman's reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix-soluble fraction for RLM, incubated and frozen in 25-75 mM TlNO3 . This increase was greater in the medium containing KNO3 regardless of the presence of Ca(2+) . It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3 . Calcium-loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl(+) with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

Tl(+) induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria.

Effects of Tl(+) were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO3 and a nitrate salt (KNO3 or NH4NO3) or TlNO3 and sucrose. Tl(+) increased permeability of the inner membrane of the RHM to K(+) and H(+). This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K(+) < NH4 (+), respectively. After succinate administration, the swollen RHM contracted. The Tl(+)-induced opening of the mitochondrial permeability pore (MPTP) in Ca(2+)-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl(+) were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoKATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoKATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl(+) effects on muscle cell mitochondria.

Influence of Tl(+) on mitochondrial permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

The Tl(+)-induced opening of the MPTP in Ca(2+)-loaded rat liver mitochondria energized by respiration on the substrates succinate or glutamate plus malate was recorded as increased swelling and dissipation of mitochondrial membrane potential as well as decreased state 4, or state 3, or 2,4-dinitrophenol-stimulated respiration. These effects of Tl(+) increased in nitrate media containing monovalent cations in the order of Li(+) < NH (4) (+) ≤ Na(+) < K(+). They were potentiated by inorganic phosphate and diminished by the MPTP inhibitors (ADP, CsA, Mg(2+), Li(+), rotenone, EGTA, and ruthenium red) both individually and more potently in their combinations. Maximal swelling of both non-energized and energized Ca(2+)-loaded mitochondria in rotenone-free media is an indication of Ca(2+) uptake driven by respiration on mitochondrial endogenous substrates. It is suggested that Tl(+) (distinct from Cd(2+), Hg(2+), and other heavy metals and regardless of the used respiratory substrates) can stimulate opening of the MPTP only in the presence of Ca(2+). We discuss the possible participation of Ca(2+)-binding sites, located near the respiratory complex I and the adenine nucleotide translocase, in inducing opening of the MPTP.

In vitro modulation of heavy metal-induced rat liver mitochondria dysfunction: a comparison of copper and mercury with cadmium.

Cadmium (Cd), mercury (Hg) and copper (Cu) are very toxic environmental pollutants that exert their cytotoxic effects as cations by targeting mitochondria. To further underscore molecular mechanism(s) underlying the heavy metal-induced mitochondrial dysfunction we continued to compare the action of Cd, Hg and Cu using a simple and convenient in vitro model, namely isolated rat liver mitochondria incubated in assay media of different ionic contents and energized by respiratory substrates, glutamate plus malate for complex I, succinate plus rotenone for complex II, and ascorbate plus tetramethylphenylenediamine for complex IV. With the help of various selective electrodes, fluorescent probes, isotope and spectrophotofluorometric techniques, significant differences were found in the modulating action of various substances affecting the activity of these respiratory chain complexes and mitochondrial Ca²+ uniporter or permeability transition pore effectors on the mitochondrial function disturbed by the heavy metals, including clear-cut substrate specificity of many effects of these cations. Sequence of events manifested in the mitochondrial dysfunction produced by the metals under test was elucidated.

Effects of Tl(+) on ion permeability, membrane potential and respiration of isolated rat liver mitochondria.

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K(+), Na(+), H(+)) but high to Tl(+). Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (DeltaPsi(mito)) of rat liver mitochondria were studied in media containing 0-75 mM TlNO(3) either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO(3), or NaNO(3), or NH(4)NO(3)). Tl(+) increased permeability of the inner mitochondrial membrane to K(+), Na(+), and H(+), that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of DeltaPsi(mito). These effects of Tl(+) increased in the order of sucrose

Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria.

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate-energized mitochondria in the TlNO3 medium. A valinomycin-induced decrease of K+-diffusion potential was also accelerated by Tl+ in the presence of P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) did not affect both the Tl+-stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4-dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of P(i). We suggested that stimulation by P(i) of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of P(i) and Ca2+.

Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin.

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.

Cd2+ versus Ca2+-produced mitochondrial membrane permeabilization: a proposed direct participation of respiratory complexes I and III.

A comparison of Cd2+ and Ca2+ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd2+ and/or Ca2+-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca2+ and Cd2+-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca2+, catalase and dibucaine did not influence on main Cd2+ effects. In NH4NO3 medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd2+-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd2+-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd2+-produced swelling in NH4NO3 medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca2+ and Pi were simultaneously present in NH4NO3 medium, DTT reversed only partially Cd2+-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca2+ or Pi were separately administered to the Cd2+-treated mitochondria. Besides, DTT added following a low Cd2+ pulse in KCl medium containing exogenous Ca2+ induced a substantial enhancing of sustained Cd2+ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd2+] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd2+ in the absence of added Ca2+ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd2+ and/or Ca2+-produced MMP was discussed.

Cd2+ -promoted mitochondrial permeability transition: a comparison with other heavy metals.

We compared action of Cd(2+), Hg(2+), and Cu(2+) on isolated rat liver mitochondria in the absence of added Ca(2+) and P(i). The heavy-metal ions produced dose-dependently: (1) enhanced membrane permeabilization manifested in mitochondrial swelling and activation of basal respiration, (2) inhibition of uncoupler-stimulated respiration, and (3) membrane potential dissipation. Among the metals, Cu(2+) exhibited maximal stimulatory effect on basal respiration and minimal inhibitory action on DNP-uncoupled respiration whilst Cd(2+) promoted the strongest depression of uncoupled respiration and the largest swelling in NH(4)NO(3) medium. Dithiothreitol induced a basal respiration release if added after high [Cd(2+)] and [Hg(2+)], and the stimulation was CsA-insensitive.