Alvaro Mordente

Defective Plasma Antioxidant Defenses and Enhanced Susceptibility to Lipid Peroxidation in Uncomplicated IDDM

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 ±131 vs. 955 ± 104 μmol/1, P < 0.001) and significantly increased levels of ROOHs (7.13 ± 2.11 vs. 2.10 ± 0.71 μmol/1, P < 0.001) and conjugated dienes (0.0368 ± 0.0027 vs. 0.0328 ± 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 ± 0.0028 vs. 0.0259 ± 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 ± 0.0011 vs. 0.0069 ± 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH‐supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe‐4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX‐Fe(II) complexes as ultimate by‐products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein‐1 (IRP‐1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP‐1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol‐dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP‐1 or to reincorporate iron within new Fe‐S motifs. This damage is mediated by DOX‐Fe(II) complexes and reflects oxidative modifications of ―SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP‐1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP‐1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.

Lycopene and Cardiovascular Diseases: An Update

Cardiovascular disease (CVD) is the leading cause of death in Western societies and accounts for up to a third of all deaths worldwide. In comparison to the Northern European or other Western countries, the Mediterranean area has lower rates of mortality from cardiovascular diseases and cancer, and this is attributed, at least in part, to the so-called Mediterranean diet, which is rich in plantderived bioactive phytochemicals. Identification of the active constituents of the Mediterranean diet is therefore crucial to the formulation of appropriate dietary guidelines. Lycopene is a natural carotenoid found in tomato, an essential component of the Mediterranean diet, which, although belonging to the carotenoid family, does not have pro-vitamin A activity but many other biochemical functions as an antioxidant scavenger, hypolipaemic agent, inhibitor of pro-inflammatory and pro-thrombotic factors, thus potentially of benefit in CVD. In particular, the review intends to conduct a systematic analysis of the literature (epidemiological studies and interventional trials) in order to critically evaluate the association between lycopene (or tomato products) supplementation and cardiovascular diseases and/or cardiovascular disease risk factors progression, and to prepare provision of evidence-based guidelines for patients and clinicians. Several reports have appeared in support of the role of lycopene in the prevention of CVD, mostly based on epidemiological studies showing a dose-response relationship between lycopene and CVD. A less clear and more complex picture emerges from the interventional trials, where several works have reported conflicting results. Although many aspects of lycopene in vivo metabolism, functions and clinical indications remain to be clarified, supplementation of low doses of lycopene has been already suggested as a preventive measure for contrasting and ameliorating many aspects of CVD.

Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers

Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically significant difference between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were significantly (p < 0.05) higher in smokers. Smoking of a single cigarette induced a significant reduction in glutathione concentration (p < 0.05). Salivary antioxidant power may affect individual sensitivity toward tobacco stress.

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer's disease.

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimers disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P < 0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P < 0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.

Defective one- or two-electron reduction of the anticancer anthracycline epirubicin in human heart. Relative importance of vesicular sequestration and impaired efficiency of electron addition

One-electron quinone reduction and two-electron carbonyl reduction convert the anticancer anthracycline doxorubicin to reactive oxygen species (ROS) or a secondary alcohol metabolite that contributes to inducing a severe form of cardiotoxicity. The closely related analogue epirubicin induces less cardiotoxicity, but the determinants of its different behavior have not been elucidated. We developed a translational model of the human heart and characterized whether epirubicin exhibited a defective conversion to ROS and secondary alcohol metabolites. Small myocardial samples from cardiac surgery patients were reconstituted in plasma that contained clinically relevant concentrations of doxorubicin or epirubicin. In this model only doxorubicin formed ROS, as detected by fluorescent probes or aconitase inactivation. Experiments with cell-free systems and confocal laser scanning microscopy studies of H9c2 cardiomyocytes suggested that epirubicin could not form ROS because of its protonation-dependent sequestration in cytoplasmic acidic organelles and the consequent limited localization to mitochondrial one-electron quinone reductases. Accordingly, blocking the protonation-sequestration mechanism with the vacuolar H+-ATPase inhibitor bafilomycin A1 relocalized epirubicin to mitochondria and increased its conversion to ROS in human myocardial samples. Epirubicin also formed ∼60% less alcohol metabolites than doxorubicin, but this was caused primarily by its higher Km and lower Vmax values for two-electron carbonyl reduction by aldo/keto-reductases of human cardiac cytosol. Thus, vesicular sequestration and impaired efficiency of electron addition have separate roles in determining a defective bioactivation of epirubicin to ROS or secondary alcohol metabolites in the human heart. These results uncover the molecular determinants of the reduced cardiotoxicity of epirubicin and serve mechanism-based guidelines to improving antitumor therapies.

Human Heart Cytosolic Reductases and Anthracycline Cardiotoxicity

Anthracyclines are a class of antitumor drugs widely used for the treatment of a variety of malignancy, including leukemias, lymphomas, sarcomas, and carcinomas. Different mechanisms have been proposed for anthracycline antitumor effects including freeradical generation, DNA intercalation/binding, activation of signaling pathways, inhibition of topoisomerase II and apoptosis. A life‐threatening form of cardiomyopathy hampers the clinical use of anthracyclines. According to the prevailing hypothesis, anthracyclines injure the heart by generating damaging free radicals through iron‐catalyzed redox cycling. Although the “iron and freeradical hypothesis” can explain some aspects of anthracycline acute toxicity, it is nonetheless disappointing when referred to chronic cardiomyopathy. An alternative hypothesis implicates C‐13 alcohol metabolites of anthracyclines as mediators of myocardial contractile dysfunction (“metabolite hypothesis”). Hydroxy metabolites are formed upon two‐electron reduction of the C‐13 carbonyl group in the side chain of anthracyclines by cytosolic NADPH‐dependent reductases. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca 2+ movements, ultimately impairing cardiac contraction and relaxation. In addition, alcohol metabolites can impair cardiac intracellular iron handling and homeostasis, by delocalizing iron from the [4Fe‐4S] cluster of cytoplasmic aconitase. Chronic cardiotoxicity induced by C‐13 alcohol metabolite might be primed by oxidative stress generated by anthracycline redox cycling (“unifying hypothesis”). Putative cardioprotective strategies should be aimed at decreasing C‐13 alcohol metabolite production by means of efficient inhibitors of anthracycline reductases, as short‐chain coenzyme Q analogs and chalcones that compete with anthracyclines for the enzyme active site, or by developing novel anthracyclines less susceptible to reductive metabolism.

Inhibition of salivary enzymes by cigarette smoke and the protective role of glutathione

Tobacco smoke is involved in the pathogenesis of several diseases regarding different body systems, mainly cardiovascular and respiratory in addition to its local toxic effect in the oral cavity. The noxious effects of smoke compounds justify the high incidence of periodontal diseases, caries, and neoplastic diseases of oral tissues in smokers. Some toxic components of tobacco smoke, unsaturated and saturated aldehydes, could interact with thiol rich compounds, leading to structural and functional modification of these molecules. Previous papers have demonstrated an in vitro significant decrease of some enzymatic activities, both in plasma and in saliva, following external addition of aldehydes or exposure to cigarette smoke (CS). Furthermore, the same studies underlined the protective effect exerted by the addition of glutathione (GSH) against the damaging role of smoke aldehydes. In this study some salivary enzymes (lactic dehydrogenase [LDH], aspartate aminotransferase [AST] and amylase), and total GSH were measured in 20 volunteers smokers, before and just after smoking a single cigarette. All enzymatic activities showed a significant inhibition following a single cigarette, probably due to the interaction between smoke aldehydes and–SH groups of the enzyme molecules. Moreover, the percentage of the enzymatic inhibition showed a negative correlation with the basal level of salivary GSH. Our results emphasize that not only one cigarette is sufficient to impair the salivary enzymatic activities but also strengthen the proposed protective role of GSH against the noxious biochemical effects of CS.

New Developments in Anthracycline-Induced Cardiotoxicity

Anthracyclines are among the most effective anticancer drugs ever developed. Unfortunately, their clinical use is severely limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure, usually refractory to conventional therapy. The pathophysiology of anthracycline-induced cardiomyopathy remains controversial and incompletely understood. The current thinking is that anthracyclines are toxic per se but gain further cardiotoxicity after one-electron reduction with ROS overproduction or two-electron reduction with conversion to C-13 alcohol metabolites. ROS overproduction can probably be held responsible for anthracycline acute cardiotoxicity, but not for all the aspects of progressive cardiomyopathy. Intramyocardial formation of secondary alcohol metabolites might play a key role in promoting the progression of cardiotoxicity toward end-stage cardiomyopathy and congestive heart failure. In this review we also discuss recent developments in: a) the molecular mechanisms underlying anthracycline-induced cardiotoxicity; b) the role of cytosolic NADPH-dependent reductases in anthracycline metabolism; c) the influence of genetic polymorphisms on cardiotoxicity outcome; d) the perspectives on the most promising strategies for limiting or preventing anthracycline-induced cardiotoxicity, focusing on controversial aspects and on recent data regarding analogues of the natural compounds, tumor-targeted formulations and cardioprotective agents.

Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. Pharmacologic and molecular reappraisal of anthracycline cardiotoxicity.

Anticancer therapy with doxorubicin (DOX) and other quinone anthracyclines is limited by severe cardiotoxicity, reportedly because semiquinone metabolites delocalize Fe(II) from ferritin and generate hydrogen peroxide, thereby promoting hydroxyl radical formation and lipid peroxidation. Cardioprotective interventions with antioxidants or chelators have nevertheless produced conflicting results. To investigate the role and mechanism(s) of cardiac lipid peroxidation in a clinical setting, we measured lipid conjugated dienes (CD) and hydroperoxides in blood plasma samples from the coronary sinus and femoral artery of nine cancer patients undergoing intravenous treatments with DOX. Before treatment, CD were unexpectedly higher in coronary sinus than in femoral artery (342 +/- 131 vs 112 +/- 44 nmol/ml, mean +/- SD; P < 0.01), showing that cardiac tissues were spontaneously involved in lipid peroxidation. This was not observed in ten patients undergoing cardiac catheterization for the diagnosis of arrhythmias or valvular dysfunctions, indicating that myocardial lipid peroxidation was specifically increased by the presence of cancer. The infusion of a standard dose of 60 mg DOX/m(2) rapidly ( approximately 5 min) abolished the difference in CD levels between coronary sinus and femoral artery (134 +/- 95 vs 112 +/- 37 nmol/ml); moreover, dose fractionation studies showed that cardiac release of CD and hydroperoxides decreased by approximately 80% in response to the infusion of as little as 13 mg DOX/m(2). Thus, DOX appeared to inhibit cardiac lipid peroxidation in a rather potent manner. Corollary in vitro experiments were performed using myocardial biopsies from patients undergoing aortocoronary bypass grafting. These experiments suggested that the spontaneous exacerbation of lipid peroxidation probably involved preexisting Fe(II) complexes, which could not be sequestered adequately by cardiac isoferritins and became redox inactive when hydrogen peroxide was included to simulate DOX metabolism and hydroxyl radical formation. Collectively, these in vitro and in vivo studies provide novel evidence for a possible inhibition of cardiac lipid peroxidation in DOX-treated patients. Other processes might therefore contribute to the cardiotoxicity of DOX.