Mitochondrial permeability transition induced by 1-hydroxyethyl radical

Free Radical Biology & Medicine, Vol. 28, No. 2, pp. 273–280, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(99)00236-1

Original Contributions MITOCHONDRIAL PERMEABILITY TRANSITION INDUCED BY 1-HYDROXYETHYL RADICAL KOICHI SAKURAI,*† DETCHO A. STOYANOVSKY,† YUKIO FUJIMOTO,*

and

ARTHUR I. CEDERBAUM†

*Department of Biochemistry, Hokkaido College of Pharmacy, 7-1 Katsuraoka-cho, Otaru Hokkaido, Japan and †Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY, USA (Received 8 April 1999; Revised 26 October 1999; Accepted 28 October 1999)

Abstract—Impairment of mitochondrial functions has been found in ethanol-induced liver injury. Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat liver microsomal systems. Experiments were carried out to evaluate the ability of HER to cause mitochondrial swelling as an indicator of the mitochondrial permeability transition (MPT). Electron spin resonance (ESR) spectroscopy was used to detect HER and to study its interaction with mitochondria. The ESR signal intensity of the spin adduct formed from ␣-(4-pyridyl-1-oxide) N-tert-butylnitrone (POBN) and HER generated from either a thermic decomposition of 1,1⬘-dihydroxyazoethane (DHAE) or a Fenton reaction system containing ethanol was markedly diminished by the addition of mitochondria, indicating an interaction between HER and mitochondria. Exposure of rat liver mitochondria to HER generated from either system caused swelling, as reflected by a decrease in absorbance at 540 nm, in a HER concentration-dependent and a cyclosporin A–sensitive manner. Mitochondrial swelling was also induced in the Fenton reaction system without ethanol. The DHAE-dependent generation of HER in mitochondrial suspension resulted in a decrease of membrane protein thiols and collapse of the membrane potential (⌬ ⌿). The swelling induced by HER was prevented by glutathione and vitamin E, but not by superoxide dismutase. Catalase did not prevent the swelling caused by the acetaldehyde/hydroxylamine O-sulfonate (HOS) system, but was inhibitory in the Fenton reaction system with or without ethanol. These results indicate that HER, as well as hydroxyl radical, can induce the MPT, and suggest the possibility that the collapse of ⌬ ⌿caused by HER may, at least in part, contribute to impairment of mitochondrial function caused by ethanol and in ethanol-induced liver injury. © 2000 Elsevier Science Inc. Keywords—Mitochondria, Ethanol, 1-hydroxyethyl, Radical, ESR, Oxidation, Thiols, Vitamin E, Free radicals

INTRODUCTION

an important role among the many factors that help to initiate or control apoptosis [9 –11]. For example, reduction in the mitochondrial membrane potential (⌬ ⌿) and formation of the permeability transition pore followed by swelling of mitochondria appear to occur during the early phase of apoptosis [10, 11]. Apoptotic cells were observed in the livers of rats that exhibited alcohol liver injury [12, 13]. The mechanism by which ethanol may cause hepatocyte apoptosis is not known; ethanol itself can not directly cause the mitochondria permeability transition (MPT). Ethanol has been shown to be oxidized to a free radical metabolite, the 1-hydroxyethyl radical (HER) by liver microsomal systems (14 –16). HER has also been detected in rat and deer mice in vivo [17, 18]. Covalent bound HER/protein adducts have been detected, and these adducts have immunological properties, leading to

There is much interest that ethanol may promote an imbalance between pro-oxidant and antioxidant systems in favor of the former and that ethanol-induced oxidative stress may play a role in the liver damage produced by ethanol [1, 2]. Mitochondria are an important target for ethanol toxicity as chronic administration of ethanol causes changes in the structure and function of liver mitochondria [3– 6]. In addition, the activity and content of enzymes related to ATP synthesis within the mitochondrial membrane are decreased in ethanol-fed rats [3, 7, 8]. Recently, mitochondrial events were shown to play Address correspondence to: Dr. Arthur I. Cederbaum, Department of Biochemistry, Box 1020, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA; Tel: (212) 241-7285; Fax: (212) 996-7214. 273

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the formation of antibodies that can specifically recognize the HER moiety of the protein adduct [19]. Such antibodies have been detected in the blood of patients with alcoholic cirrhosis [20]. However, the role of HER in alcohol liver injury has not been clearly defined. The goal of the current study was to evaluate the ability of HER to interact with mitochondria. To minimize the effect of various species of oxygen radicals, HER generated via thermic decomposition of DHAE was utilized in these experiments and results compared with that produced by HER generated from a Fenton reaction system in the presence of ethanol. The effect of HER on the MPT, membrane protein thiols, and the ⌬ ⌿, and modulation of these effects by antioxidants was the focus of this study. MATERIALS AND METHODS

Acetaldehyde, vitamin E, SOD (from bovine erythrocytes), catalase (from bovine liver, thymol free) and rhodamine 123 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hydroxylamine-O-sulfonic acid (HOS) was from Aldrich Chemical Co. (Milwaukee, WI, USA). Cyclosporin A (Cys A), reduced glutathione, and mannitol were from Wako Pure Chemical Ltd., (Osaka, Japan). Vitamin E and Cys A were dissolved in ethanol. All other reagents used were of the highest grade from commercial suppliers. Isolation of mitochondria Male rats of the Wistar strain, weighting about 190 g were starved overnight before being killed. All steps of the mitochondrial isolation were carried out at 4°C. The liver was cut into small pieces with scissors, and homogenized in 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and 0.1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N⬘N⬘tetraacetic acid (EGTA) with two strokes of a homogenator at 800 rpm; 20 ml of medium was used per g of liver. The homogenate was centrifuged at 600 ⫻ g for 10 min. The pellet was discarded, and the supernatant was centrifuged at 7700 ⫻ g for 10 min. The mitochondrial pellet was washed once again under the same conditions except an EGTA-free medium was used. Finally, the mitochondrial pellet was resuspended in 10 mM TrisHCl buffer (pH 7.4) containing 0.25 M sucrose at a concentration of about 40 mg of mitochondrial protein per ml. The protein concentration was determined using the Micro BCA kit (Pierce, Rockford, IL, USA), with bovine serum albumin as a standard. ESR measurements DHAE was prepared as previously described in [21]. Briefly, HOS (0.13 g) was dissolved in 50% ethanol

(1.57 ml; 4°C) containing 10% KOH. Acetaldehyde (0.3 ml) was added to form DHAE and the reaction mixture was incubated at 4 –10°C for 5 min. Immediately before the experiments, the DHAE solution was titrated with HCl to pH 7.4 (final volume of the reaction solution is 3 ml). The other HER-generating system was the Fenton reaction system consisting of 0.1 mM H2O2 and 20 ␮M ferrous ammonium sulfate in the presence of ethanol (0.2 M). Fenton reactions were initiated by the addition of Fe2⫹ (20 ␮M, unless otherwise indicated) in the absence or presence of 0.2 M ethanol. H2O2 was always present at a final concentration of 0.1 mM for Fenton reactions. ESR observations were performed with a Brucker ECS 106 (Bruker Instruments, Billerica, MA, USA) or a JEOL model JES-RE1X (JEOL USA, Peabody, MA, USA) spectrometer at room temperature. ESR spectrum settings were: modulation amplitude 0.8 gauss, scan time 20 s, time constant 10 ms, and microwave power 20 mW. For spin trapping of HER, POBN was used at a final concentration of 0.05 M in 10 mM Tris-HCl buffer. Mitochondrial swelling This was estimated from the decrease in the absorbance at 540 nm [22, 23]. Briefly, mitochondria (1 mg protein/ml) were equilibrated in a total volume of 3 ml in a medium consisting of 0.25 M sucrose and 10 mM Tris-HCl, pH 7.4, at 37°C for 10 min. The suspension was then preincubated with 5 mM succinate for 5 min. Mitochondrial swelling was initiated by the addition of DHAE (25, 40, or 75 ␮l, prepared as described in the ESR Measurements section) or in a Fenton-like system (3, 5, or 10 ␮M Fe2⫹, 100 ␮M H2O2, and 0.2 M ethanol). The decrease of the absorbance at 540 nm was determined with a Hitachi model U-2000 spectrophotometer (Hitachi, San Jose, CA, USA). Membrane thiol contents The experimental condition was the same as described for the swelling determination except that the incubation time was 10 min and 50 ␮g protein/ml of mitochondria was used. The mitochondria were washed twice at 7700 ⫻ g for 10 min and the thiol content was determined using Ellman’s reagent (DTNB) as described in Kowaltowski et al. [24]. Mitochondrial membrane potential (⌬ ⌿) The experimental condition was the same as described for the swelling determination except that 0.2 mg mitochondrial protein/ml was used. ⌬ ⌿ was measured with the fluorescent dye, rhodamine 123, by a modification of

Permeability transition induced by HER

Fig. 1. ESR detection of POBN spin adducts formed in an acetaldehyde/HOS system or in a Fenton reaction system containing ethanol. (A) Scans 1 to 4 ESR spectra of a DHAE solution (0.1 ml, prepared as described in the method section) after 4 (spectrum 1) and 8 min (spectrum 2) of incubation in the presence of POBN (0.05 M); trace 3, minus HOS; trace 4, minus acetaldehyde. Trace 5, H2O2 (0.1 mM), Fe2⫹ (20 ␮M) and POBN (0.05 M) after 2 min of incubation; trace 6, H2O2, Fe2⫹, POBN and ethanol (0.2 M) after 8 min of incubation. All spin trapping experiments were conducted in 0.3 ml Tris/HCL (10 mM; pH ⫽ 7.4). Other conditions are described in Materials and Methods. (B) ESR signal intensity as a function of the amount of DHAE added to POBN (0.05 M). Spectra were recorded after 8 min of reaction.

the method of Emaus et al. [25]. Briefly, mitochondria were incubated with 0.2 ␮M rhodamine 123 at 37°C. The change of fluorescence at an excitation wavelength of 420 –520 (emission, 528 nm) or at an emission wavelength of 520 – 650 nm (excitation 503 nm) was recorded continuously using a Hitachi F-2000 fluorescence spectrophotometer with constant temperature control and stirring.

RESULTS

When an aliquot of a DHAE-containing solution and POBN were mixed, or ethanol was added to a hydroxyl radical generating system, the Fenton reaction, a typical ESR spectra of the POBN/HER spin adduct was produced (Fig. 1A, spectra 1 and 2, and 6). The spectrum of spin adducts was identified as the POBN/HER nitroxide from the hyperfine splitting constants (in G) AN ⫽ 15.6 and AH ⫽ 2.7, which is in agreement with that reported by other workers [21, 26]. No POBN/HER spin adduct was formed when HOS or acetaldehyde was omitted from the DHAE reaction system (Fig. 1A, spectra 3 and 4). The ESR spectra of the POBN/•OH spin adduct (AN ⫽ 14.9 and AH ⫽ 1.7) was observed when ethanol was omitted from the Fenton reaction (Fig. 1A, spectrum 5); this adduct, however, is unstable and its ESR spectra disappeared within 2–3 min (data not shown). Increasing concentrations of spin adduct were produced as the content of DHAE was elevated (Fig. 1B), and as time of incubation increased (Fig. 1A, spectra 1 and 2).

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Fig. 2. Effect of mitochondria on the formation of POBN/HER adduct. (A) Mitochondria (1 mg protein/ml) and POBN (0.05 M) were added to either a solution of DHAE (0.1 ml) or an ethanol (0.2 M)-containing Fenton reaction system and incubated for 8 min before the recording of the spectra. Spectrum 1, DHAE; spectrum 2, plus mitochondria; spectrum 3, Fenton reaction system including ethanol; spectrum 4, 3 plus mitochondria. All other experimental conditions were as indicated in Figure 1A. (B) Effect of mitochondrial protein concentration on formation of POBN/HER adduct in DHAE (0.1 ml; open circles) and ethanol-containing Fenton systems (closed circles). Other conditions were the same as described in (A).

As shown in Figure 2, ESR spectroscopy was used to study the interaction of HER with mitochondria. The addition of mitochondria to reaction mixtures containing both HER-generating systems caused a marked decrease in the intensity of the POBN/HER signal. The decrease of signal intensity was dependent on the concentration of the mitochondria, indicating the interaction of HER and mitochondria (Fig. 2B). There was no effect if mitochondria were added to the already formed POBN/HER spin adduct. As shown in Fig. 3A, when rat liver mitochondria that had been preincubated with 5 mM succinate for 5 min were exposed to DHAE, there was a decrease in absorbance at 540 nm. Increasing concentrations of DHAE caused a concentration-dependent swelling. Similarly, when increasing amounts of HER were produced by the Fenton plus ethanol system (by adding increasing amounts of ferrous ammonium sulfate), there was increased mitochondrial swelling (Fig. 3B). When acetaldehyde or HOS was omitted from the DHAE system (Fig. 3A), or ferrous ions or H2O2 was omitted from the Fenton reaction including ethanol (Fig. 3B), the mitochondrial swelling was not observed. In the absence of ethanol, the Fenton reaction also caused mitochondrial swelling, most likely reflecting a •OH-dependent reaction (Fig. 3C). In the presence of 0.001 mM Cys A, an inhibitor of the MPT, HER generated from both systems and •OH generated from the Fenton reaction did not produce swelling (Fig. 3, dashed lines). The sensitivity to Cys A suggests that the mitochondrial swelling induced by HER and hydroxyl radical is reflective of “opening” of the permeability transition pore. The addition of vitamin E and glutathione (GSH) to

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Fig. 3. Mitochondrial swelling induced by HER and •OH. After preincubation in medium containing 5 mM succinate, 0.25 M sucrose, and 10 mM Tris-HCl, pH 7.4, mitochondria (1 mg protein per ml) were incubated with 0.02, 0.04, or 0.075 ml of DHAE (A) or Fenton reaction system with (B) or without (C) 0.2 M ethanol. The broken trace shows an experiment in which 1 ␮M cyclosporine A was present in the medium before the addition of 75 ␮l of DHAE or •OH (10 ␮M Fe2⫹ )-generating system. Other conditions were the same as described in Materials and Methods. In (B), the different amounts of HER were generated by adding increasing amounts of ferrous ammonium sulfate (3, 5, or 10 ␮M) to the Fenton reaction containing ethanol (0.2 M). In (A), the top solid line was the same for no addition or when either HOS or acetaldehyde was omitted from the DHAE preparation. In (B) and (C), no swelling was observed if either Fe2⫹ or H2O2 (0.1 mM) was omitted from the Fenton system.

the mitochondrial suspension before the generation of HER almost completely prevented the swelling induced by HER (Fig. 4). However, SOD and the •OH scavenger, mannitol, did not prevent the swelling. Catalase pre-

Fig. 4. Effect of antioxidants on HER-induced mitochondrial swelling. Mitochondria (1 mg protein per ml) were incubated in standard medium with the indicated antioxidants that were present from the beginning of the incubation prior to the addition of 0.1 ml DHAE (A) or the 20 ␮M Fe2⫹ Fenton plus 0.2 M ethanol system (B). The experimental conditions (control) of panel (A) and (B) were the same as described in the legend to Figs. 3 A and 3B. GSH ⫽ 2 mM; Vitamin E ⫽ 0.025 mM; catalase ⫽ 2000 U/ml; SOD ⫽ 1000 U/ml; mannitol ⫽ 10 mM.

vented the swelling in the Fenton reaction system containing ethanol but had no effect in the DHAE-containing system. These results suggest that reactive oxygen species (ROS), such as superoxide anion, H2O2 or •OH were not directly involved in the mitochondrial swelling induced by DHAE. GSH and dithiothreitol (DTT) when added shortly after initiation of the MPT by HER, prevented further swelling, i.e., inhibited further interaction of HER with the mitochondria (Fig. 5). However, addition of GSH or DTT after completion of the MPT did not reverse the swelling (Fig. 5). POBN at a concentration of 0.03 M significantly prevented the swelling induced by

Table 1. HER-induced Decrease in Membrane Thiol Content ⫹ Vitamin E

Control

A.

B.

DHAE (␮L)

Thiol content (nmol/50 ␮g protein)

Control (%)

Thiol content (nmol/50 ␮g protein)

Control (%)

0 (ctrl) 8 25 50 75 0 (ctrl) DHAE (0.1 ml) -HOS -Acetaldehyde

2.01 ⫾ 0.10 1.93 ⫾ 0.21 1.78 ⫾ 0.21 1.55 ⫾ 0.16* 1.28 ⫾ 0.17* 2.04 ⫾ 0.21 1.04 ⫾ 0.17 2.19 ⫾ 0.11 1.73 ⫾ 0.11

100 96.0 88.6 77.1 63.7 100 51 108 85

2.17 ⫾ 0.16 — — — 1.91 ⫾ 0.24

100 — — — 88.2

Mitochondria were incubated for 10 min with varying concentrations of DHAE. Where indicated, vitamin E (0.025 mM) was present from the beginning of the incubation. Each value represents the mean ⫾ SE of three through nine experiments. *p ⬍ .05 compared with control mitochondria. In (B), either the complete DHAE generating system was used, or HOS or acetaldehyde was omitted from the system.

Permeability transition induced by HER

Fig. 5. Effect of GSH and DTT on HER-induced mitochondrial swelling. After preincubation in medium containing 5 mM succinate, 0.25 M sucrose, and 10 mM Tris-HCl, pH 7.4, mitochondria (1 mg protein per ml) were incubated with 0.1 ml DHAE (trace a). Two millimolar DTT (traces b and d) or 2 mM GSH (traces c and e) were added where indicated by arrows. The broken trace shows an experiment in which mitochondria were incubated in standard medium with DTT or GSH without DHAE addition.

HER generated from DHAE or from Fenton plus ethanol system (data not shown). We previously demonstrated that HER amplified the oxidation of GSH and production of HER in HepG2 cells was associated with consumption of GSH [21]. To characterize the activity of HER with mitochondrial membrane thiols, which could be involved in the MPT, the effect of HER on the content of membrane protein thiols was examined. Table 1 shows that the amount of protein thiols decreased with increasing concentration of HER generated from DHAE. The addition of vitamin E, which directly interacts with HER, strongly prevented the thiol oxidation. Omission of either HOS or acetaldehyde from the DHAE preparation system prevented the loss of mitochondrial thiols as compared with the complete DHAE preparation (Table 1, part B). Figure 6 shows that rhodamine 123 in aqueous solution had a fluorescence excitation maximum at 503 nm and emission maximum at 528 nm (line 2). Mitochondria were able to build up the ⌬ ⌿ in a reaction medium (pH 7.4) when succinate was used as a substrate as indicated by the decreased fluorescence spectrum (Fig. 6, trace 1). The fluorescence intensity returned towards the nonenergized value (minus succinate, Fig. 6, trace 3) upon the addition of HER (Fig. 6, trace 5) or especially the protonophore (Fig. 6, trace 4), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to the energized mitochondria. DISCUSSION

Experiments were carried out to initiate studies on the interaction of HER with mitochondria because impairment of mitochondrial function is frequently observed after ethanol treatment, and HER is a reactive metabolite produced in small amounts from ethanol metabolism. The ESR intensity of HER/POBN adducts was markedly

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Fig. 6. Effect of HER on the mitochondrial membrane potential. DHAE (75 ␮l) was added to a previously preincubated (5 min at 25°C) mitochondrial suspension (0.2 mg protein/ml), containing 5 mM succinate, 0.25 M sucrose, 0.2 ␮M rhodamine 123, and 10 mM Tris-HCl (pH 7.4). The control reaction mixture (trace 1) consisted of mitochondria, succinate, sucrose, rhodamine 123, and Tris-HCl buffer. Trace 2, 1 ⫺ mitochondria; Trace 3, 1 ⫺ succinate; 4 ⫽ 1 ⫹ 20 ␮M CCCP; 5 ⫽ 1 ⫹ DHAE (75 ␮l). Other conditions were as indicated in Materials and Methods.

decreased in the DHAE system or Fenton reaction system containing ethanol in the presence of mitochondria, and HER stimulated mitochondrial swelling in a Cys A–sensitive manner indicative of stimulation of the MPT. GSH and vitamin E, which directly react with HER [21], prevented the MPT induced by HER. These results suggest that HER is itself an inducer of MPT. In addition, based on the mechanism of MPT inhibition by Cys A [27, 28], it is interesting to speculate that cyclophilin, which normally regulates or catalyzes pore opening, may be involved in the process of MPT induced by HER. Exogenous SOD, catalase, and a classical •OH scavenger, mannitol, did not significantly prevent the DHAEdependent swelling, indicating that HER itself and not ROS produced from the interaction of HER with oxygen, e.g. superoxide anion, H2O2, is responsible for induction of the MPT. In contrast to the DHAE-induced mitochondrial swelling, exogenous catalase did prevent the swelling when HER was produced from the Fenton-like reaction system; in this system HER is generated from the reaction of ethanol with •OH and the latter is produced through the decomposition of H2O2 by ferrous ions [14, 15]. In the Fenton reaction system containing ethanol, mannitol did not prevent the MPT, and a similar lack of protection was observed with other •OH scavengers, such as thiourea and dimethylsulfoxide. The second order rate constants of these scavengers for reaction with •OH is similar to that of ethanol (k2 for mannitol ⫽ 2.7 ⫻ 108; for thiourea ⫽ 4.7 ⫻ 109 ; and for ethanol ⫽ 7.2 ⫻ 108) [29]. Under our experimental conditions, the concentration of ethanol is about 30 times higher than that of mannitol and thiourea. Therefore, most of the generated

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OH is likely to react with ethanol to produce HER than with other •OH scavengers, explaining the lack of an inhibitory effect by these scavengers. These results suggest that HER to a greater extent than •OH is involved in the induction of the MPT under these Fenton reaction conditions. The reactivity of •OH is so great that, if •OH is generated in living cells, it will interact with biomolecules in the immediate environment. In view of the ability of HER as well as •OH to induce mitochondrial swelling, it is interesting to speculate that HER with a longer biological half-time than •OH may be especially effective in enhancing toxicity by diffusing to critical targets that are some distance away from the production site of •OH. Oxidation of protein thiols could play an important role in the mechanism by which HER induces the MPT, because both the swelling and thiol oxidation were strongly prevented by vitamin E, which interacts with HER. The mitochondrial thiols related to MPT have proven to be complex. It has been reported that the reaction of mitochondria with thiol oxidants affects the permeability properties of the inner mitochondrial membrane by changing the conformation of proteins in the membrane [30, 31]. Adenine nucleotide translocator (ANT) in the inner mitochondrial membrane, which has been considered a likely candidate as the pore-forming protein, has three critical cysteines required for induction of the MPT [32]. The ANT is highly abundant, constituting 14% of the inner membrane protein. However, much about the location of HER-reactive sites on mitochondria remains to be determined. One important regulator of the MPT is ⌬ ⌿ because a decrease in membrane potential induces MPT [33]. HER was found to collapse the ⌬ ⌿ almost as effectively as the classical protonophore CCCP. We wondered whether an uncoupling activity of HER may be responsible for its ability to induce the MPT, because the MPT can be induced by uncoupling agents under several conditions [27, 34, 35]. HER generated via decomposition of DHAE did not affect the rate of mitochondrial respiration using substrates that donate electrons to complex I (malate/glutamate) and complex II (succinate) of the respiratory chain (data not shown), dissociating HER from a true uncoupling effect. Petronilli et al. have reported that the oxidation-reduction state of vicinal thiols of cysteinyl residues play a critical role in acting as a voltage sensor of the transition pore [36]. Interestingly, there are reports that the conformation of ANT is influenced by ⌬ ⌿ [27, 31]. From these findings, we speculate that the MPT induced by HER involves collapse of ⌬ ⌿ as a consequence of the oxidation of the membrane thiols critical for mitochondrial structure and function. A long-term goal of these studies is to further define the role of mitochondria in ethanol-induced hepatotox-

icity. A large body of evidence has accumulated to suggest that oxidative stress may play a role as a common mediator of apoptosis [9, 11] and of CYP2E1dependent apoptosis [37–39]. For example, antioxidants or molecules that enhance the endogenous antioxidant defense system of the cell can inhibit or delay apoptosis to varying extents [37– 40]. Ethanol has been also shown to produce liver apoptosis after long-term consumption by mice or rats or when added to suspensions of isolated rat hepatocytes [12, 13, 41]. Apoptotic cells were observed in the livers of rats that exhibited alcohol liver injury [13]. Recent results suggest that damage to mitochondria may be important in the overall mechanism by which cells undergo apoptosis as apoptotic-inducing factors, such as cytochrome c are released [8 –11]. These apoptotic-inducing factors are often associated with the activation of a family of cysteine proteases, which subsequently act as effectors of the apoptotic process [42, 43]. It is proposed that the MPT constitutes an early and irreversible feature of the apoptotic effector phase. The free radical metabolite of ethanol HER may contribute to mechanisms responsible for ethanol hepatotoxicity via immune response, potentiation of oxidative stress, interaction with antioxidants, adduct formation, and so on. HER can induce a MPT, which may play a role in ethanol-induced apoptosis. This will require further evaluation. Further studies are also required to disclose the mechanism of MPT induced by HER, e.g. modulation of Ca2⫹, possible protection by bcl-2, and whether the HER induced MPT is associated with release of apoptotic-inducing agents. Acknowledgements —This work was supported by United States Public Health Service Grant AA-09460 from The National Institute on Alcohol Abuse and Alcoholism.

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ABBREVIATIONS

HER—1-hydroxyethyl radical MPT—mitochondrial permeability transition ESR— electron spin resonance

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POBN—alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone HOS— hydroxylamine O-sulfonate SOD—superoxide dismutase Cys A— cyclosporine A DTNB— dithionitrobenzoic acid ANT—adenine nucleotide translocator

ROS—reactive oxygen species DHAE—1,1⬘-dihydroxyazoethane DTT— dithiothreitol CCCP— carbonyl cyanide m-chlorophenyl hydrazone A.U.—absorbance units a.u.—arbitrary units