Leucoisoprenochrome-o-semiquinone Formation in Freshly Isolated


Nov 20, 2004 - Los Angeles, California 90089-9121, and University of Southern California Research Center for. Liver Disease, Keck School of Medicine, ...
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Leucoisoprenochrome-o-semiquinone Formation in Freshly Isolated Adult Rat Cardiomyocytes Fernando Remia˜o,*,† Daniel Rettori,‡ Derick Han,§ Fe´lix Carvalho,† Maria L. Bastos,† and Enrique Cadenas‡ REQUIMTE/Servic¸ o de Toxicologia, Faculdade de Farma´ cia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4099-030 Porto, Portugal, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90089-9121, and University of Southern California Research Center for Liver Disease, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90089-9121 Received March 4, 2004

Sustained high levels of circulating catecholamines can lead to cardiotoxicity. There is increasing evidence that this process may result from metal-catalyzed catecholamine oxidation into semiquinones, quinones, and aminochromes. We have previously shown that Cu2+-induced oxidation of isoproterenol into isoprenochrome induces toxic effects in isolated cardiomyocytes. The aim of this study was to characterize the isoproterenol oxidation process and to locate the formation of semiquinone radicals in cardiomyocyte suspensions. Freshly isolated rat cardiomyocytes were incubated with 1 or 10 mM isoproterenol and 20 µM Cu2+ for 4 h. The formation of an isoproterenol oxidation radical was detected in the extracellular medium, cells, membranes, and heavy organelles by electron spin resonance spectroscopy. An electron spin resonance signal assigned to leucoisoprenochrome-o-semiquinone increased in a time-dependent manner in the extracellular medium. A second electron spin resonance signal, characteristic of an immobilized radical, was also found in the cardiomyocytes. The latter was attributed to leucoisoprenochrome-o-semiquinone immobilized on cellular components such as membranes, cytoskeleton, nucleus, and heavy organelles. In addition, the levels of leucoisoprenochromeo-semiquinone decreased in the presence of glutathione. Computer simulations of the experimental spectra indicate the formation of two distinct isomeric leucoisoprenochrome-osemiquinone radicals during isoproterenol oxidation. The present study shows that the isoproterenol oxidation in isolated rat cardiomyocytes correlates with the formation of leucoisoprenochrome-o-semiquinone in the cells and in the extracellular medium, suggesting that it might be involved in cardiotoxicity induced by the oxidation of catecholamines.

Introduction Sympathetic activity has been associated with cardiotoxic effects (1). Excessive release of catecholamines into the interstitial space and circulation may result from stress due to the lifestyle of Western societies (2), reduced physical activity and hypercaloric nutrition (3), or pathologies such as ischemia (4) and pheochromocytoma (5). The isoproterenol (or isoprenaline, ISO)1 is a synthetic β-agonist that has been used as a model compound for cardiotoxicity studies of catecholamines. In these studies, ISO has been reported to induce myocardial necrosis (2, 3), which could result from alterations in Ca2+ homeostasis (2), lipid peroxidation (2, 3, 6), and decrease in ATP and creatine phosphate stores (2). It was suggested that these effects might result from adrenoceptors activation and subsequent intracellular Ca2+ overload (2, 7). However, recent evidences suggest that oxidative stress is also implicated in ISO-mediated cardiotoxic effects (1, 8-10). * To whom correspondence should be addressed. Tel: 351-222078979. Fax: 351-222003977. E-mail: [email protected] † Universidade do Porto. ‡ School of Pharmacy, University of Southern California. § Keck School of Medicine, University of Southern California. 1 Abbreviations: ISO, isoproterenol; chromium oxalate, potassium chromium(III) oxalate trihydrate; ESR, electron spin resonance

Moreover, catecholamine oxidative products have been implicated in poor prognosis of patients with heart failure (11). Furthermore, catecholamines oxidation, such as dopamine oxidation in the brain, seems to be involved in other pathologies, as in Parkinson’s disease (12-14). The oxidation of catecholamines can occur by autoxidation, enzymatic, or metal catalysis, and these pathways may involve the formation of reactive oxygen species. The oxidative stress induced by catecholamines results at least partially from their capacity to oxidize into oquinones. These quinones are reactive intermediates that undergo irreversible 1,4-intramolecular cyclization into leucoaminochromes that further oxidize to aminochromes (2,3-dihydroindole-5,6-diones; Scheme 1) (3, 15). During this process, two catecholamine radicals can be formed: catecholamine-o-semiquinone and leucoaminochrome-osemiquinone. The aminochromes are extremely reactive and rapidly disappear from blood probably as results of adduct, aminolutins, and polymers of aminochromesaminolutins formation (3, 10, 16, 17). The oxidation of catecholamines has been thoroughly investigated by electron spin resonance (ESR) spectroscopy, particularly with the spin stabilization technique, using a diamagnetic cation (e.g., Zn2+, Mg2+) to stabilize oxidatively generated semiquinones (18, 19). By stabilizing the

10.1021/tx049924g CCC: $27.50 © 2004 American Chemical Society Published on Web 11/20/2004

Cardiotoxicity of Isoproterenol/Cu2+ Scheme 1. Proposed Mechanism for Catecholamines Oxidation to Aminochromes (Adapted from Bindoli et al. (Ref 15))

radical species, higher concentrations are obtained to reach the detection limit necessary for ESR analysis. The presence of transition elements such as copper seems to enhance catecholamines toxicity by promoting their oxidation (20-22). In fact, our recent results show that the oxidative stress induced by the simultaneous incubation of ISO and Cu2+ in cardiomyocyte suspensions contributes to catecholamines cardiotoxicity (8, 9). The ISO oxidation process and/or the products of ISO oxidation induced loss of cardiomyocyte viability, intracellular GSSG formation with subsequent release into the extracellular medium, and total glutathione depletion, probably by conjugation with o-quinones. Furthermore, the intracellular activity of selenium-dependent glutathione peroxidase, glutathione-S-transferase, and glutathione reductase decreased after ISO oxidation, reflecting oxidation or covalent adduct formation at thiol groups. These toxic effects can be attributed to isoproterenol-o-quinone, isoprenochrome, and to the radical species generated during these oxidation processes (8, 9). The present study attempts to identify and locate the formation of semiquinones in isolated cardiomyocytes by using ESR spectroscopy, to elucidate the molecular mechanism of ISO oxidation in the presence of Cu2+ and its related toxic effects.

Materials and Methods Adult Rat Cardiomyocytes. Isolation of calcium-tolerant cardiomyocytes from the adult rat was performed by collagenase type II (Worthington, Lakewood, NJ) and protease type XIV (Sigma, St. Louis, MO) perfusion as previously described (23, 24). Adult male Wistar rats, weighing 175-225 g, were used. Incubations were performed at 37 °C, using 2.5 × 105 cells/mL in modified Krebs-Henseleit solution containing (mM) 102 NaCl, 4.2 KCl, 1.0 MgSO4, 10.0 glucose, 12.5 NaHCO3, 0.9 KH2PO4, 11.0 HEPES, supplemented with 1.8 CaCl2 (pH 7.2) and saturated with a gaseous stream of carbogen. After 30 min of preincubation, ISO (1 or 10 mM) and 20 µM Cu2+ (final concentrations) were added to the cell suspensions. Control samples were treated with only 20 µM Cu2+. Incubations were performed for up to 4 h. Cell viability was determined by the trypan blue exclusion assay.

Chem. Res. Toxicol., Vol. 17, No. 12, 2004 1585 Sample Treatments. After 1, 2, 3, and 4 h of incubation, aliquots of cell suspensions (1 mL) were centrifuged at 300g for 2 min to obtain different types of samples for ESR analysis. (1) Extracellular sample: The supernatant (200 µL) was used for extracellular determinations. (2) Wash solution sample: The pellet was washed with 1 mL of fresh incubating solution and centrifuged at 300g for 2 min. This process was repeated three times, and the last supernatant (200 µL) was used as the wash solution sample. (3) Cellular sample: The pellet was resuspended in 200 µL of fresh incubating solution and was used as the cellular sample. (4) Membrane sample: Cellular sample was sonicated (50 W; 20 kHz) for 1 min on ice and centrifuged at 16 000g for 2 min. The pellet was resuspended in 200 µL of fresh incubating solution and was used as the membrane sample. (5) Membrane sample supernatants: The supernatant obtained after the membrane sample preparation was used for ESR analysis (membrane sample supernatant I). In addition, the membrane sample was sonicated (50 W; 20 kHz) for 1 min on ice and centrifuged at 16 000g for 2 min, and the supernatant was subsequently used for ESR analysis (membrane sample supernatant II). In the ESR analysis performed in this study, 5 mM Mg2+ (final concentration) was used to stabilize the semiquinone radicals formed during ISO oxidation in cardiomyocyte suspensions. The Mg2+ was added immediately before ESR analysis and was shown not to affect cell viability during the time of ESR analysis. This concentration of Mg2+ was sufficient for stabilization and detection of the studied radicals. However, occasionally, 10 mM of Mg2+ was used in extracellular samples to amplify the signal, but this concentration was shown to be too aggressive to the cells and therefore could not be used for the other samples. When used, potassium chromium(III) oxalate trihydrate (chromium oxalate) and GSH were added to the extracellular and cellular samples at final concentrations of 20 and 2 mM, respectively, immediately before ESR analysis. The studies were performed on cell suspensions obtained from five rats. The studies using membrane samples, membrane sample supernatants, chromium oxalate, or GSH were performed on cell suspensions obtained from two rats. ESR Spectroscopy. ESR spectra were recorded with a Bruker ECS 106 spectrometer (operating at X-band) equipped with a cylindrical room-temperature cavity operating in TM110 mode (Bruker Biospin, Billerica, MA). Aliquots of 200 µL of the samples were transferred to bottom-sealed Pasteur pipets and measured at room temperature with the following instrument settings: microwave frequency, 9.77 GHz; microwave power, 20 mW; field modulation frequency, 100 kHz; field modulation amplitude, 1.0 G; receiver gain, 8 × 105; scan rate, 1.4 G s-1; time constant, 21 ms; number of scans accumulated, 14. Computer simulations of spectra were performed using the program WinSIM (EPR calculations for MS-Windows NT 95, version: 0.96, from P.E.S.T. - Public EPR Software Tools) written by Duling (25). The ESR spectra presented in the figures are representative scans of the experiments.

Results Extracellular samples were analyzed by ESR with 5 or 10 mM Mg2+, every hour during the 4 h of ISO (1 or 10 mM) and 20 µM Cu2+ incubation. A signal between 3475 and 3490 G with roughly three bands of similar intensities (1 × I ) 1; e.g., coupling with one nitrogen nucleus) could be observed in the extracellular samples (Figure 1). Each band seems to be a triplet with intensity ratios of 1:2:1 (2 × I ) 1/2; e.g., coupling with two hydrogen nuclei) as shown by the better defined lower field band. Samples treated with 1 mM ISO and 20 µM Cu2+ generated very weak, low-resolution ESR spectra, which were better discernible with 10 mM Mg2+ and after

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Figure 1. ESR spectra of extracellular samples from cardiomyocyte suspensions: in the presence of 10 mM Mg2+ obtained after 4 h of ISO (1 mM) and Cu2+ (20 µM) incubation; in the presence of 5 mM Mg2+ obtained after 2 and 3 h of ISO (10 mM) and Cu2+ (20 µM) incubation.

Figure 2. ESR spectra of cellular and wash solution samples in the presence of 5 mM Mg2+ from a cardiomyocyte suspension obtained after 3 or 4 h of ISO (10 mM) and Cu2+ (20 µM) incubation. An ESR spectrum of a control cellular sample obtained after 4 h of Cu2+ (20 µM) incubation is also shown.

4 h of incubation. In extracellular samples obtained after incubation with 10 mM ISO and 20 µM Cu2+, a signal, in the presence of 5 mM Mg2+, was already observed after 1 h of incubation (data not shown). However, the ESR signal was intensified in these extracellular samples after 2 and 3 h of incubation (Figure 1). Similar results were obtained by ESR analysis with 5 mM of Mg2+ in the cellular samples analyzed during the 4 h of 10 mM ISO and 20 µM Cu2+ incubation. ESR spectra of cellular samples after 3 and 4 h of incubations are shown in Figure 2. The obtained signal was apparently the same observed in the extracellular samples, albeit with a lower spectral resolution, that is, with broader ESR lines. The signal increased between 3 and 4 h of incubation, and cell viabilities at the time of these spectra analysis were 29% and 18%, respectively. Furthermore, no signals were observed in the wash solution and in the cellular control samples after 4 h of incubation (Figure 2). Similar ESR results were obtained in the control extracellular sample (data not shown). In Figure 3, the ESR spectra of the membrane sample and of the membrane sample supernatants I and II after

Remia˜ o et al.

Figure 3. ESR spectra of membrane and membrane sample supernatants I and II in the presence of 5 mM Mg2+ from a cardiomyocyte suspension incubated for 3 h with ISO (10 mM) and Cu2+ (20 µM).

3 h of 10 mM ISO and 20 µM Cu2+ incubation can be observed. It is clearly shown that the membrane sample presented an ESR signal with a similar pattern when compared to the ESR signal observed in cellular samples. Furthermore, the membrane sample supernatants I and II presented a weak signal that seemed analogous to those observed in the extracellular samples. The localization of the radicals was further studied using chromium oxalate (Figure 4). Extracellular samples treated with chromium oxalate showed signal broadening (Figure 4A), confirming the freely tumbling nature of the detected radicals. Conversely, chromium oxalate did not broaden the ESR signal present in the cellular and membrane samples (Figure 4B). The structural identification of the radical responsible for the ESR signal present in extracellular samples was achieved by computer simulation using the program WinSIM. Computer simulation of the experimental spectrum shown in Figure 5 indicates the formation of two isomeric radicals of leucoisoprenochrome-o-semiquinone. For the nitrogen (1 × I ) 1) and the two phenylic hydrogens (2 × I ) 1/2) of one of the o-semiquinones, the simulation approach provided hyperfine splitting constants of 4.17, 1.12, and 1.50, respectively. This osemiquinone species accounted for 70% of the total radical concentration, and its g value was 2.0046. The hyperfine splitting constants for the nitrogen and the two phenylic hydrogens of the other o-semiquinone were 4.32, 1.21, and 1.24, respectively. This o-semiquinone species accounted for 30% of the total radical concentration, and its g value was 2.0052. The generation of leucoisoprenochrome-o-semiquinone in cellular suspensions suggested possible interactions with GSH. This was confirmed in the experiments performed in the presence of 2 mM GSH, which showed a decrease in the intensity of the detected ESR signals in cellular samples (Figure 6). Similar results were observed in extracellular samples (data not shown). Radical concentrations were determined by double integration of spectra using TEMPOL as standard. The values obtained were 2.4 nmol mL-1 for the extracellular sample, and 2.8 nmol in the cellular sample after 3 h of 10 mM ISO and 20 µM Cu2+ incubation.

Cardiotoxicity of Isoproterenol/Cu2+

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Figure 6. ESR spectra of cellular samples in the presence of 5 mM Mg2+, treated or untreated with GSH (2 mM), from a cardiomyocyte suspension obtained after 3 h of ISO (10 mM) and Cu2+ (20 µM) incubation.

Figure 4. ESR spectra of extracellular (A), and cellular and membrane (B) samples in the presence of 5 mM Mg2+, treated or untreated with chromium oxalate, from a cardiomyocyte suspension obtained after 3 h (A) or 4 h (B) of ISO (10 mM) and Cu2+ (20 µM) incubation.

Figure 5. (A) ESR spectrum of an extracellular sample in the presence of 5 mM Mg2+ from a cardiomyocyte suspension obtained after 4 h of ISO (10 mM) and Cu2+ (20 µM) incubation. (B) Computer simulation of an ESR spectrum of a 7:3 molar ratio mixture of leucoisoprenochrome-o-semiquinone isomers (r ) 0.963).

Discussion The results presented in this study demonstrate that, in suspensions of isolated cardiomyocytes, radical species are formed from the Cu2+-mediated oxidation of isopro-

terenol. At the cellular and extracellular levels, ESR detected two isomers of leucoisoprenochrome-o-semiquinone, which can react with GSH. The observation of a broad ESR spectrum in cells indicates restricted mobility of the radical and strongly supports the notion that these radicals interact with cellular components such as membranes, cytoskeleton, nucleus, and heavy organelles. It has been shown that ESR detection and identification of semiquinones produced during oxidation of catecholamines can be greatly facilitated through the use of the spin stabilization approach (18). Several metals such as Mg2+, Zn2+, Cd2+, Sc3+, Y3+, and Al3+ have been used for spin stabilization of semiquinones (19, 26-29). The half-lives of the semiquinones are increased due to complexation with these metals and electron delocalization (14). However, these metals have been used in chemical models and in concentrations between 0.1 and 0.5 M, which are toxic for the cells. In this study, it was found that 5 mM Mg2+ was sufficient to spin stabilize the studied radicals, without affecting cell viability during the ESR analysis. The results presented in Figures 1 and 2 show the presence of an ESR signal in the extracellular medium and in cells. The absence of such a signal in wash solution samples confirms that the signal observed in the cellular samples was not a contamination from the extracellular medium. The signals, between 3475 and 3490 G, showed to be ISO dependent (not present in control samples) and were strong in extracellular and cellular samples from cardiomyocyte suspensions incubated with 10 mM ISO and 20 µM Cu2+ for 3 h. Samples analyzed before the 3 h of incubation or treated with a lower concentration of ISO also generated a signal. However, these ESR spectra were weaker and had lower resolution. Although having a lower spectral resolution, the signal present in the cellular samples seemed to be the same signal appearing in the extracellular samples. The anisotropic character of this signal can be explained by the slow tumbling of the radicals because of their interactions with cellular components. The ESR spectra in Figures 1 and 2 also demonstrate that the signals in both extracellular and cellular samples were time-dependent. Although a correlation between the cell viability and the intensity of the signal was not determined in this study, we have previously observed

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the cytotoxicity of ISO oxidation in the presence of Cu2+ in isolated cardiomyocytes, and it increased in a timedependent manner (8). In that study, 1 mM ISO and 20 µM Cu2+ induced membrane disruption in 35% and 50% of the cardiomyocytes after 3 and 4 h of incubation, respectively (after 4 h of incubation, viability values in the control and in 20 µM Cu2+ samples were about 72% and 68%, respectively). Glutathione depletion, due to GSH oxidation or GSH-adduct formation, and the inhibition of GSH dependent enzymes were also reported to increase in a time-dependent manner during incubation of 1 mM ISO and 20 µM Cu2+ in isolated cardiomyocyte suspensions (8, 9). Thus, the combination of these results indicates that the observed radical formation and cytotoxicity are strongly correlated. In fact, as reported before, cardiomyocyte death can be related not only to the formation of the toxic metabolite isoprenochrome but also to the free radicals formed during ISO oxidation (9). One of these radicals seems to be the leucoisoprenochrome-o-semiquinone. Noteworthy, in ESR studies, a similar radical, the leucodopaminochrome-o-semiquinone, showed to be extremely reactive (30) and responsible for the degenerative process occurring in dopaminergic neurons in Parkinson’s disease (20). In addition, dopamine-o-semiquinone, another radical that can result from nitric oxide-mediated dopamine oxidation, was also detected by ESR and implicated in nigral degeneration (28). The presence of an ESR signal in cells that had low viability suggests that the radical interacts with cellular components of viable and dead cells. To clarify this, the cells were completely destroyed by sonication and the obtained membrane samples were analyzed by ESR (Figure 3). The similarity of signals present in membrane and cellular samples clearly indicates that the same radical is present in both samples. Furthermore, the membrane sample supernatants showed ESR signals resembling those observed in extracellular samples. Because the signal present in membrane sample supernatants seems to result from the release of the radicals attached to the pellet of the cellular and the membrane samples after sonication and centrifugation, one may conclude that the observed signal corresponds to the same radicals. Taken together, these results indicate that free radicals were attached to cellular components of cardiomyocytes, such as membranes, cytoskeleton, nucleus, and heavy organelles, resulting in poorly resolved ESR spectra due to immobilization of the radicals. By sonication and centrifugation, the radicals gained mobility due to detachment and transference to the supernatant, and, as a consequence, a narrowing of the ESR lines was observed. Furthermore, the studies using chromium oxalate (Figure 4) support the notion that the ESR signals detected in the cellular and membrane samples are due to radicals that are interacting with an immobilized on cellular components; otherwise, signal broadening should have been observed. In fact, this chemical does not cross membranes and has the ability to broaden ESR signals from organic radicals (31, 32). The computer simulation of the ESR signal present in the extracellular samples allowed the identification of two isomeric leucoisoprenochrome-o-semiquinone radicals with g values of 2.0046 (70%) and 2.0052 (30%) (Figure 5). These data did not permit one to distinguish the relative attribution of each radical to the percentages mentioned above. The poorly resolved ESR spectra obtained from cellular and membrane samples made the identification

Remia˜ o et al. Scheme 2. Proposed Mechanism for the Copper-Catalyzed Oxidation of ISO in Cardiomyocyte Suspensions

of the radicals difficult. Nevertheless, the ESR results strongly suggest that it is the same extracellular radical, the leucoisoprenochrome-o-semiquinone, which is responsible for the nonresolved signals. In addition, it seems to be present in concentrations similar to the radical found in the extracellular sample. Furthermore, the interaction between these radicals and GSH (Figure 6) agrees with the intracellular and extracellular GSH depletion previously observed in cardiomyocyte suspensions incubated with ISO and Cu2+ (8, 9). As a proposed mechanism, it is expected that the formation of the leucoisoprenochrome-o-semiquinone radical due to ISO oxidation in cardiomyocyte suspensions is highly catalyzed by Cu2+ (Scheme 2). It is known that Cu2+ forms a complex with catechols (Cu2+-catechol) (20, 21, 33) and the oxidation of the catechol into o-quinone seems to be initially catalyzed by the reaction of a second Cu2+ with the complex (Scheme 2) (33). Chain propagation is promoted by the species CuO2+, formed by O2 addition to Cu+ (33), which can oxidize either the catecholamine or the Cu2+-isoproterenol complex. Both pathways proceed via a two-electron oxidation mechanism, which explains the absence of any ESR signal of isoproterenol-o-semiquinone. Isoproterenol-o-quinone can undergo an irreversible 1,4-intramolecular cyclization involving a nucleophilic attack of the nitrogen at the 6-position of the quinone ring, leading to the formation of the unstable leucoisoprenochrome (15). Leucoisoprenochrome autoxidizes rapidly into isoprenochrome via the intermediate leucoisoprenochrome-o-semiquinone. Alternatively, the o-quinones can undergo intermolecular (nonradical) Michael addition reactions with suitable nucleophiles, particularly thiol and amino residues, or GSH, forming protein-bound catecholamine or catecholamine-GSH adducts, respectively (14, 19, 34, 35). This kind of reaction can explain the observed interaction

Cardiotoxicity of Isoproterenol/Cu2+

between ISO oxidation products and the cellular components. The protein-bound ISO may undergo oxidative pathways similar to those for free ISO, as was suggested for protein-bound dopa (14, 36). Thus, taking into account the present results, the formation of protein-bound leucoisoprenochrome-o-semiquinone resulting from oxidation of ISO bound to proteins present in cardiomyocyte membranes may be suggested. However, we are not able to know with certainty which kind of interaction exists between the semiquinone formed and the proteins. In addition, the isoprenochrome can form adducts with thiol or amino residues of proteins (1, 10). The formation of leucoisoprenochrome-o-semiquinones can also occur via isoprenochrome reduction. In fact, the one-electron reduction of aminochromes by antioxidants (37) and enzymes (30, 31, 38, 39) and the two-electron reduction of quinones by DT-diaphorase (40) have been reported. The leucoisoprenochrome-o-semiquinones present in the cellular suspensions seem to easily interact with cellular components, which is in accordance with the high reactivity described for leucodopaminochrome-o-semiquinone (20, 30). Thus, the leucoisoprenochrome-o-semiquinone seems to be an important part of the cardiotoxicity induced by ISO oxidation. The isoproterenol-oquinone and the isoprenochrome are the other ISO oxidation products that may also be responsible for this effect (8, 9). Furthermore, the catecholamine oxidation process easily generates superoxide radicals, which in the presence of transition metals such as Cu2+ form hydroxyl radicals by Fenton-like chemistry (10, 14). Thus, reactive oxygen substances can also contribute to the cardiotoxicity. The importance of leucoaminochrome-o-semiquinones has been corroborated by a report of an increase in the cardiotoxicity of adrenochrome after heart perfusion in the presence of cysteine or ascorbic acid, which may induce one-electron reduction of adrenochrome (37). In conclusion, the ISO oxidation was correlated with the formation of the radical species leucoisoprenochromeo-semiquinone detected by ESR in isolated rat cardiomyocytes. Our work therefore suggests that these radical species may be involved in the cardiotoxicity induced by the oxidation of catecholamines.

Acknowledgment. This work was supported by Fundac¸ a˜o Luso-Americana (Proj. 488/2001), by Fundac¸ a˜o Calouste Gulbenkian, and by FCT/POCTI/FEDER European Community project (POCTI/36099/FCB/2000).

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