Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H2O2 from DNA damage

Free Radical Biology & Medicine, Vol. 30, No. 12, pp. 1357–1364, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00537-8

Original Contribution TRIMETAZIDINE PROTECTS LOW-DENSITY LIPOPROTEINS FROM OXIDATION AND CULTURED CELLS EXPOSED TO H2O2 FROM DNA DAMAGE ALEXANDROS TSELEPIS,* PASCHALIS-THOMAS DOULIAS,† EVAGGELIA LOURIDA,* GEORGIOS GLANTZOUNIS,‡ EVANGELOS TSIMOYIANNIS,‡ and DIMITRIOS GALARIS† *Laboratory of Biochemistry, Department of Chemistry and †Laboratory of Biological Chemistry, School of Medicine, University of Ioannina, Ioannina, Greece; and ‡Department of Surgery, “G. Hatzikosta” General Hospital, Ioannina, Greece (Received 2 November 2000; Accepted 8 March 2001)

Abstract—Trimetazidine is a well-established anti-ischemic drug, which has been used for long time in the treatment of pathological conditions related with the generation of reactive oxygen species. However, although extensively studied, its molecular mode of action remains largely unknown. In the present study, the ability of trimetazidine to protect low-density lipoproteins (LDL) from oxidation and cultured cells from H2O2-induced DNA damage was investigated. Trimetazidine, tested at concentrations 0.02 to 2.20 mM, was shown to offer significant protection to LDL exposed to three different oxidizing systems, namely copper, Fe/ascorbate, and met-myoglobin/H2O2. The oxidizability of LDL was estimated by measuring, (i) the lag period, (ii) the maximal rate of conjugated diene formation, (iii) the total amount of conjugated dienes formed, (iv) the electrophoretic migration of LDL protein in agarose gels (REM), and (v) the inactivation of the enzyme PAF-acetylhydrolase present in LDL. In addition, the presence of trimetazidine decreased considerably the DNA damage in H2O2-exposed Jurkat cells in culture. H2O2 was continuously generated by the action of glucose oxidase at a rate of 11.8 ⫾ 1.5 ␮M per min (60 ng enzyme per 100 ␮l), and DNA damage was assessed by the single cell gel electrophoresis assay (also called comet assay). The protection offered by trimetazidine in this system (about 30% at best) was transient, indicating modification of this agent during its action. These results indicate that trimetazidine can modulate the action of oxidizing agents in different systems. Although its mode of action is not clarified, the possibility that it acts as a lipid barrier permeable transition metal chelator is considered. © 2001 Elsevier Science Inc. Keywords—LDL, Oxidized-LDL, PAF-acetylhydrolase, Hydrogen peroxide, Single cell gel electrophoresis (comet assay), Glucose oxidase, Jurkat cells, Free radicals

INTRODUCTION

others including AIDS and cancer [1–3]. It is well known that ROS are able to induce damage to all the basic constituents of the cell, i.e., lipids, proteins, DNA, and other. However, the molecular mechanisms underlying the induction of damage remain partly controversial or even elusive. It seems likely that redox active transition metal ions are mainly involved in mediating ROS-induced damage to cell constituents and consequently cell toxicity. Organisms have evolved effective protective mechanisms in order to withstand oxidative stress, but these defenses are often overwhelmed leading to development of serious diseases. In such cases the administration of exogenous protective compounds (e.g., therapeutic agents, food constituents, and others) may be beneficial.

Reactive oxygen species (ROS) of various kinds are continuously generated in aerobic organisms but their rate of production and steady state concentrations are considerably increased under a variety of pathological conditions [1]. Examples of pathological conditions in which ROS have been proposed to be implicated are cardiovascular diseases (mainly of atheroslerotic origin), ischemia/reperfusion syndromes, neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and many Address correspondence to: Dimitrios Galaris, Ph.D., Laboratory of Biological Chemistry, University of Ioannina Medical School, 451 10 Ioannina, Greece; Tel: ⫹30 (651) 97562; Fax: ⫹30 (651) 97868; E-Mail: [email protected]. 1357

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These agents can act in several different ways such as: (i) direct chain-breaking antioxidants or free radical scavengers, (ii) inhibitors of ROS formation, (iii) transition metal chelators, and (iv) inducers of enzymes involved in detoxification of ROS or damage repair and others. Trimetazidine [1-(2,3,4-trimethoxy-benzyl)-piperazine dihydrochloride, TMZ] is a well established drug that has been extensively used in the treatment of pathological conditions related with the generation of ROS, such as ischemia/reperfusion, heart surgery, brain disorders, and others [4 –7]. Based on the observed actions, it was suggested that TMZ may possess properties related to defenses against ROS-induced toxicity [8 –11]. However, it was recently shown that TMZ was not effective as a free radical scavenger [12]. Consequently, the purpose of this study was to reinvestigate the antioxidant role of TMZ. Two different systems were used, (i) the oxidation of low-density lipoproteins (LDL) by three different initiating agents, namely, copper, Fe/ascorbate, and met-myoglobin/H2O2; and (ii) the induction of DNA damage in cultured cells exposed to continuous generation of H2O2. It is well established that oxidative modification of LDL plays an important role in the processes of initiation and progression of atherosclerosis [13,14]. Several studies have shown that oxidation of LDL proceeds through a complex series of events involving formation of lipid hydroperoxides and modification of apolipoprotein B by products of their breakdown [15]. In addition, the oxidative modification of LDL involves the hydrolysis of its content of oxidized phosphatidylcholine into lysophosphatidylcholine, which is mediated by the LDL-associated enzyme “platelet-activating factor acetylhydrolase” (PAF-acetylhydrolase, EC 3.1.1.47) [16 –18]. PAFacetylhydrolase is a Ca2⫹-independent phospholipase A2, highly specific for the hydrolysis of phospholipids containing a short acyl group at the sn-2 position, such as the potent mediator of inflammatory reactions, plateletactivating factor (PAF) [19]. During oxidative modification of LDL, PAF-acetylhydrolase is progressively inactivated and may represent an index for estimating the degree of LDL oxidation [16,20]. On the other hand, in order to study the DNA damage, a new, extremely sensitive assay called single cell gel electrophoresis or comet assay, able to detect formation of single strand breaks (SSB) in individual cells, was employed [21,22]. By using this technique our group has previously studied the induction of SSBs in the DNA of human lymphocytes as induced by continuously generated H2O2 from the action of glucose oxidase [23]. The results of the present study indicate that TMZ was able to offer considerable protection toward both LDL oxidation and cellular DNA damage. Although the exact mechanism of its protective action remains elusive and

needs further investigation, the results seem to support the idea that TMZ acts as a lipid barrier-permeable transition metal chelator. MATERIALS AND METHODS

Chemicals Growth medium RPMI-1640 supplemented with Lglutamine, sodium bicarbonate, gentamicin, and the enzymes glucose oxidase (from Aspergillus niger, 18,000 units/g) were from Sigma Chemical Company (St. Louis, MO, USA). Fetal bovine calf serum, Nunc tissue culture plastics, and low melting point agarose were obtained from Gibco BRL (Grand Island, NY, USA). Normal melting point agarose was obtained from Serva GmbH (Heidelberg, Germany). Gentamicine sulphate was from Garamycin, Schering-Plough, USA. Microscope glass super frosted slides were supplied by Menzel-Glaset and 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) by Boehringer Mannheim (Indianapolis, IN, USA). PAF (hexadecyl) was from Bachem (Zurich, Switzerland) and 1-O-hexadecyl-2-[3H-acetyl]-snglycero-3-phosphocholine (10 Ci/mmol) was from DuPont-New England Nuclear (Boston, MA, USA). All other chemicals used were of analytical grade. TMZ (Servier), was dissolved in 10 mM PBS, stored in the dark at 4 °C and were used within 2 d after preparation. Isolation of LDL LDL (d ⫽ 1.019 –1.063 g/ml) was isolated from freshly prepared human plasma supplemented with 0.01% EDTA (w/v) and 5 mg/ml gentamicine sulphate (Garamycin, Schering-Plough, USA) by sequential centrifugation in a Beckman L7-65 ultracentrifuge at 40,000 rpm, 14°C with a Type NVT 65 rotor [16]. The duration of each ultracentrifugation was 10 h. The LDL preparation was dialyzed against 10 mM PBS containing 0.01% EDTA (pH 7.4) for 24 h at 4°C. Then it was filtersterilized (0.22 mM, Millipore, USA) and stored in the dark at 4°C under nitrogen for up to 2 weeks. LDL protein was determined by the bicinchoninic acid method. Purity of the LDL preparation was assessed by agarose gel electrophoresis (Hydragel Lipo and Lp(a) kit, Sebia). Measurement of antioxidant capacity TMZ solutions of various concentrations were tested for their antioxidant capacity by using a commercial kit (Randox Laboratories Ltd., UK). The principle of the assay is based on the ability of H2O2-oxidized met-

The role of trimetazidine as antioxidant

myoglobin to produce ABTS•⫹ free radicals from ABTS. Furthermore, the ability of TMZ to inhibit H2O2-induced oxidation of myoglobin was tested by following the changes in the difference spectra of metmyoglobin versus ferryl myoglobin in the visible area [24,25]. LDL oxidation Before oxidation, LDL was extensively dialyzed (to remove EDTA) against two changes of a 200-fold volume of 10 mM PBS for 24 h at 4°C in the dark. LDL (100 ␮g protein/ml) was incubated in the presence of copper sulphate (5 ␮M final concentration) [26]. Oxidations were performed in the presence or absence of TMZ concentrations ranging from 0.02 to 2.2 mM. The kinetics of the oxidation was determined by monitoring the increase in absorbance at 234 nm every 10 min for 3 h. The lag time, the maximal rate of conjugated diene formation, and the total amount of dienes formed were calculated as previously described [27]. The electrophoretic migration of Ox-LDL was studied on agarose gels and expressed as relative to native LDL electrophoretic mobility (REM). In selected experiments LDL oxidation was performed either in the presence of metmyoglobin/H2O2 (10 and 50 ␮M, respectively) and monitored as above, or dialyzed in 0.15 M NaCl (pH 7.4) and exposed for 6 h, 37°C in 10 ␮M FeSO4 and 1 mM ascorbate. In the latter case thiobarbituric acid reactive substances (TBARS) [28] and REM values were estimated. PAF-acetylhydrolase PAF-AH activity was measured by the tricloroacetic acid precipitation procedure [29]. A 1 mM [3H]-PAF solution in 0.25% (w/v) BSA/saline (specific activity 2000 cpm/nmol) containing PAF and 1-O-hexadecyl-2[3H-acetyl]-sn-glycero-3-phosphocholine, prepared as previously described [29], was used as a substrate for PAF-AH. Four ␮g of protein from either native or Cu2⫹oxidized LDL, in 90 ␮l of HEPES buffer, pH 7.4, were used as the source of the enzyme. The reaction was initiated by adding 10 ␮l of [3H]-PAF solution (100 ␮M final concentration in the reaction mixture) and performed for 10 min at 37°C. In preliminary experiments the effect of TMZ on PAF-AH activity was evaluated by using native LDL (4 ␮g protein) as a source of the enzyme. PAF-AH activity was expressed as nmol PAF degraded per min per mg of LDL protein. Cell culture and treatment One tenth ml of Jurkat cells (a human T-lemphocytic cell line, ATCC, clone E6-1) containing 1.5 ⫻ 105 cells,

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were placed into each of 96 wells of ELISA plastic plates and incubated for 1 h at 37°C, 95% air, 5% CO2. In the end of this period, cells were treated with 60 ng glucose oxidase (releasing 11.8 ⫾ 1.5 ␮M H2O2 per min) in the presence or absence of the indicated concentrations of TMZ (added 15 min prior to the addition of glucose oxidase). Cells were collected 10 min later by centrifugation (250 ⫻ g, 4°C, 5 min) for further analysis. Measurement of hydrogen peroxide The amount of hydrogen peroxide generated by the action of glucose oxidase in PBS containing 5.0 mM glucose was estimated either directly by following the increase in the absorbance at 240 nm (Molar Extinction Coefficient ⫽ 43.6 M⫺1 cm⫺1), or indirectly by following the oxidation of OPD in the presence of horseradish peroxidase at 492 nm. In separate experiments, it was found that TMZ was not able to directly inhibit glucose oxidase in the H2O2 generating system (not shown). Single cell gel electrophoresis The assay used was an adaptation of the method described first by Ostling and Johanson [30] and later by Singh et al. [31]. Cells were suspended in 1% low melting point agarose (LMP agarose) in PBS, pH 7.4, and pipetted onto super frosted glass microscope slides precoated with a layer of 1% normal melting point agarose (warmed to 37°C prior to use). The agarose was allowed to set for 10 min at 4°C and then the slides were immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris at pH 10, 1% Triton X-100 v/v) at 4°C for 1 h in order to remove cellular proteins. Slides were then placed in single rows in a 30 cm wide horizontal electrophoresis tank containing 0.3 M NaOH and 1 mM EDTA, pH ⬎ 13 for 40 min at 4°C in order to allow the separation of the two DNA strands (alkaline unwinding). Electrophoresis was performed in the unwinding solution at 30 Volts (1 V/cm), 300 mAmps for 30 min. The slides were then washed three times for 5 min each with 0.4 M Tris, pH 7.5 at 4°C before staining with DAPI (5 mg/ml). Image analysis and scoring DAPI-stained nucleoids were examined under a UV microscope with an excitation filter of 435 nm and a magnification of 400. Visual scoring of the cellular DNA on each slide was based on characterization of 100 randomly selected nucleoids. The comet-like DNA formations were placed into five classes (0, 1, 2, 3, and 4) representing an increasing extent of DNA damage seen as a “tail” [23]. If each comet is assigned a value ac-

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Table 1. Effect of Trimetazidine on Cu2⫹-induced LDL Oxidation

TMZ (mM)

Lag time (min)

Rate of oxidation (nmol/mg protein/ min)

Control 0.02 0.05 0.11 0.22 0.55 1.10 2.20

65 ⫾ 10 61 ⫾ 8 63 ⫾ 12 60 ⫾ 9 72 ⫾ 8a 81 ⫾ 11b 93 ⫾ 10b 115 ⫾ 12c

6.2 ⫾ 1.5 6.3 ⫾ 0.8 6.8 ⫾ 0.9 6.4 ⫾ 1.0 5.5 ⫾ 0.9a 3.7 ⫾ 0.6c 2.2 ⫾ 0.5c 1.4 ⫾ 0.4c

Total dienes (nmol/mg protein)

REM values

408 ⫾ 35 391 ⫾ 42 493 ⫾ 51 415 ⫾ 39 311 ⫾ 48a 228 ⫾ 36b 116 ⫾ 30c 70 ⫾ 18c

2.8 ⫾ 0.5 2.9 ⫾ 0.2 3.0 ⫾ 0.3 2.9 ⫾ 0.4 2.5 ⫾ 0.3 2.0 ⫾ 0.2a 1.6 ⫾ 0.3b 1.2 ⫾ 0.2c

Oxidation was performed by incubating of LDL (100 ␮g of protein) with 5 ␮M CuSO4 at 37°C. The kinetics of oxidation were determined by monitoring the increase in absorbance at 234 nm every 10 min for 3 h. The Relative Electrophoretic Mobility (REM) of oxidized LDL was studied on agarose gels. Values reprsent the mean ⫾ SD, n ⫽ 5. a p ⬍ .05, bp ⬍ .03, cp ⬍ .01 compared to the control values (in the absence of TMZ).

cording to its class, the overall score for 100 comets ranges from 0 (100% of comets being in class 0) up to 400 (100% of comets in class 4). In this way the overall DNA damage of the cell population can be expressed in arbitrary units. Visual scoring expressed in this way correlates in a near-linear way with other parameters such as percent of DNA in the tail estimated after computer image analysis using a specific software package [32,33]. Observation and analysis of the results were always carried out by the same experienced person in a blind way. A specific pattern was always followed moving along the slide. Statistical analysis A Student’s paired t-test was used in order to examine statistically significant differences. RESULTS AND DISCUSSION

Effects of trimetazidine in LDL oxidative modification TMZ at concentrations higher than 0.22 mM protected significantly against Cu2⫹-induced LDL oxidation (5 ␮M CuSO4, 100 ␮g LDL protein) as indicated by: (i) prolongation of lag time, (ii) decrease of the maximal rate and total amount of conjugated dienes formed, and (iii) decreased REM values (Table 1). Essentially the same results were obtained when LDL (100 ␮g of protein in 0.15 M NaCl, pH 7.4) was exposed in FeSO4/ascorbate (10 ␮M and 1mM respectively) and the oxidation was estimated by measuring TBARS and REM values. Lower amounts of TBARS and decreased REM values were obtained at concentrations

Table 2 Effect of Trimetazidine on LDL Oxidation Induced by FESO4 Ascorbate

TMZ (mM) Control 0.55 1.10 2.20

TBARS (nmol MDA/mg protein)

REM values

20 ⫾ 7.4 18 ⫾ 9.2 11.8 ⫾ 4.3a 5.5 ⫾ 1.2b

1.9 ⫾ 0.3 2.0 ⫾ 0.1 1.5 ⫾ 0.2a 1.2 ⫾ 0.1c

LDL (100 ␮g protein/ml in 0.15 M NaCl, pH 7.4) was oxidized for 6 h in the presence of 10 ␮M FeSO4/1 mM ascorbate. The rate of oxidation was estimated at the end of oxidation by measuring the thiobarbituric acid reactive substances (TBARS) and the Relative Electrophoretic Mobility (REM) on agarose gels. Values represent the mean ⫾ SD, n ⫽ 4. ap ⬍ .04, bp ⬍ .02, cp ⬍ .03 compared to the control values (in the absence of TMZ).

higher than 1.1 mM, indicating a clear protective effect (Table 2). The effect of TMZ on the activity of the LDL-associated enzyme PAF-acetylhydrolase during Cu2⫹-induced oxidation was also studied. As shown in Fig. 1, PAF-acetylhydrolase activity was significantly reduced by about 45% after 3 h of LDL oxidation (p ⬍ .01). In the presence of 1.1 to 2.2 mM TMZ the decrease in the enzyme activity was significantly lower compared to that in the absence of TMZ (p ⬍ .05 and p ⬍ .03, respectively). It has to be noted that in the presence of 2.2 mM TMZ, PAF-acetylhydrolase activity in LDL was not significantly different than in native LDL (Fig. 1). The mechanism of this enzyme inactivation is presently not known. However, it has been shown that it is not a consequence of oxidative changes of LDL itself and it is not due to a decrease in the enzyme affinity for the substrate or to modifications of amino acid residues at (or near) the active site [34]. Although it is plausible to think that the protection offered by TMZ against LDL oxidation in particular, and lipid oxidation in general, may be related to its wellestablished action as anti-ischemic agent, the molecular mechanism(s) of its action remains controversial. Probable explanations could be related either to a chainbreaking antioxidant action or to transition metal chelating capacity of this agent. However, the antioxidant capacity of TMZ was zero when tested with a commercial kit (Total Antioxidant Capacity, Randox Laboratories) (results not shown). This kit is based on the formation of ABTS•⫹ free radicals from ferryl myoglobin, which is generated after interaction of met-myoglobin with H2O2. Moreover, the presence of TMZ was ineffective in decreasing the rate of ferryl myoglobin formation after interaction of met-myoglobin with H2O2 as assessed by following the changes in difference spectra of met- versus ferryl myoglobin [24,25]. These results, in accordance with others recently reported [12], clearly

The role of trimetazidine as antioxidant

Fig. 1. Effect of TMZ on LDL-associated PAF-acetylhydrolase activity during Cu2⫹-induced LDL oxidation. Oxidation was performed by incubation of LDL (100 ␮g of protein) with 5 ␮M CuSO4 at 37°C for 3 h. PAF-acetylhydrolase activity was determined as described in Materials and Methods by the TCA precipitation procedure, using 40 ␮g/ml of LDL protein as the source of the enzyme and [3H]-PAF (100 ␮M final concentration) as a substrate. Each value represents the mean ⫾ SD from three different LDL preparations. $p ⬍ .01, compared to native LDL (n-LDL). *p ⬍ .05 and **p ⬍ .03 compared to oxidized LDL (ox-LDL) in the absence of TMZ (0.0 mM).

indicate that TMZ by itself is unable to act as a direct electron-donating typical antioxidant. Moreover, the ability of TMZ to decrease the rate of propagation of LDL oxidation (Table 1) clearly indicates that it does not act as a chain-breaking antioxidant. In order to test the possibility of TMZ acting as a transition metal chelator, a system devoid of free iron ions (chelation of contaminating iron with 0.1 mM DTPA) was used to induce the oxidation of LDL. In this case, the interaction of met-myoglobin (10 ␮M) with H2O2 (50 ␮M), in the presence of DTPA (0.1 mM), leads to formation of ferryl myoglobin free radicals able to oxidize vulnerable targets, like unsaturated lipids [24]. As anticipated, LDL was rapidly oxidized in this system. The lag time observed was 44 ⫾ 12 min (n ⫽ 3), the maximal rate of diene formation was 5.8 ⫾ 0.6 nmol/mg protein/min, the total amount of dienes formed was 468 ⫾ 67 nmol/mg protein and the REM value 2.4 ⫾ 0.3. TMZ, at concentrations ranged from 1.1 to 2.2 mM, completely inhibited met-myoglobin-induced LDL oxidation (results not shown). Since TMZ is not a typical

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electron-donating scavenger, a plausible explanation of this action might be that it is able to penetrate into the lipid layer of LDL and chelate transition metals that are inaccessible by DTPA. In this case, transition metals positioned deeper into the lipid components of LDL may participate in decomposition of lipid hydroperoxides formed during the initial action of either the ferryl-iron or the protein free radical on LDL polyunsaturated fatty acids. This proposal, although attractive, needs further investigation. However, other possible explanations, like chelation of free copper ions contaminating the reaction medium, or a lipid stabilizing action of this agent must also be considered. The levels in which TMZ was effective in the experiments of the work presented above were rather high (about 10⫺4–10⫺3 M). Concentrations of this drug about 10⫺7–10⫺6 M have been previously reported in patients 2–3 h after oral administration of 60 mg [7,35]. Doses of 120 mg per day are currently administrated in humans, while higher doses were found to be effective in animal studies. Although the effective doses in the present study were rather high in comparison to serum levels, one has to take into account TMZ incorporation in cells and tissues. Effects of trimetazidine on DNA damage in H2O2-exposed cultured cells In a subsequent set of experiments, the ability of TMZ to protect H2O2-exposed cells in culture from DNA damage was investigated. It has been reported previously that H2O2 generated by the action of glucose oxidase induces a time and dose dependent formation of single strand breaks (SSBs) in isolated and cultured lymphocytes from human peripheral blood [23]. In the same manner, it was observed in this work that exposure of Jurkat cells (a T-lymphocytic cell line) to continuously generated H2O2 by the action of glucose oxidase (between 20 to 100 ng/100 ␮l generating about 5–20 ␮M H2O2 per min) induced a rapid and concentration-dependent rise in the DNA damage as assessed by the single cell gel electrophoresis assay (comet assay) (results not shown). In the following experiments, 1.5 ⫻ 105 cells dispersed in 100 ␮l of growth medium containing 10% fetal calf serum were exposed in 60 ng glucose oxidase (releasing 11.8 ⫾ 1.5 ␮M H2O2 per min) in the presence or absence of TMZ and the formation of SSBs was evaluated 10 min after the addition of glucose oxidase. In order to avoid extracellular iron ion involvement (extracellular interaction of H2O2 in Fenton-type reactions), the ability of DTPA (a nonpermeable iron chelator) was checked. No differences in the level of DNA damage were observed in the presence or absence of 1.0 mM DTPA when the treatment was performed in growth

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Fig. 2. Effect of TMZ on H2O2-induced single strand break formation in cellular DNA. Jurkat cells (1.5 ⫻ 105 cells/100 ␮l) were exposed for 15 min to continuously generated H2O2 by the action of the enzyme glucose oxidase (60 ng/100 ␮l) in the presence of the indicated concentrations of TMZ (added 15 min before the addition of glucose oxidase). The DNA damage of individual cells was estimated by the comet assay and expressed in arbitrary units, as described under Materials and Methods. Each value represents the mean ⫾ SD of triplicate measurements from two different experiments (*p ⬍ .05).

medium containing 10% of fetal calf serum (results not shown). As can be seen in Fig. 2, pretreatment of the cells with increasing concentrations of TMZ before the addition of glucose oxidase (60 ng/100 ␮l, inducing 305 ⫾ 15 arbitrary units of SSB compared to 102 ⫾ 7 of controls) protected these cells from H2O2-induced DNA damage. The protection offered by TMZ was dose dependent and offered about 15 and 30% protection for 0.5 and 1mM of TMZ, respectively (p ⬍ .05). Pretreatment with lower concentrations of TMZ (0.1 mM) offered no protection, rather a slight, nonsignificant increase in the level of SSBs was apparent. Moreover, when TMZ was added 60 rather than 15 min before the addition of glucose oxidase, the protection offered was always somewhat higher (although not significantly), indicating possibly an increased import of TMZ into the cells at longer incubation times. Another characteristic of the protective action of TMZ was that it was transient, lasting less than half an hour after the addition of glucose oxidase (Fig. 3). Shortly after exposure to H2O2 (5 and 10 min), when SSB formation had reached almost its highest level (315 ⫾ 10 arbitrary units), TMZ pretreated cells shown about 25–30% lower SSBs (p ⬍ .05). At 20 min exposure, the protection offered by TMZ was lower (15–20%) but still significant (p ⬍ .05). At 30 min or later the protective action of TMZ was totally abolished. The fact that the action of TMZ was transient may indicate that it is modified during its action, giving rise to inactive products. The fact that 60 min preincubation did

Fig. 3. Time course of the effect of TMZ on H2O2-induced single strand break formation in cellular DNA. Conditions were as in Fig. 2, except that DNA damage was estimated at the time points indicated after the addition of glucose oxidase in the presence (shaded bars) or absence (empty bars) of 1.0 mM TMZ (15 min before the addition of glucose oxidase). The results are presented as % of DNA damage observed in the presence versus the absence of TMZ (*p ⬍ .05).

not decrease its protective capacity (Fig. 2), indicates that the presence of H2O2 (rather than cells alone) is responsible for the observed inactivation of TMZ. Unpublished results from our laboratory have shown that compounds with iron (but not copper) chelating ability, able to penetrate cell membranes, are potent inhibitors of DNA damage in the above cell system. In contrast, typical antioxidants, like lipoic acid or trolox, were almost completely ineffective (Galaris et al., manuscript submitted for publication). Although it needs further investigation, we are tempted to speculate— based on the results of the present work—that TMZ may possess transition metal chelating properties. This ability in combination with its lipid barrier-penetrating capacity may allow the binding and inactivation of redox active transition metals associated with lipids in both LDL or cellular membranes. Such an explanation of the antioxidant action of TMZ is in accordance with its well established anti-ischemic properties, because the concentration of transition metal ions is thought to increase intracellularly during ischemia together with concomitant generation of H2O2 and other ROS. This situation

The role of trimetazidine as antioxidant

may become detrimental for cells and surrounding tissues in the absence of protective mechanism(s) and TMZ may be able to interfere in this process.

CONCLUSIONS

In summary, the results of the present work clearly indicate that the presence of TMZ (though at relatively high concentrations) can protect LDL oxidation initiated by three different systems, namely copper ions, Fe/ascorbate, and met-myoglobin/H2O2. Because TMZ is not likely to assess any direct antioxidant capacity by itself, a possible explanation of this action is that it is able to chelate transition metal ions. The fact that the commonly used iron chelator DTPA—in contrast to TMZ—was unable to offer any protection in LDL oxidation initiated by the met-myoglobin/H2O2 system, may indicate that TMZ may be able to penetrate and act deeper into the lipid layer of LDL, which is not possible for DTPA. This postulation is further substantiated by experiments in which DNA damage in H2O2-exposed Jurkat cells was estimated by the alkaline single cell gel electrophoresis technique (comet assay) in the presence of increasing concentrations of TMZ. The protection offered by TMZ in this case might also be explained by metal chelating and lipid barrier penetrating properties of this agent. Acknowledgements — This research was partly supported from grant PENED 99, No. 99ED181 of General Secretariat of Research and Technology, Athens, Greece to D.G., and from Biomed 2 program BMH-CT98-3161 to A.T.

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