Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA Damage

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Free Radical Biology & Medicine, Vol. 31, No. 4, pp. 490 – 498, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00608-6

Original Contribution INTRACELLULAR IRON, BUT NOT COPPER, PLAYS A CRITICAL ROLE IN HYDROGEN PEROXIDE-INDUCED DNA DAMAGE ALEXANDRA BARBOUTI,* PASCHALIS-THOMAS DOULIAS,* BEN-ZHAN ZHU,† BALZ FREI,† DIMITRIOS GALARIS*

and

*Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina, Greece; and †Linus Pauling Institute, Oregon State University, Corvallis, OR, USA (Received 25 July 2000; Accepted 15 May 2001)

Abstract—The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or “comet” assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1–5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copperchelating agents, did not protect against H2O2-induced cellular DNA damage. In fact, membrane permeable copperchelating agents induced DNA damage in the absence of H2O2. These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H2O2-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid acetoxymethyl ester), an intracellular Ca2⫹-chelator, also protected against H2O2-induced DNA damage, it is likely that intracellular Ca2⫹ changes are involved in this process as well. The exact role of Ca2⫹ and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated. © 2001 Elsevier Science Inc. Keywords—Hydrogen peroxide, Single strand breaks, Single cell gel electrophoresis (comet assay), Iron, Copper, Calcium, Transition metal chelators, Glucose oxidase, Free radicals

INTRODUCTION

indirect targets of intracellular H2O2. Protein kinases and phosphatases, proteins containing sulfhydryl groups or iron sulfur clusters, lipids, DNA, and others are among the potential targets [3– 6]. Cellular DNA seems to be especially sensitive to the action of H2O2. H2O2-induced damage to cellular DNA is widely believed to be mediated by transition metal ions, mainly iron and/or copper, which are able to catalyze the formation of hydroxyl radicals (•OH) by Fentontype reactions [7–9]. The location of redox-active metals is of utmost importance for the ultimate effect, because • OH, due to its extreme reactivity, interacts exclusively in the vicinity of the bound metal [10]. Formation of •OH close to DNA results in its damage, including base modifications, single and double strand breakage, and sister chromatid exchange. On some occasions, single strand

Controlled generation of hydrogen peroxide (H2O2) appears to be a common phenomenon among different cell types and may play an important role in signaling pathways [1,2]. Since H2O2 is able to pass freely across cell membranes, it can transfer information to nearby cells or tissues acting in a paracrine fashion, similar to nitric oxide. Although extensively studied, the exact molecular mechanisms of the generation as well as the mode of action of H2O2 remain elusive. A large number of molecules have been proposed or identified as direct or 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]. 490

The role of iron in H2O2-induced DNA damage

break (SSB) formation has been linked to intracellular Ca2⫹ increases, indicating an obligatory intermediary role for Ca2⫹ [11,12]. We have previously shown that H2O2-induced DNA damage is Ca2⫹-dependent at low rates of continuous H2O2 production, although it seems likely that a Ca2⫹-independent mechanism operates at higher rates of H2O2 production [12]. When H2O2 is added directly as a bolus, as is the case in most studies [11,13], cells are initially exposed to relatively high concentrations followed by a fast decrease as H2O2 is consumed. If the mode of action of H2O2 is concentration-dependent [13], the results regarding Ca2⫹-dependence may appear inconsistent, unless special attention is given to H2O2 concentrations. In the present work, the H2O2-generating enzyme glucose oxidase (GO) was employed to expose the cells to constant, relatively low concentrations of H2O2. Formation of SSBs was detected by the single cell gel electrophoresis or “comet” assay [14,15]. Preincubation of the cells with several chelating agents of varying specificity, before the challenge with H2O2, revealed that the redox active pool of intracellular iron— but not copper—is pertinent to H2O2-induced DNA damage. All the Cu-chelating agents used in this study, although structurally different, induced SSB formation by themselves in the absence of exogenous H2O2. MATERIALS AND METHODS

Materials RPMI 1640 growth medium supplemented with Lglutamine and gentamicin, glucose oxidase (from Aspergillus niger, 18,000 units/g), and catalase (from bovine liver) were from Sigma Chemical Company (St. Louis, MO, USA). 1,7- and 1,10-phenanthroline, diethylenetriaminepentaacetic acid (DTPA), neocuproine, bathocuproine disulfonate, diethyldithiocarbamate, and pyrrolidine dithiocarbamate were purchased from Aldrich (Milwaukee, WI, 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). Desferrioxamine mesylate (Desferal; Novartis, Basel, Switzerland) was obtained from Ciba Geigy. Microscope glass super frosted slides were supplied by Menzel-Glaset, 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) by Boehringer Mannheim (Indiana, IN, USA) and BAPTA/AM (1,2-bis(2aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid acetoxymethyl ester) by Calbiochem (Schwalbach, Denmark). All other chemicals used were of analytical grade.

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Cell culture and treatment Growth medium (100 `ıl RPMI 1640) containing 1.5 ⫻105 Jurkat cells (ATCC, clone E6-1) were placed into each of 96 wells of ELISA plastic plates and incubated for 1 h at 37°C, 95% air, 5% CO2. Cells were subsequently treated with the indicated amounts of glucose oxidase for the time periods indicated. Additions of metal chelators were done prior to the addition of glucose oxidase for the periods indicated. Finally, at the time points indicated, cells were collected by centrifugation (250 ⫻ g for 5 min at 4°C) for further analysis. Single cell gel electrophoresis The assay used was essentially the same as described first by Ostling and Johanson [16] and later by Singh et al. [17]. Cells were suspended in 1% low-melting-point agarose in PBS, pH 7.4, and pipetted onto superfrosted glass microscope slides precoated with a layer of 1% normal-melting-point agarose (warmed at 37°C prior to use). The agarose was allowed to set at 4°C for 10 min 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 at 4°C for 40 min in order to allow for separation of the two DNA strands (alkaline unwinding). Electrophoresis was performed in the unwinding solution at 30 V (1 V/cm), 300 Amps 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. The damage was not homogeneous and visual scoring of the cellular DNA on each slide was based on characterization of 100 randomly selected nucleoids. The comet-like DNA formations were categorized into 5 classes (0, 1, 2, 3, and 4) representing an increasing extent of DNA damage seen as a “tail.” Each comet was assigned a value according to its class. Accordingly, the overall score for 100 comets ranged from 0 (100% of comets in class 0) 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 [12]. Visual scoring expressed in this way correlates near linearly with other parameters such as percent of DNA in the tail estimated after computer image analysis using a specific software package [18,19]. Observation and anal-

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Fig. 1. Time course of H2O2-induced cellular DNA damage following addition of glucose oxidase. Jurkat cells (1.5 ⫻ 105 cells/100 ␮l) were exposed to increasing rates of H2O2 generated by the action of glucose oxidase for the indicated periods of time ranging from 0 to 45 min. The final concentrations of glucose oxidase were 0.1 (-}-), 1.0 (-■-), 5.0 (-Œ-), and 50 (-X-) ng per 100 ␮l. The DNA damage of individual cells was estimated by the comet assay, as described in Materials and Methods. Each value represents the mean of duplicate measurements, which differed no more than 10%. The same experiment was repeated several times with essentially the same results.

ysis of the results were always carried out by the same experienced person, using a specific pattern when moving along the slide. The analysis was conducted in a blinded way, i.e., the observer had no knowledge of the identity of the slide. Flow cytometric analysis Flow cytometric analysis was performed on a FACScan Becton Dickinson apparatus. Cells were incubated for 5 min with 50 ␮g/ml propidium iodide before being analyzed. 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, all chelators used in this study were found not to be able to directly inhibit glucose oxidase in the H2O2 generating system (not shown). Statistical analysis Student’s paired t-test was used in order to examine statistically significant differences.

RESULTS AND DISCUSSION

Time course of H2O2-induced SSB-formation in cellular DNA We have reported previously that H2O2 generated by the action of glucose oxidase (GO) induces time- and dose-dependent formation of single strand breaks (SSB) in isolated, cultured lymphocytes from human peripheral blood [12]. Similarly, exposure of Jurkat cells (1.5 ⫻ 105 cells per 100 ␮l) in growth medium containing 10% fetal calf serum to continuously generated H2O2 caused timeand dose-dependent induction of SSB, as assessed by the comet assay (Fig. 1). At low rates of H2O2 generation (0.1 ng GO per well) there was no observable increase in SSB formation. However, when the rate of H2O2 generation reached submicromolar levels (0.2 ␮M/min, 1 ng of GO per well) an initial increase of nuclear SSB was observed, followed by a decline and return to basal levels. This transient increase in SSB occurred despite the fact that H2O2 was continuously generated throughout the experiment. At intermediate rates of H2O2 generation (1.0 –2.0 ␮M H2O2/min, 5–10 ng GO) the same profile with an initial increase and a subsequent gradual decrease was observed, but this was followed by an increase toward maximal DNA damage after 45 min of incubation (Fig. 1). This profile of a transient decrease in the level of SSB was repeatedly observed in several separate experiments (results not shown). It seems likely from these experiments that intracellular defense mechanisms initially protect against or repair DNA damage, but subsequently become exhausted or overwhelmed,

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Fig. 2. Effect of DTPA on H2O2-induced single strand break formation in cellular DNA. Conditions were as in Fig. 1 using 50 ng glucose oxidase. DTPA, at the concentrations indicated, was added to the cells 15 min before the addition of glucose oxidase. Ten minutes after the addition of glucose oxidase, DNA damage was determined by the comet assay as described in Materials and Methods. Each value represents the mean of triplicate measurements in two different experiments ⫾ SD. Scheme 1. Chemical structures of iron chelators used in this study.

leaving the nuclear DNA unprotected against H2O2. At even higher rates of H2O2 production (10.1 ⫾ 1.3 ␮M H2O2/min, 50 ng GO) DNA damage rapidly reached the maximum level observable by the “comet” assay (Fig. 1). In all experiments shown in Fig. 1 the cells remained viable for at least 60 min of incubation, as determined by propidium iodide exclusion using flow cytometry (results not shown). Involvement of iron in H2O2-induced cellular DNA damage It has been proposed previously that iron plays a crucial role in H2O2-induced DNA damage and cytotoxicity [20 –22]. In order to evaluate the role of iron in H2O2-induced SSB formation in our system, a number of specific iron chelators were used (Scheme 1). When cells were pre-incubated with the membrane-impermeable iron chelator DTPA in growth medium containing 10% fetal calf serum, no differences in the level of DNA damage were observed either in the presence or absence of 50 ng glucose oxidase (Fig. 2). In contrast, treatment of the cells with increasing concentrations of desferrioxamine (DFO, 0.1 to 5 mM) prior to the addition of GO protected the cells in a dose- and time-dependent manner from H2O2-induced DNA damage (Fig. 3). The higher the concentration of DFO, the faster it provided maximal protection against DNA damage. In combination with the results obtained with DTPA, it appears plausible that

DFO offers its protection by binding intracellular iron ions. The fact that the degree of protection by DFO increased with the preincubation time suggests a slow rate of penetration across the cell membrane. The involvement of intracellular iron in H2O2-induced DNA damage was further supported by the results of experiments using 1,7- and 1,10-phenanthroline. These compounds have the same chemical composition but differ in the position of a single nitrogen atom (Scheme 1). This structural difference renders 1,7phenanthroline unable to chelate transition metal ions, while 1,10-phenanthroline is a good ferrous ion chelator [23]. As shown in Fig. 4, 1,10-phenanthroline, but not 1,7-phenanthroline, dose-dependently protected against cellular DNA damage. In contrast to DFO, the effects of 1,10-phenanthroline were not time-dependent, suggesting fast penetration of this compound into the cell. This notion is also in agreement with the relative lipophilicity of DFO and 1,10-phenanthroline [24]. The inability of 1,7-phenanthroline to protect the cells from SSB formation indicates that the metal binding capacity—and not other properties— of 1,10-phenantroline is responsible for the observed effect. It should be noted that when very low concentrations of 1,10-phenanthroline were used (0.1 to 1.0 ␮M) a small nonsignificant increase in SSB formation was observed (results not shown), indicating that at low 1,10-phenanthroline/iron ratios, 1,10-phenanthroline may enhance H2O2-induced DNA damage. None of the above agents (DFO, DTPA, 1,7-, and 1,10phenanthroline), when tested in the absence of H2O2,

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Fig. 3. Effect of desferrioxamine on H2O2-induced single strand break formation in cellular DNA. Conditions were as in Fig. 2, except that desferrioxamine (DFO) was added to the cells at the concentrations and for the time periods indicated before the addition of 50 ng glucose oxidase. Ten min after the addition of glucose oxidase, DNA damage was determined by the comet assay. Each value represents the mean of duplicate measurements, which differed no more than 10%. The same experiment was repeated one more time with essentially the same results.

induced DNA damage by themselves at the concentrations used (Fig. 2, and results not shown). In another set of experiments, the 8-hydroxyquinoline-ferrous ion complex (8-HQ:Fe2⫹, ratio 2:1) was used to load the cells with Fe2⫹ and thus potentially increase H2O2-induced DNA damage. However, low micromolar concentrations of 8-HQ, in the absence of exogenously added iron and H2O2, induced SSB formation.

Fig. 4. Effect of 1,10- and 1,7-phenanthroline on H2O2-induced single strand break formation in cellular DNA. Conditions were as in Fig. 2, except that 1,10- and 1,7-phenanthroline were added to the cells at the concentrations indicated 15 min before the addition of 50 ng glucose oxidase. Ten min after the addition of glucose oxidase, DNA damage was determined by the comet assay. Each value represents the mean of duplicate measurements.

In the presence of H2O2, increased DNA damage was observed only at concentrations of 8-HQ:Fe2⫹ that were toxic by themselves (results not shown). Thus, the interpretation of these data is uncertain. The role of copper in cellular DNA damage The second-order rate constants for the iron- and copper-mediated Fenton reaction are 76 and 4,700 M⫺1 s⫺1, respectively, thus copper is 62-fold more efficient than iron in producing •OH under the same conditions [25,26]. However, the copper(I)-specific chelating agent neocuproine (NC, 2,9-dimethyl-1,10-phenanthroline) (Scheme 2) was ineffective at preventing H2O2-induced DNA damage, while at the same time it induced SSB formation by itself in the absence of exogenous H2O2 (Fig. 5A). On the other hand, bathocuproine disulfonate (BCS, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate), the water-soluble, membrane-impermeable analog of neocuproine, also was ineffective at preventing H2O2-induced DNA damage, but did not induce SSB formation in the absence of H2O2 (Fig. 5B). These results suggest that copper is not involved in H2O2-induced SSB formation. Copper chelators have previously been shown to be cytotoxic, likely due to facilitated copper transport from the growth medium into the cell [27,28]. To further investigate this possibility, two dithiocarbamates (diethyldithiocarbamate (DDC) and pyrrolidine dithiocarbamate (PDTC)), which are well-known copper chelators,

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Scheme 2. Chemical structures of copper and calcium chelators used in this study.

were examined for their ability to protect against cellular DNA damage. As shown above, these agents were ineffective at preventing H2O2-induced cellular DNA damage, while they induced the formation of SSB in the absence of H2O2 (Fig. 6A,B). These data are consistent with previous suggestions that dithiocarbamate-induced cytotoxicity is due to facilitated copper transport across the plasma membrane [29 –31]. However, incubation of cells with the membrane-impermeable copper chelator BCS (0.5 mM) before the addition of copper chelators (NC, PDTC, and DDC, 0.5 mM each), did not provide any protection against H2O2induced DNA damage (results not shown). In contrast, pre-incubation of the cells with 1 mM 1,10-phenanthroline 15 min before the addition of the copper chelators (NC, PDTC, and DDC, 0.5 mM each) offered complete protection in all cases (Fig. 7). These data indicate the involvement of intracellular iron in the mechanism(s) of copper chelator-induced DNA damage. The role of calcium in H2O2-induced DNA damage The role of intracellular Ca2⫹ in H2O2-induced DNA damage and cell toxicity is controversial. Although several authors have proposed the involvement of intracellular Ca2⫹ in H2O2-induced DNA damage and cell toxicity [11,12], there are recent papers postulating the

Fig. 5. Effect of neocuproine and bathocuproine disulfonate on H2O2induced single strand break formation in cellular DNA. Conditions were as in Fig. 2, except that (A) neocuproine (NC) and (B) bathocuproine disulfonate (BCS) were added to the cells at the concentrations indicated 15 min before the addition of 50 ng glucose oxidase. Ten min after the addition of glucose oxidase, DNA damage was determined by the comet assay. Each value represents the mean of duplicate measurements in two different experiments ⫾ SD.

opposite [32]. We recently reported that the Ca2⫹ chelator BAPTA/AM at low concentrations (1.0 to 5 ␮M) strongly inhibits H2O2-induced SSB formation in human lymphocytes [12]. Similarly, in this study we observed that BAPTA/AM protects Jurkat cells from H2O2-induced DNA damage (Fig. 8). The fact that relatively low concentrations of BAPTA/AM were effective supports the idea that it acted by chelating Ca2⫹. However, since BAPTA/AM accumulates in cells after its hydrolysis, the possibility exists that it can reach high intracellular concentrations, which may be able to prevent DNA damage by chelating redox active iron. Another interesting possibility is that the interaction of H2O2 and Fe2⫹ may be responsible for the elevation of intracellular Ca2⫹, which subsequently activates endonucleases leading to SSB formation. Such an interplay between iron and calcium has been proposed previously [33,34] and is currently under investigation in our laboratory.

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Fig. 7. Effect of 1,10-phenanthroline pretreatment on copper chelatorinduced DNA damage. Conditions were as in Figs. 5 and 6, except that cells were preincubated with 1.0 mM 1,10-phenanthroline before the addition of NC, PDTC, and DDC (0.5 mM each). Ten min later, DNA damage was determined by the comet assay technique as described in Materials and Methods. Each value represents the mean of triplicate measurements in two different experiments ⫾ SD.

Fig. 6. Effect of dithiocarbamate on H2O2-induced single strand break formation in cellular DNA. Conditions were as in Fig. 2, except that (A) diethyldithiocarbamate (DDC) and (B) pyrrolidine dithiocarbamate (PDTC) were added to the cells at the concentrations indicated 15 min before the addition of 50 ng glucose oxidase. Ten min after the addition of glucose oxidase, DNA damage was determined by the comet assay. Each value represents the mean of duplicate measurements in two different experiments ⫾ SD.

efficient than iron in catalyzing H2O2-induced DNA damage in noncellular systems [7,10,25,26,35,36], as far as we know, there are no published data showing that intracellular copper plays a critical role in H2O2-induced DNA damage in cellular systems. This notion is consistent with recent findings that intracellular free copper is undetectable [37], while intracellular free iron can be detected in low micromolar concentrations [38]. Based on these considerations and our own experimental results of the present study, we believe that intracellular iron, not copper, plays a critical role in H2O2-induced cellular DNA damage.

CONCLUSIONS

The results presented in this study demonstrate the involvement of intracellular redox-active iron ions in H2O2-induced SSB formation. This conclusion is based on the ability of a number of iron chelators to reduce the levels of H2O2-induced SSB. On the other hand, specific copper chelators were ineffective at preventing H2O2induced DNA damage, indicating that iron represents the main redox-active transition metal inside the cell. In addition, the copper-specific chelators enhanced SSB in the cellular DNA in the absence of H2O2. This inherent toxicity of copper chelating agents has been proposed to be related to their copper-binding ability and formation of the respective lipophilic copper complexes [25–29]. This notion, however, is not supported by the results of the present study, since the iron-specific chelator 1,10-phenanthroline was shown to be able to prevent DNA damage induced by these copper chelating agents (Fig. 7). Although it has been shown that copper is more

Fig. 8. Effect of BAPTA/AM on H2O2-induced single strand break formation in cellular DNA: Conditions were as in Fig. 2, except that BAPTA/AM was added to the cells at the concentrations indicated 15 min before the addition of 50 ng glucose oxidase. Ten min after the addition of glucose oxidase, DNA damage was determined by the comet assay. Each value represents the mean of duplicate measurements in two different experiments ⫾ SD.

The role of iron in H2O2-induced DNA damage

The observation that the intracellular Ca2⫹-chelator BAPTA/AM was also an effective inhibitor of H2O2induced SSBs, at least at low fluxes of H2O2, indicates the involvement of intracellular Ca2⫹ changes in this process. Although not experimentally established, we are tempted to speculate that the increase in the intracellular Ca2⫹ concentration may be a consequence of the interaction of Fe2⫹ with H2O2 and subsequent formation of • OH, causing oxidative damage to Ca2⫹-regulating proteins. It has to be stressed, however, that this may take place in cells only at low steady-state concentrations of H2O2, as in this work by the use of glucose oxidase. At high concentrations, H2O2 may overwhelm the scavenging systems in the cytoplasm and reach the nucleus to directly induce site-specific DNA damage. This mechanism may apply to most of the previously published data [11,13,20,22,30], since H2O2 was added as a bolus. Whether such conditions are relevant to in vivo situations remains to be investigated. Acknowledgements — This research was supported by grants from the program EPET II, No 11480 and PENED 99, No 99ED181 of General Secretariat of Research and Technology, Athens, Greece. The authors would like to thank Prof. O. Tsolas for his support and comments. REFERENCES [1] Forman, H. J.; Cadenas, E., eds. Oxidative stress and signal transduction. New York: Chapman and Hall; 1997. [2] Remacle, J.; Raes, M.; Toussaint, O.; Renard, P.; Rao, G. Low levels of reactive oxygen species as modulators of cell function. Mutat. Res. 316:103–122; 1995. [3] Wolin, M. S.; Mohazzab, K. M. Mediation of signal transduction by oxidants. In: Scandalios, J. G., ed. Oxidative stress and the molecular biology of antioxidant defenses. New York: Cold Spring Harbor Laboratory Press; 1997:21– 48. [4] Hansen, L. L.; Ikeda, Y.; Olsen, G. S.; Busch, A. R.; Mosthaf, L. Insulin signaling is mediated by micromolar concentrations of H2O2: evidence for a role of H2O2 in tumor necrosis factor alpha-mediated insulin resistance. J. Biol. Chem. 274:25078 – 25084; 1999. [5] Hampton, M. B.; Orrenius, S. Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett. 414:552–556; 1997. [6] Carballo, M.; Conde, M.; El Bekay, R.; Martin-Nieto, J.; Camacho, M. J.; Monteseirin, J.; Conde, J.; Bedoya, F. J.; Sorbino, F. Oxidative stress triggers STAT3 tyrosine phosphorylation and nuclear translocation in human lymphocytes. J. Biol. Chem. 274: 17580 –17586; 1999. [7] Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18:321–336; 1995. [8] Halliwell, B.; Aruoma, O. I. DNA damage by oxygen-derived species: its mechanism and measurement in mammalian systems. FEBS Lett. 281:9 –19; 1991. [9] Henle, E. S.; Han, Z.; Tang, N.; Rai, P.; Luo, Y.; Linn, S. Sequence-specific DNA cleavage by Fe2⫹-mediated Fenton reactions has possible biological implications. J. Biol. Chem. 274: 962–971; 1999. [10] Chevion, M. A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals. Free Radic. Biol. Med. 5:27–37; 1988. [11] Cantoni, O.; Sestili, P.; Cattabeni, F.; Bellomo, G.; Pou, S.; Cohen, M.; Cerutti, P. Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity. Eur. J. Biochem. 182:209 –212; 1989.

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ABBREVIATIONS

BAPTA/AM—1,2-bis(2-aminophenoxy)ethane-N,N,N⬘, N⬘-tetraacetic acid acetoxymethyl ester BCS— bathocuproine disulfonate DDC— diethyldithiocarbamate DFO— desferrioxamine, Desferal, deferoxamine DTPA— diethylenetriaminepentaacetic acid GO— glucose oxidase NC—neocuproine PDTC—pyrrolidine dithiocarbamate SSB—single stand break

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