Free radical scavenging action of the natural polyamine spermine in rat liver mitochondria

Free Radical Biology & Medicine 41 (2006) 1272 – 1281 www.elsevier.com/locate/freeradbiomed

Original Contribution

Free radical scavenging action of the natural polyamine spermine in rat liver mitochondria Irina G. Sava, Valentina Battaglia, Carlo A. Rossi, Mauro Salvi, Antonio Toninello ⁎ Unità per lo Studio delle Biomembrane, Istituto di Neuroscienze del CNR, Dipartimento di Chimica Biologica, Università di Padova, Viale G. Colombo 3, 35121 Padova, Italy Received 14 February 2006; revised 30 June 2006; accepted 8 July 2006 Available online 15 July 2006

Abstract The isoflavonoid genistein, the cyclic triterpene glycyrrhetinic acid, and salicylate induce mitochondrial swelling and loss of membrane potential (ΔΨ) in rat liver mitochondria (RLM). These effects are Ca2+-dependent and are prevented by cyclosporin A and bongkrekik acid, classic inhibitors of mitochondrial permeability transition (MPT). This membrane permeabilization is also inhibited by N-ethylmaleimide, butylhydroxytoluene, and mannitol. The above-mentioned pro-oxidants also induce an increase in O2 consumption and H2O2 generation and the oxidation of sulfhydryl groups, glutathione, and pyridine nucleotides. All these observations are indicative of the induction of MPT mediated by oxidative stress. At concentrations similar to those present in the cell, spermine can prevent swelling and ΔΨ collapse, that is, MPT induction. Spermine, by acting as a free radical scavenger, in the absence of Ca2+ inhibits H2O2 production and maintains glutathione and sulfhydryl groups at normal reduced level, so that the critical thiols responsible for pore opening are also consequently prevented from being oxidized. Spermine also protects RLM under conditions of accentuated thiol and glutathione oxidation, lipid peroxidation, and protein oxidation, suggesting that its action takes place by scavenging the hydroxyl radical. © 2006 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Spermine; Hydroxyl radical; Oxidative stress; Mitochondrial permeability transition; Free radicals

Natural polyamines are ubiquitous metabolites in prokaryotic and eukaryotic cells and are universally known as essential molecules for physiological processes such as cell growth and differentiation (for a review see [1]). Several reports address specific, important activities undertaken by polyamines, including activation of kinases involved in signal transduction pathways, regulation of ion channel gating, and modulation of oxidative processes [2–4]. Polyamines are aliphatic amines with three or four methylene carbon chains connecting the amino and/or imino groups; they are positively charged at physiological pH, thus behaving as polycationic molecules, and exhibit increased hydrophilicity and flexibility with increasing Abbreviations: ΔΨ, membrane potential; BHT, butylhydroxytoluene; CsA, cyclosporin A; GEN, genistein; GLYC, glycyrrhetinic acid; MPT, mitochondrial permeability transition; NEM, N-ethylmaleimide; RLM, rat liver mitochondria; ROS, reactive oxygen species; SAL, salicylate; SPM, spermine. ⁎ Corresponding author. Fax: +39 49 8276133. E-mail address: [email protected] (A. Toninello). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.07.008

numbers of charged amino and imino groups [5]. These characteristics mediate the binding of polyamines to negatively charged cellular macromolecules such as nucleic acids [6] and components of membranes such as acidic (phospho)lipids and negatively charged residues of membrane-bound proteins [7]. In this regard, it has been demonstrated that spermine and spermidine bind to the mitochondrial membrane at two specific binding sites and putrescine to one site, all having high binding capacity and low affinity and exhibiting monocoordination. This binding represents the first step of polyamine transport in mitochondria, which has electrophoretic behavior and requires high ΔΨ values in order to operate. This transport system is composed of a channel with two asymmetric energy barriers (for a review on polyamine binding and transport, see [8]). The interaction between polyamines, in particular spermine, and mitochondria has several physiological implications, as it activates the transport of Ca2+, phosphate, and several enzymes. Spermine also stimulates the activity of pyruvate

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

dehydrogenase and citrate synthase, inhibits ATP hydrolysis by F0F1ATPase, and maintains the ATP concentration at high levels [8].

1273

Polyamines are inhibitors of mitochondrial permeability transition (MPT) in isolated mitochondria. In particular, spermine may be considered as one of the most powerful physiological

Fig. 1. Spermine prevents mitochondrial swelling induced by pro-oxidants. RLM were incubated for 15 min in standard medium under conditions described under Materials and methods. (A) 50 μM genistein (GEN), (B) 10 μM glycyrrhetinic acid (GLYC), and (C) 0.5 mM salicylate (SAL) were present. Concentrations used were 1 μM CsA, 10 μM NEM, 25 μM BHT, 100 μM mannitol (MAN), and 100 μM spermine (SPM). NEM has to be used with care because higher concentrations, e.g., 100 μM, cause GSH depletion. Control curves: absence or presence of 50 μM Ca2+ or pro-oxidants without Ca2+. Downward deflection: mitochondrial swelling. Insets: calculations of IC50 value for SPM evaluated as percentage of ΔA extent after 15 min, with respect to that of control. Assays were performed four times, with comparable results.

1274

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

inhibitors. MPT is caused by the opening of the transition pore, a large proteinaceous pore, which takes place when mitochondria are incubated in the presence of supraphysiological Ca2+ concentrations in combination with a wide variety of inducing agents or inducing conditions. The transition pore is a dynamic multiprotein complex, containing the translocase of adenine nucleotides, located most probably at the gap junctions between the outer and the inner membranes [9–11]. Opening of the pore, which exhibits several levels of conductance and negligible ion selectivity, leads to colloid-osmotic matrix swelling; collapse of the electrochemical gradient; loss of glutathione, Ca2+, and other endogenous cations; and oxidation of thiol groups and pyridine nucleotides, which break down and release nicotinamide in the medium. All these events compromise cellular energy metabolism and Ca2+ homeostasis to the point of provoking cell death by apoptosis [12]. The protective effect of spermine on MPT in various types of mitochondria has been investigated using, in addition to Ca2+,

several other inducers such as phosphate, long-chain acyl CoA, tert-butylhydroperoxide, phenylarsine oxide, and carboxyatractyloside [8]. An important event which occurs during the establishment of MPT is efflux of glutathione from the matrix [13]. The action of mitochondrial glutathione is most probably directed against oxidative stress [14], and its deficiency is generally associated with mitochondrial damage [15]. However, spermine is able to block this efflux from liver mitochondria, but at a concentration which is four times higher than that necessary to prevent MPT [16]. To explain these results, the authors suggested that spermine has an antioxidant property, as previously proposed [17]. Other authors had observed this property in biological systems, although not in mitochondria [18]. Taking into account these considerations, the first aim of this study was to establish if spermine can protect against MPT inducers such as genistein, glycyrrhetinic acid, and salicylate, whose mechanisms of interaction with the respiratory

Fig. 2. Spermine protects RLM against ΔΨ collapse induced by pro-oxidants. Experimental conditions and reagent concentrations were as in Fig. 1. ΔE value: electrode potential. Assays were performed four times with comparable results.

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

chain and the consequent generation of ROS are well documented (see [19–21], respectively). If this were so, another aim—the more important—was to study the mechanism by means of which spermine carries out its protective effect against MPT inducers. Materials and methods Rat liver mitochondria (RLM) were isolated by conventional differential centrifugation in a buffer containing 250 mM sucrose, 5 mM Hepes (pH 7.4), and 1 mM EGTA; EGTA was omitted from the final washing solution [22]. Protein content was measured by the biuret method, with bovine serum albumin as a standard [23]. Mitochondria (1 mg protein/ml) were incubated in a water-jacketed cell at 20°C. The standard medium contained 250 mM sucrose, 10 mM Hepes (pH 7.4), 5 mM succinate, 50 μM Ca2+, and 1.25 μM rotenone. Variations and/or other additions are given with each experiment. Mitochondrial swelling was determined by measuring the apparent absorbance change of mitochondrial suspensions at 540 nm on a Uvikon Model 922 spectrophotometer (Kontron, Eching, Germany) equipped with thermostatic control. Membrane potential was calculated on the basis of distribution of the lipid-soluble cation tetraphenylphosphonium through the inner membrane, measured using a tetraphenylphosphonium-specific electrode prepared in our laboratory according to published procedures [24]. The protein sulfhydryl oxidation assay was performed according to Santos et al. [25]. The oxidation of glutathione was performed as in Tietze [26]. The redox state of endogenous pyridine nucleotides was followed fluorometrically in an Aminco–Bowman 4-8202 spectrofluorometer, with excitation at 354 nm and emission at 462 nm. Oxygen uptake was measured with a Clark electrode. The production of H2O2 in mitochondria was measured fluorometrically by the scopoletin method in an Aminco– Bowman 4-8202 spectrofluorometer. It should be noted that the measured amount of H2O2 refers to that diffusing out of mitochondria, as the enzyme for its determination (horseradish peroxidase) cannot enter mitochondria. This measurement gives a qualitative indication but cannot be considered for rigorous quantitative evaluations [27]. Lipid peroxidation was determined by monitoring the formation of thiobarbituric acid-reactive species (TBARS) according to Wills and Wilkinson [28]. TBARS were determined spectrophotometrically at 532 nm using the extinction coefficient of 1.56 × 10 5 M −1 cm−1. Protein carbonyls were measured spectrophotometrically at 360 nm using the extinction coefficient of 22,000 M−1 cm−1 according to Reznick et al. [29].

1275

the presence of Ca2+, greatly decrease the apparent absorbance of mitochondrial suspension, indicative of mitochondrial swelling. This colloid-osmotic alteration is paralleled by rapid and almost complete collapse of ΔΨ (Figs. 2A–2C). These alterations in membrane permeability are completely preserved by the immunosuppressant cyclosporin A (CsA), a well-known inhibitor of MPT, and by the alkylating agent N-ethylmaleimide (NEM), the antioxidant butylhydroxytoluene (BHT), and mannitol (Figs. 1A–1C and 2A–2C). That is, the results shown in Figs. 1 and 2 summarize the induction of MPT by various agents, highlighting the fact that the phenomenon is dependent not only on Ca2+, but also on the presence of a prooxidizing agent. It should be taken into account that, in the reported experiments, no mitochondrial alteration is observable in the absence of pro-oxidants or in their presence but without Ca2+ (see control traces). Figs. 1 and 2 also show that mitochondrial swelling and ΔΨ collapse are completely prevented

Results The results shown in Figs. 1 and 2 show the known effects of various pro-oxidizing agents such as genistein (A), glycyrrhetinic acid (B), and salicylate (C), in inducing the phenomenon of MPT. Figs. 1A–1C show that the above compounds, in

Fig. 3. Spermine inhibits mitochondrial (A) thiols and (B) glutathione oxidation. Experimental conditions and concentrations of GEN, GLYC, and SAL were as in Fig. 1. When present, spermine was 100 μM. Mean values ± SD of four experiments are shown.

1276

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

Fig. 4. Spermine inhibits pyridine nucleotide oxidation. Experimental conditions and reagent concentrations were as in Fig. 1. Three additional experiments gave almost identical results.

by 100 μM spermine. The insets of Fig. 1 show the dosedependent effect of spermine with calculation of the IC50. Swelling and ΔΨ collapse are accompanied by oxidative stress, evidenced by strong oxidation of sulfhydryl groups (36% with genistein, 37% with glycyrrhetinic acid, 54% with salicylate) (Fig. 3A), glutathione (18% with genistein, 20% with glycyr-

Fig. 5. Stimulation of oxygen uptake by pro-oxidants in the absence of Ca2+. RLM were incubated in standard medium, deprived of Ca2+, under conditions described under Materials and methods. Concentrations of GEN, GLYC, and SAL were as in Fig. 1. Control curve, absence of pro-oxidant. Four additional experiments exhibited the same trend.

rhetinic acid, 27% with salicylate) (Fig. 3B), and pyridine nucleotides (about −200, −250, and −300 arbitrary units of fluorescence change for the pro-oxidants, respectively, as above) (Fig. 4). All these effects, as previously reported [19–21], are almost completely prevented by NEM and BHT (results not shown). Also spermine, at 100 μM concentration, similarly inhibits the oxidation of thiol, glutathione, and pyridine nucleotides (Fig. 3). The observed effects of spermine, although previously explained in various ways by several authors (e.g., see [30]), led

Fig. 6. Effects of spermine on hydrogen peroxide generation in the absence of Ca2+. Experimental conditions and reagent concentrations were as in Fig. 3, except that Ca2+ was absent. In order to statistically analyze the continuous curves of this array, the amounts of H2O2, generated at 1, 3, and 5 min, were calculated by standard calibration for each of the experiments (see data points). Mean values ± SD of four experiments are shown.

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

1277

performed in the absence of Ca2+, a condition under which the transition pore does not open (see Figs. 1 and 2). However, the observed effects are necessary to predispose mitochondria toward pore opening and its amplification. Fig. 7A shows that all the pro-oxidants are able to cause oxidation of thiol groups (25% with genistein, 23% with glycyrrhetinic acid, 26% with salicylate). An almost similar effect is observed in Fig. 7B, the oxidation of glutathione (13% with genistein, 15% with glycyrrhetinic acid, 17% with salicylate). Effects on both thiols and glutathione are prevented by NEM and BHT (data not reported), but also by spermine, which exhibits full protection at 100 μM concentration. It should be emphasized that, under these conditions, pyridine nucleotides are not oxidized (data not shown). Spermine at 100 μM can also protect RLM against oxidative stress under more severe conditions than those of Fig. 8. This figure shows that a concentration of glycyrrhetinic acid higher than that normally used (10 μM), i.e., 50 μM, can induce thiol and glutathione oxidation by more than 60 and 50%, respectively. The presence of spermine strongly inhibits this oxidation. In order to obtain other convincing results about spermine protection on oxidative stress and in particular against the effect of the hydroxyl radical, experiments on lipid peroxidation and protein oxidation were performed. The results reported in Fig. 9A show that glycyrrhetinic acid, at the same concentration that induces MPT (10 μM), can induce lipid peroxidation by increasing the generation of TBARS by about 60% with respect to the control. This effect is entirely prevented in the presence of spermine. If glycyrrhetinic acid is substituted for by ascorbate plus Fe2+ lipid peroxidation is strongly enhanced by increasing TBARS production by 300%. Spermine exhibits significant protection also in this case (Fig. 9B). Glycyrrhetinic acid at 10 μM also induces protein oxidation by increasing the generation of carbonyl groups by about 50% with respect to the control. The presence of spermine Fig. 7. Spermine inhibits mitochondrial (A) thiols and (B) glutathione oxidation in the absence of Ca2+. RLM were incubated for 15 min in standard medium in the absence of Ca2+ under conditions described under Materials and methods. Concentrations of GEN, GLYC, and SAL were as in Fig. 1. Mean values ± SD of four experiments are shown.

us to take into account another possibility not yet considered— that is, the free radical scavenging action of this polyamine. Figs. 5 and 6 also reveal some typical effects of pro-oxidants at the mitochondrial level, i.e., increase in oxygen uptake and generation of ROS (hydrogen peroxide is detected in this case). The effects of spermine on the stimulation of oxygen uptake (most probably inhibitory) are not reported, because they are difficult to evaluate precisely, as spermine is contemporaneously transported into the matrix by an electrophoretic mechanism [8] requiring an increase in oxygen uptake. Instead, spermine partially inhibits H2O2 production by pro-oxidants (Fig. 6). The production of ROS is responsible for the oxidative stress in RLM evidenced by Figs. 7A and 7B, showing the oxidation of thiol groups and glutathione, respectively. It should be emphasized that the experiments of Figs. 5–7 were

Fig. 8. Spermine inhibits high levels of thiol and glutathione oxidation induced by high concentration of glycyrrhetinic acid. Experimental conditions were as in Fig. 1, in the absence of Ca2+. GLYC was present at 50 μM concentration. Mean values ± SD of three experiments are shown.

1278

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

gical Ca2+ concentrations. Spermine can also maintain the reduced state of sulfhydryl groups (Fig. 3A), glutathione (Fig. 3B), and pyridine nucleotides (Fig. 4), oxidized by the above pro-oxidants, at very high levels (see controls). All these results confirm the known protective action of spermine on MPT [8,28], here also evidenced against the above drugs, of which the reaction mechanisms have been demonstrated. As also reported in previous papers describing the induction of MPT by genistein [19], glycyrrhetinic acid [20], and salicylate [21], ROS are responsible for oxidative stress, with oxidation of critical thiol groups (Fig. 3A) located on adenine nucleotide translocase (AdNT), which leads to the opening of the transition pore when Ca2+ is also present [32]. This oxidative stress is the result not only of the reactivity of ROS produced by pro-oxidants, but also of ROS produced by pore opening [12]. In this regard, it is to be emphasized that pore opening establishes the so-called redox catastrophe [12], which is characterized by ROS hyperproduction, loss of oxidized glutathione, and depletion of oxidized pyridine nucleotides by their rupture and release of nicotinamide. All this is accompanied by bioenergetic collapse, with depletion of ATP and ADP. To induce ROS production, the above inducers target the mitochondrial respiratory chain at the level of the iron–sulfur center N-2 of Complex I (salicylate) and cytochrome bH heme of the bc1 complex (glycyrrhetinic acid and genistein) by producing hydrogen peroxide by means of a different mechanism [19–21]. Subsequently hydrogen peroxide, by interacting with the transition metals of the respiratory chain, such as iron, can produce the potent toxic hydroxyl radical, by means of a U Fenton-type reaction: H2O2 + Fe(II) → HO + OH− + Fe(III). Glutathione and pyridine nucleotide oxidation (Figs. 3B and 4), which demonstrates the involvement of the glutathione peroxidase/glutathione reductase system, further confirms the above statement on ROS effects. Fig. 9. Spermine inhibits lipid peroxidation induced by (A) glycyrrhetinic acid and (B) ascorbate plus Fe2+. Experimental conditions were as in Fig. 1, in the absence of Ca2+. GLYC was present at 10 μM, ascorbate at 100 μM, and Fe2+ at 20 μM concentrations. Mean values ± SD of four experiments are shown.

completely prevents oxidation (Fig. 10). Spermine protection against a stronger protein oxidation induced by ascorbate/Fe2+ was not possible to assess due to an interaction of the polyamine with the assay reagents which alters the measurement. All experiments were conducted in a sucrose-based medium in order to compare results with other studies on MPT. However, the use of a saline-based medium gave almost identical results. Discussion The experimental results described above show that, at 100 μM concentration, very close to that present in free form in the cytosol of rat liver [31], spermine can prevent the mitochondrial swelling and ΔΨ collapse induced by genistein (Figs. 1A and 2A), glycyrrhetinic acid (Figs. 1B and 2B), and salicylate (Figs. 1C and 2C) in the presence of supraphysiolo-

Fig. 10. Spermine inhibits protein oxidation induced by glycyrrhetinic acid. Experimental conditions were as in Fig. 1, in the absence of Ca2+. GLYC was present at 10 μM concentration. Mean values ± SD of five experiments are shown.

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

Although Figs. 1–4 clearly confirm that spermine is an inhibitor of MPT induction, they do not elucidate its protective mechanism. Several mechanisms have been proposed to explain this, including triggering of the protein phosphorylation/ dephosphorylation process [30] and interaction with a specific MPT inhibitor site in the matrix or with negatively charged head groups of inner membrane phospholipids, in particular the cardiolipin annular domain of AdNT [33]. It has also been suggested that spermine exerts its effect by increasing the affinity of ADP for its inhibitory binding [34]. Another proposal is that the binding of spermine to the so-called S1 site, responsible for polyamine transport in RLM, is also competent in preventing MPT [35]. Figs. 5–7 show that oxidative stress, although of reduced extent, is also induced by the above agents in the absence of Ca2+—that is, without pore opening (see also Figs. 1 and 2) and without the establishment of a redox catastrophe and bioenergetic collapse (compare Figs. 7A and 7B with Figs. 3A and 3B). Production of ROS is predictable by considering the increase in oxygen uptake observed in Fig. 5. Fig. 6 confirms that all the compounds produce hydrogen peroxide. Note that the real amounts of H2O2 generated by the various agents are probably higher than those reported because, as soon as it is generated, H2O2 reacts with its targets and is also transformed into other ROS (see also description of method under Materials and methods). The establishment of oxidative stress is demonstrated by the oxidation of sulfhydryl groups (Fig. 7A) and glutathione (Fig. 7B). The observation that, under these conditions, pyridine nucleotides are not oxidized demonstrates that their oxidation, detected in the presence of Ca2+ (Fig. 4), is an effect of pore opening rather than a cause. Furthermore, the failure of pyridine nucleotides to oxidize, when considered together with the oxidation of glutathione, demonstrates that glutathione peroxidase activity is not accompanied by that of

1279

glutathione reductase. Note that the observed effects represent preliminary steps predisposing the membrane to subsequent opening of the pore. Figs. 7A and 7B, showing that spermine can prevent oxidation of thiols and glutathione, also under conditions under which these species are strongly oxidized (Fig. 8), thus behaving like the antioxidant BHT and the alkylating agent NEM, strongly supports the hypothesis that spermine acts as a free radical scavenger. Other convincing results concerning this proposal are reported in Figs. 9 and 10, showing a protective effect of spermine on lipid peroxidation and protein oxidation, respectively. As these oxidations are due to the effect of hydroxyl radical, a suggestion is that this type of ROS is involved in these processes. The observations that mannitol, a well-known scavenger of hydroxyl radical [36], inhibits mitochondrial swelling and protects ΔΨ drop (Figs. 1 and 2) strongly support this proposal. The incomplete inhibition is most probably due to the delayed entry of mannitol in the matrix as the molecule can cross the membrane only after the pore begins to open. Further confirmation of the above-mentioned hypothesis is given by Casero's group, who demonstrated “in vitro” DNA protection by spermine against radical attack by means of a scavenging action [37]. This scavenging is due to the reaction of spermine with the hydroxyl radical and the production of spermine dialdehyde (1,12-bis1,12-dioxo-4,9-diazododecane) and is consistent with EPR, NMR, and mass spectrometry results [37]. Fig. 11 shows the proposed steps of this reaction. The partial inhibition by spermine of H2O2 generation in the absence of Ca2+ (Fig. 6) demonstrates that this ROS can also be scavenged by spermine, although to a lesser extent. It is noteworthy that the production of spermine dialdehyde could potentially have some toxic effects [38], although far lower in intensity than those of the hydroxyl radical. In fact, as shown in Figs. 1 and 2, spermine completely prevents

Fig. 11. Proposed mechanism for hydroxyl radical scavenging by spermine.

1280

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281

mitochondrial swelling and ΔΨ drop, thus demonstrating that the produced dialdehyde is ineffective. In any case, possible overproduction of this dialdehyde can be scavenged by the general detoxifiers of mitochondria, including glutathione and glutathione peroxidase [37]. As Figs. 3B and 7B show, the level of reduced glutathione remains very high, like that of controls, after treatment with spermine. In view of the importance of the number of electric charges in the molecules of polyamines required for inducing the protection against MPT induction [39], among the various mechanisms previously proposed is electrostatic interaction of the polyamine with anionic charges located on pore-forming structures [33,39]. However, it is predictable that strong electrostatic interactions are necessary for this effect to take place. Instead, it has been demonstrated that spermine binds to mitochondrial membranes by means of weak interactions [35], thus raising several doubts about the above proposal. As the generation of ROS by the above-mentioned prooxidants takes place in the inner mitochondrial compartment, prevention of the consequent oxidative stress by spermine requires transport of the polyamine into the mitochondrial matrix. This observation highlights a new physiological role for electrophoretic spermine transport in mitochondria, which requires membrane energization to be operating [40]. Thus, the importance of the number of electrical charges for polyamine protection, as mentioned above, may be related to the facilitation of highly charged molecules (like spermine) entering the mitochondrial matrix. This transport also protects mitochondrial DNA against free radical attack, as observed in the ΦX-174 plasmid DNA and Cu(II)/H2O2-dependent oxygen radical-generating systems [37]. Acknowledgment The authors are grateful to Mr. Mario Mancon for his skilful technical assistance. References [1] Tabor, C. W.; Tabor, H. Polyamines. Annu. Rev. Biochem 53:749–790; 1984. [2] Neel, B. G.; Tonks, N. K. Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell. Biol. 9:193–204; 1997. [3] Ficker, E.; Taglialatela, M.; Wible, B. A.; Henley, C. M.; Brown, A. M. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 266:1068–1072; 1994. [4] Schuber, F. Influence of polyamines on membrane functions. Biochem. J. 260:1–10; 1989. [5] Weiger, T. M.; Langer, T.; Hermann, A. External action of di- and polyamines on maxi calcium-activated potassium channels: an electrophysiological and molecular modeling study. Biophys. J. 74:722–730; 1998. [6] Martin-Sanz, P.; Hopewell, R.; Brindley, D. N. Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect. FEBS Lett. 179:262–266; 1985. [7] Gonzalez-Bosch, C.; Miralles, V. J.; Hernandez-Yago, J.; Grisolia, S. Spermidine and spermine stimulate the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria. Biochem. Biophys. Res. Commun. 149:21–26; 1987.

[8] Toninello, A.; Salvi, M.; Mondovì, B. Interaction of biologically active amines with mitochondria and their role in the mitochondrial-mediated apoptosis. Curr. Med. Chem. 11:2349–2374; 2004. [9] Beutner, G.; Ruck, A.; Riede, B.; Welte, W.; Brdiczka, D. Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett. 396:189–195; 1996. [10] Beutner, G.; Ruck, A.; Riede, B.; Brdiczka, D. Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore: implication for regulation of permeability transition by the kinases. Biochim. Biophys. Acta 1368:7–18; 1998. [11] O'Gorman, E.; Beutner, G.; Dolder, M.; Koretsky, A. P.; Brdiczka, D.; Wallimann, T. The role of creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett. 414:253–257; 1997. [12] Susin, S. A.; Zamzami, M.; Kroemer, G. Mitochondria as regulators of apoptosis: doubt no more. Biochim. Biophys. Acta 194:1276–1281; 1998. [13] Savage, M. K.; Jones, D. P.; Reed, D. J. Calcium- and phosphatedependent release and loading of glutathione by liver mitochondria. Arch. Biochem. Biophys. 290:51–56; 1991. [14] Reed, D. J. Glutathione: toxicological implications. Annu. Rev. Pharmacol. Toxicol. 30:603–631; 1990. [15] Martensson, J.; Meister, A. Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects. Proc. Natl. Acad. Sci. USA 88:4656–4660; 1991. [16] Rigobello, M. P.; Toninello, A.; Siliprandi, D.; Bindoli, A. Effect of spermine on mitochondrial glutathione release. Biochem. Biophys. Res. Commun. 194:1276–1281; 1993. [17] Tadolini, B. Polyamine inhibition of lipoperoxidation. Biochem. J. 249:33–36; 1988. [18] Das, K. C.; Misra, H. P. Hydroxyl radical scavenging and singlet oxygen quenching by naturally occurring polyamines. Mol. Cell. Biochem. 262:127–133; 2004. [19] Salvi, M.; Brunati, A. M.; Clari, G.; Toninello, A. Interaction of genistein with the mitochondrial electron transport chain results in opening of the membrane transition pore. Biochim. Biophys. Acta 1556:187–196; 2002. [20] Fiore, C.; Salvi, M.; Palermo, M.; Sinigaglia, G.; Armanini, D.; Toninello, A. On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid. Biochim. Biophys. Acta 1658:195–201; 2004. [21] Battaglia, V.; Salvi, M.; Toninello, A. Oxidative stress is responsible for mitochondrial permeability transition induction by salicylate in liver mitochondria. J. Biol. Chem. 280:33864–33872; 2005. [22] Schneider, W. C.; Hogeboon, H. G. Intracellular distribution of enzymes: XI. Glutamic dehydrogenase. J. Biol. Chem. 204:233–238; 1953. [23] Gornall, A. G.; Bardawill, C. J.; David, M. M. Determination of serum proteins by means of the biuret method. J. Biol. Chem. 177:751–766; 1949. [24] Toninello, A.; Salvi, M.; Colombo, L. The membrane permeability transition in liver mitochondria of the great green goby Zosterisessor ophiocephalus (Pallas). J. Exp. Biol. 203:3425–3434; 2000. [25] Santos, A. C.; Uyemura, S. A.; Lopes, J. L.; Bazon, J. N.; Mingatto, F. E.; Curti, C. Effect of naturally occurring flavonoids on lipid peroxidation and membrane permeability transition in mitochondria. Free Radic. Biol. Med. 24:1455–1461; 1998. [26] Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27:502–522; 1969. [27] Loschen, G.; Azzi, A.; Flohè, L. Mitochondrial H2O2 formation: relationship with energy conservation. FEBS Lett. 33:84–87; 1973. [28] Wills, E. D.; Wilkinson, A. E. Release of enzymes from lysosomes by irradiation and the relation of lipid peroxide formation to enzyme release. Biochem. J. 99:657–664; 1966. [29] Reznick, Z.; Packer, L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 233:357–363; 1994. [30] Lapidus, R. G.; Sokolove, P. M. Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria. FEBS Lett. 313:314–318; 1992. [31] Watanabe, S.; Kusama-Eguchi, K.; Kobayashi, H.; Igarashi, K. Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver. J. Biol. Chem. 266:20803–20809; 1991.

I.G. Sava et al. / Free Radical Biology & Medicine 41 (2006) 1272–1281 [32] McStay, G. P.; Clarke, S. J.; Halestrap, A. P. Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem. J. 367:541–548; 2002. [33] Lapidus, R. G.; Sokolove, P. M. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: an investigation of mechanism. Arch. Biochem. Biophys. 306:246–253; 1993. [34] Lapidus, R. G.; Sokolove, P. M. The mitochondrial permeability transition: interactions of spermine, ADP, and inorganic phosphate. J. Biol. Chem. 269:18931–18936; 1994. [35] Dalla Via, L.; Di Noto, V.; Siliprandi, D. Spermine binding to liver mitochondria. Biochem. Biophys. Acta 1284:247–253; 1996. [36] Goldstein, S.; Czapski, G. Mannitol as an OHU scavenger in aqueous solutions and in biological systems. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 46:725–729; 1984.

1281

[37] Ha, H. C.; Sirisoma, N. S.; Kuppusamy, P.; Zweier, J. L.; Woster, P. M.; Casero, R. A. Jr. The natural polyamine spermine functions directly as a free radical scavenger. Proc. Natl. Acad. Sci. USA 95:11140–11145; 1998. [38] Morgan, D. M. Oxidized polyamines and the growth of human vascular endothelial cells: prevention of cytotoxic effects by selective acetylation. Biochem. J. 242:347–352; 1987. [39] Tassani, V.; Biban, C.; Toninello, A.; Siliprandi, D. Inhibition of mitochondrial permeability transition by polyamines and magnesium. Biochem. Biophys. Res. Commun. 207:661–667; 1995. [40] Toninello, A.; Dalla Via, L.; Siliprandi, D.; Garlid, K. D. Evidence that spermine, spermidine and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter. J. Biol. Chem. 267:18393–18397; 1992.