QUARTERLY Communication Cd 2+-promoted mitochondrial permeability transition:

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.

tochondrial respiration and MMP are involved in hepatorenal toxic effects of the metals (Zaba & Harris, 1976;Weinberg et al., 1982a;1982b;Leblondel & Allain, 1984;Rasheed et al., 1984;Chavez et al., 1988;Chavez & Holquin, 1989;Micadei & Floridi, 1993).However, the mechanism(s) underlying the heavy metal-produced mitochondrial dysfunction is not completely understood.As recognized, MPT pore-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vitro and in vivo (Zoratti & Szabo, 1995;Haouzi et al., 2002), with heavy metals among them (Broekemeier et al., 1989;Zazueta et al., 1998;2000;Garcia et al., 2000;Belyaeva et al., 2001;2002;Pourahmad et al., 2001).Besides, there are data indicating that MPT pores opened by some inducers (including Hg 2+ ) operate in two modes, a regulated one that is activated by Ca 2+ and inhibited by cyclosporin A (CsA) and Mg 2+ , and an unregulated mode that is Ca 2+ -independent and insensitive to CsA and Mg 2+ (He & Lemasters, 2002).The objective of the present study was to compare mechanism(s) of action of heavy metals on in vitro rat liver mitochondrial function in the absence of exogenous Ca 2+ , P i and Mg 2+ in assay media.In consequence, the primary targets and the sequence of events of the heavy metal-induced mitochondrial impairment were elucidated.The involvement of regulated and unregulated pores in the mitochondrial dysfunction is discussed as well.

Isolation of mitochondria.
Rat liver mitochondria (RLM) were prepared according to a standard differential centrifugation procedure in a medium containing 250 mM sucrose, 3 mM Tris/HCl (pH 7.4) and 0.5 mM EGTA that was omitted in two last washings as described previously (Belyaeva et al., 2001).Protein content was determined by the Bradford method.The endogenous Ca 2+ content of these mitochondria was 10-15 nmol/ mg of protein as determined by flame photometry.
Mitochondrial swelling.Heavy metal-induced membrane permeabilization of isolated mitochondria was determined from their swelling in a medium containing 125 mM NH 4 NO 3 and 5 mM Tris/NO 3 (pH 7.4) after addition of Cd 2+ , Hg 2+ , or Cu 2+ (as chloride salts).As known, NH 4 + ions are capable of crossing the membrane only as protons and freely diffusing molecules of NH 3 .The nitrate anion (NO 3 -) is also freely permeant through the inner mitochondrial membrane, so the monitoring of mitochondrial swelling in NH 4 NO 3 medium makes it possible to estimate the proton membrane permeability of the mitochondria.Rotenone (5 mM) and oligomycin (5 mg/ml), if not otherwise mentioned, were present in all the media; 5 mM Tris/succinate (Succ) was administered where indicated in figures.Reactions were carried out at 20°C in a 2.5 ml chamber filled with incubation medium and contained 2.5 mg of mitochondrial protein.The swelling was monitored as apparent absorbance changes at 520 nm with the help of recording spectrophotometer, as before (Belyaeva et al., 2001).The results shown are representative of a series of four experiments.Mitochondrial respiration.The oxygen consumption rate of mitochondria was monitored at 26°C with a Clark oxygen electrode in a thermostatic closed chamber of 1.2 ml with magnetic stirring.Mitochondria (1 mg/ml of protein) were added to a medium containing 100 mM KCl, 20 mM Tris/HCl, 5 mM Tris/ succinate (pH 7.3), rotenone (5 mM), and oligomycin (5 mg/ml).Cd 2+ , Hg 2+ , or Cu 2+ (as chloride salts) at the concentrations tested was added to the medium one to two minutes after mitochondria; the basal respiration rate was estimated both in the absence and in the presence of different MPT effectors, which were added to the assay medium either from the start of the experiment or during incubation.Besides, at the end of each experiment, 32 mM DNP was administered to the medium and the uncoupled respiration rate was determined.The oxygen consumption rate values are presented as ng atom O/min per mg of mitochondrial protein.The results shown are representative or average of a series of at least three experiments.
Radiometric assay.The influence of heavy metals on the uptake of 137 Cs + by valinomycin-treated and succinate-energized mitochondria was determined by the Millipore filtration method, the details of which were described earlier (Belyaeva et al., 2002).In particular, the kinetics of the latter process is well characterized by coefficients of distribution (r t ) of the tracer between energized mitochondria and medium.These coefficients are calculated from equation: r = A MT /A MD , where A MT is radioactivity in 1 g of mitochondrial protein and A MD is radioactivity in 1 ml of the medium.Efflux of 137 Cs + from isolated mitochondria was estimated by the time (t 1/2 ) required for the coefficients (r t ) to decrease twice compared to those in the steady state.As known, the uptake of 137 Cs + by energized mitochondria in the presence of valinomycin (estimated by the coefficient r t ) characterizes the energetic condition of isolated mitochondria and indirectly the mitochondrial membrane potential (DY mito ) value.This method of DY mito monitoring is especially convenient for determination of the dynamics of DY mito dissipation.The assay medium contained 140 mM sucrose, 50 mM Tris/acetate (pH 7.4), 2.5 mM Tris/succinate, 4 mM rotenone, 1 mg/ml oligomycin, and 0.01 mM valinomycin.Mitochondria (0.5 mg protein/ml) were incubated at 20°C.Experiments were repeated five times, with data presented being representative.For preparation of the assay media, we used, when necessary, sucrose refined with the help of a cationite KU-2-8 column, and twice recrystalized KCl.All other chemicals were purchased from Sigma. 137Cs + was provided by ISOTOPE (Moscow, Russia).

RESULTS AND DISCUSSION
Using isolated organelles as a model, we compared the action of low (5-10 mM) and high (20-50 mM) concentrations of several heavy metals (Cd 2+ , Hg 2+ , and Cu 2+ ) on rat liver mitochondria function.As seen from Fig. 1, all the metals induced swelling of non-energized mitochondria in NH 4 NO 3 medium in a dose-dependent manner; however, they did it in a different way.In particular, at equimolar concentrations of the heavy-metal ions applied (20 mM), Cd 2+ produced maximal proton MMP (Fig. 1, trace 3).In comparison with Cd 2+ , Hg 2+ showed a smaller amplitude and much slower kinetics of the swelling (Fig. 1, trace 5).In turn, Cu 2+ action was characterized by a slow initial mitochondrial swelling rate followed by its further rapid release (Fig. 1, trace 7).
A comparison of the influence of the heavy metals on the mitochondrial respiratory function was conducted as well (Fig. 2).As found, the metals affected differently the basal and DNP-uncoupled respiration of the mitochondria, and all the effects were dose-dependent.Already at 10 mM, Cd 2+ strongly inhibited the uncoupler-stimulated respiration rate (Fig. 2A, trace 3), whilst the same concentrations of Hg 2+ (Fig. 2B, trace 3) and Cu 2+ (Fig. 2C, trace 3) decreased it less than by half (compare with trace 4).At this concentration, Cu 2+ produced the highest stimulation of the basal respiration rate.The sustained activation of the resting state respiration obtained under these conditions was equal to 150-200% for Cu 2+ and 30-50% for Cd 2+ and Hg 2+ (relative to corresponding control).20 mM Cu 2+ (Fig. 2C, trace 2) and Hg 2+ (Fig. 2B, trace 2) produced sustained activation of the basal respiration rate as well and for Cu 2+ it exceeded 300%.At the same [Cd 2+ ], only transient stimulation of the basal respiration was obtained (Fig. 2A, trace 2).At a higher (50 mM) concentration, Cu 2+ produced a sharp transient stimulation of the resting state respiration (comparable to that observed at 20 mM Cu 2+ ), and then a moderate sustained activation took place (Fig. 2C, trace 1).In the case of Cd 2+ , a transient potent activation of the basal respiration was followed by its depression (Fig. 2A, trace 1).Hg 2+ at 50 mM induced a strong inhibition of the resting state respiration, without any transient stimulation observed (Fig. 2B, trace 1).In turn, the uncoupler-stimulated respiration was practically stopped by this concentration of Cd 2+ or Hg 2+ , and was substantially depressed by Cu 2+ .
It was found also that all the metals promoted a sharp dose-dependent decrease in steady-state 137 Cs + uptake level of valinomycin-treated and succinate-energized mitochondria (reflecting DY mito dissipation) with different kinetics of 137 Cs + efflux (Fig. 3).Values of t 1/2 (see Materials and Methods) averaged from five different mitochondrial preparations and describing the kinetics for r t (i.e., the tracer distribution coefficients) were equal to 1, 2, and 4.5 min in experiments using 5 mM of Hg 2+ , Cd 2+ , and Cu 2+ , respectively (Fig. 3, traces 3), with deviation of t 1/2 not exceeding 10%.It is evident that the data correlate well with the findings obtained in the swelling and respiratory experiments (see above).
Thus, the heavy metal-produced mitochondrial dysfunction is attributed to (1) an enhanced membrane permeabilizaton that manifested in mitochondrial swelling and activation of basal respiration, (2) an inhibition of uncoupler-stimulated respiration, and (3) a DY mito dissipation.Among the metals, Cu 2+ exhibited the maximal stimulatory effect on the basal respiration and the minimal inhibitory action on DNP-uncoupled respiration whilst Cd 2+ promoted the strongest depression of the uncoupled respiration and the largest swelling in NH 4 NO 3 medium.Hg 2+ induced similar effects as Cd 2+ but all of them were less profound, except the DY mito loss.Hence, it is clear that both the mitochondrial respiratory chain and the membrane perme- ability are the primary targets of the deleterious influence of the heavy metals.Moreover, the data obtained here underscore the sequence of events taking place during the disturbance by the metals of the mitochondrial function.In particular, it looks like DY mito collapse is the primary event in the Cd 2+ and Hg 2+ -induced mitochondrial dysfunction in which the direct inhibitory effect on the respiratory chain components seemingly plays a dominant role, whereas the Cu 2+ -promoted DY mito dissipation is likely a consequence of its direct enhancing effect on the membrane permeability that prevails in the Cu 2+ -produced mitochondrial dysfunction.Moreover, our preliminary data as well as reports by other authors (Leblondel & Allain, 1984;Wojtczak et al., 1996;Garcia et al., 2000) indicate that mitochondrial ATP-sensitive K + channel could be involved in Cu 2+ -induced mitochondrial dysfunction.This issue is under investigation in our group.What is the main cause of the MMP promoted by the metals tested under the applied experimental conditions (i.e., in the absence of added Ca 2+ , P i and Mg 2+ in assay medium)?Previously, we showed that Cd 2+ -produced MMP checked under identical experimental conditions was partially sensitive to CsA, a potent pharmacological inhibitor of regulated MPT pores (Belyaeva et al., 2001;2002).Recently, our evidence has been confirmed and extended by Curti and colleagues (Dorta et al., 2003).As to the Cu 2+ -and Hg 2+ -induced MMP, we found here that it was completely insensitive to CsA as estimated polarographically (Fig. 4) and by measuring the swelling of both non-respiring and succinate-energized mitochondria in NH 4 NO 3 medium in the presence and in the absence of CsA (not shown).Markedly, DTT, a dithiol reductant and MPT effector, produced an additional stimulatory effect on the basal respiration disturbed by the heavy metals only if it was supplemented after high [Cd 2+ ] and [Hg 2+ ], and the activation was insensitive to CsA (Fig. 5).Besides, the DNP-uncoupled respiration rate depressed by high [Hg 2+ ] and [Cu 2+ ] was not restored significantly by DTT administration either in the absence (Fig. 5A) or in the presence (Fig. 5B) of CsA in the assay medium.This is in opposite to the situa-tion observed by us before in the case of Cd 2+ (Belyaeva & Korotkov, 2003).Hence, both regulated and unregulated (i.e., Ca 2+ -independent and insensitive to CsA and Mg 2+ , see He & Lemasters, 2002) MPT pores contribute in the heavy-metal produced MMP but the involvement differs substantially depending on the metal used.This aspect of the toxicity of the heavy metals will be discussed elsewhere (Belyaeva et al., in preparation).

Figure
Figure 3. Action of heavy metals on 137Cs+ distribution between respiring rat liver mitochondria and in-