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Graphical abstract, Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. Surface Plasmon Resonance assays have been performed to determine the binding affinity between PDZ domain of wild type human PALS1 protein and tetradecapeptides representing the SLiMs sequences of SARS-CoV-1 and SARS-CoV-2 E proteins (E-SLiMs). SARS-CoV-2 E-SLiM binds to the human target protein with a higher affinity compared to SARS-CoV-1, showing a difference significantly greater than previously reported using the F318W mutant of PALS1 protein and shorter target peptides. Moreover, molecular dynamics simulations have provided clear evidence of the structural determinants driving this binding process. Specifically, the Arginine 69 residue in the SARS-CoV-2 E-SLiM is the key residue able to both enhance the specific polar interaction with negatively charged pockets of the PALS1 PDZ domain and reduce significantly the mobility of the viral peptide. These experimental and computational data are reinforced by the comparison of the interaction between the PALS1 PDZ domain with the natural ligand CRB1, as well as the corresponding E-SLiMs of other coronavirus members such as MERS and OCF43. Our results provide a model at the molecular level of the strategies used to mimic the endogenous SLiM peptide in the binding of the tight junctions of the host cell, explaining one of the possible reasons of the severity of the infection and pulmonary inflammation by SARS-CoV-2.

Traumatic brain injury (TBI) is the leading cause of death and disability in people less than 40 years of age in Western countries. Currently, there are no satisfying pharmacological treatments for TBI patients. In this study, we subjected rats to severe TBI (sTBI), testing the effects of a single subcutaneous administration, 30 min post-impact, of a new low molecular weight dextran sulfate, named ILB&reg, at three different dose levels (1, 5, and 15 mg/kg body weight). A group of control sham-operated animals and one of untreated sTBI rats were used for comparison (each group n = 12). On day 2 or 7 post-sTBI animals were sacrificed and the simultaneous HPLC analysis of energy metabolites, N-acetylaspartate (NAA), oxidized and reduced nicotinic coenzymes, water-soluble antioxidants, and biomarkers of oxidative/nitrosative stress was carried out on deproteinized cerebral homogenates. Compared to untreated sTBI rats, ILB&reg, improved energy metabolism by increasing ATP, ATP/ adenosine diphosphate ratio (ATP/ADP ratio), and triphosphate nucleosides, dose-dependently increased NAA concentrations, protected nicotinic coenzyme levels and their oxidized over reduced ratios, prevented depletion of ascorbate and reduced glutathione (GSH), and decreased oxidative (malondialdehyde formation) and nitrosative stress (nitrite + nitrate production). Although needing further experiments, these data provide the first evidence that a single post-injury injection of a new low molecular weight dextran sulfate (ILB&reg, ) has beneficial effects on sTBI metabolic damages. Due to the absence of adverse effects in humans, ILB&reg, represents a promising therapeutic agent for the treatment of sTBI patients.

Adult growth hormone deficiency (GHD), a condition characterized by increased oxidative stress, is related to augmented cardiovascular, metabolic and oncological risk. A case-control observational study has been performed to evaluate DNA oxidative damage analysing the production of thymidine-glycol in lymphocytes and its correlation with plasma antioxidant levels, evaluated as Total Antioxidant Capacity (TAC). GHD was diagnosed using GHRH 50μg iv+arginine 0,5 g/Kg test, with peak GH response 2 or 30 kg/m2. Three groups were identified: total GHD (n = 16), partial GHD (n = 11), and controls (n = 12). Thymidine-glycol, TAC and IGF-1 have been determined respectively in lymphocytes, plasma and serum samples. When considering thymidine-glycol, we found a significant difference between total vs partial GHD and controls. Unexpectedly thymidine-glycol was lower in total GHD, also accompanied with a significant increase in plasmatic TAC. Our results showed that in adult GHD condition, the production of antioxidant species, in response to increased oxidative stress, could exert a protective effect on thymidine-glycol formation, and consequently on DNA intracellular damages. This pilot study could be inserted in the complex scenario of oxidative damage of GHD, a subtle, yet poorly defined condition, worthy of further insights.

Selenium is a trace element, nutritionally classified as an essential micronutrient, involved in maintaining the correct function of several enzymes incorporating the selenocysteine residue, namely the selenoproteins. The human selenoproteome including 25 proteins is extensively described here. The most relevant selenoproteins, including glutathione peroxidases, thioredoxin reductases and iodothyronine deiodinases are required for the proper cellular redox homeostasis as well as for the correct thyroid function, thus preventing oxidative stress and related diseases. This review summarizes the main advances on oxidative stress with a focus on selenium metabolism and transport. Moreover, thyroid-related disorders are discussed, considering that the thyroid gland contains the highest selenium amount per gram of tissue, also for future possible therapeutic implication.

Topoisomerases are ubiquitous enzymes involved in maintaining genomic stability of the cell by regulating the over- or underwinding of DNA strands. Besides their customary functions, topoisomerases are important cellular targets of widely used anticancer drugs. In particular, topoisomerase IIα (Top2α) has been postulated as the primary molecular target of anthracycline's anticancer activity, whereas topoisomerase IIβ (Top2β), the only Top2 present in heart tissue, seems to be involved in the development of anthracycline-induced cardiotoxicity. Noteworthy, cardiotoxicity is the most frequent adverse effect of both conventional and modern anticancer targeted therapy, representing the leading noncancer-related cause of morbidity and mortality in long-term survivors. The molecular mechanisms of anthracyclineinduced cardiotoxicity have been investigated for decades and, despite the numerous mechanistic hypotheses put forward, its aetiology and pathogenesis still remain controversial. This review is aimed at focusing on the double edge sword of topoisomerase-anthracycline interaction, and, in particular, on the potential role of topoisomerases in anthracyclines anticancer activity as well as in the pathogenesis of anthracycline-induced cardiotoxicity.

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.

Irisin, a recently discovered myokine, has been considered a prognostic factor in several cardiovascular diseases. Nevertheless, no data are available on the role of irisin in patients with heart failure (HF), both with preserved (HFpEF) or reduced (HFrEF) ejection fraction. We have therefore evaluated the circulating irisin levels in HFpEF and HFrEF patients, correlating them with metabolic parameters and total antioxidant capacity (TAC), as index of oxidative stress. Irisin was significantly higher in HFpEF than in HFrEF patients (7.72 ± 0.76 vs 2.77 ± 0.77 ng/ml, respectively). An inverse correlation between irisin and TAC was found in HFpEF, but not in HFrEF. Conversely, no correlation was present with HOMA index. These data support the hypothesis that a different pathophysiological mechanism is involved in the two HF subtypes, and oxidative stress modulates irisin secretion.

Antineoplastic therapy with anthracyclines like doxorubicin (DOX) and daunorubicin (DNR) is limited by the possible development of a dose-related cardiomyopathy. Secondary alcohol metabolites like doxorubicinol (DOXol) and daunorubicinol (DNRol), formed by cytoplasmic two-electron reductases, have been implicated as potential mediators of anthracycline-induced cardiomyopathy. In the present study, we characterized the effects of 12 chalcones on the formation of anthracycline secondary alcohol metabolites by rabbit or human heart cytosol and compared them with those of quercetin and other flavonoids. Both chalcones and flavonoids inhibited DOXol or DNRol formation in isolated rabbit heart cytosol. Structure--activity relationships showed that inhibition by chalcones was determined primarily by the position of hydroxyl groups in their phenolic A and B rings. In particular, the presence of a hydroxyl group at C-4' in the A ring was an important determinant of the inhibitory activity of chalcones. Among chalcones, 2',4',2-trihydroxychalcone exhibited the highest inhibition of both DOXol and DRNol formation, but it proved less efficient than quercetin. Different results were obtained with isolated human heart cytosol: in the latter, 2',4',2-trihydroxychalcone and other hydroxychalcones inhibited both DOXol and DNRol formation, whereas quercetin and other flavonoids inhibited DNRol formation but failed to inhibit or slightly stimulated DOXol formation. These results identify chalcones as versatile inhibitors of the cytoplasmic reductases that convert anthracyclines to cardiotoxic secondary alcohol metabolites.

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.

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

The clinical efficacy of anthracyclines (e.g., doxorubicin and daunorubicin) in cancer therapy is limited by their severe cardiotoxicity, the etiology of which is still not fully understood. The development of anthracycline-induced cardiomyopathy has been found to correlate with myocardial formation and accumulation of anthracycline secondary alcohol metabolites (e.g., doxorubicinol and daunorubicinol) that are produced by distinct cytosolic NADPH-dependent reductases. The aim of the current study is to identify chemical compounds capable of inhibiting myocardial reductases implied in anthracycline reductive metabolism in an attempt to decrease the production of cardiotoxic C-13 alcohol metabolites. Among the variety of tested compounds (metal chelators, radical scavengers, antioxidants, β-blockers, nitrone spin traps, and lipid-lowering drugs), ebselen, cyclopentenone prostaglandins, nitric oxide donors, and short-chain coenzyme Q analogs resulted in being effective inhibitors of both doxorubicinol and daunorubicinol formation. In particular, ebselen (as well as ebselen diselenide, its storage form in the cells) was the most potent inhibitor of cardiotoxic anthracycline alcohol metabolites with 50% inhibition of doxorubicinol formation at 0.2 mol Eq of ebselen with respect to doxorubicin concentration. The high efficacy, together with its favorable pharmacological profile (low toxicity, lack of adverse effects, and metabolic stability) portends ebselen as a promising cardioprotective agent against anthracycline-induced cardiotoxicity.

The anthracycline doxorubicin (DOX) is an exceptionally good antineoplastic agent, but its use is limited by formation of metabolites which induce acute and chronic cardiac toxicities. Whereas the acute toxicity is mild, the chronic toxicity can produce a life-threatening cardiomyopathy. Studies in laboratory animals are of limited value in predicting the structure and reactivity of toxic metabolites in humans; therefore, we used an ethically acceptable system which is suitable for exploring DOX metabolism in human myocardium. The system involves cytosolic fractions from myocardial samples obtained during aorto-coronary bypass grafting. After reconstitution with NADPH and DOX, these fractions generate the alcohol metabolite doxorubicinol (DOXol) as well as DOX deoxyaglycone and DOXol hydroxyaglycone, reflecting reduction of the side chain carbonyl group, reductase-type deglycosidation of the anthracycline, and hydrolase-type deglycosidation followed by carbonyl reduction, respectively. The efficiency of each metabolic route has been evaluated at low and high DOX:protein ratios, reproducing acute, single-dose and chronic, multiple-dose regimens, respectively. Low DOX:protein ratios increase the efficiency of formation of DOX deoxyaglycone and DOXol hydroxyaglycone but decrease that of DOXol. Conversely, high DOX:protein ratios facilitate the formation of DOXol but impair reductase- or hydrolase-type deglycosidation and uncouple hydrolysis from carbonyl reduction, making DOXol accumulate at levels higher than those of DOX deoxyaglycone and DOXol hydroxyaglycone. Structure-activity considerations have suggested that aglycones and DOXol may inflict cardiac damage by inducing oxidative stress or by perturbing iron homeostasis, respectively. Having characterized the influence of DOX:protein ratios on deglycosidation or carbonyl reduction, we propose that the benign acute toxicity should be attributed to the oxidant activity of aglycones, whereas the life-threatening chronic toxicity should be attributed to alterations of iron homeostasis by DOXol. This picture rationalizes the limited protective efficacy of antioxidants against chronic cardiomyopathy vis-à-vis the better protection offered by iron chelators, and forms the basis for developing analogues which produce less DOXol.

Coenzyme Q and trace element contents of two antarctic fishes (Pagothenia bernacchii and Chionodraco hamatus) were investigated and compared with that of one temperate marine fish (Mugil cephalus). In particular, muscle and erythrocyte concentrations of coenzyme Q, vitamin E, selenium, copper and zinc were determined. Antarctic fishes showed, at the level of their muscles, higher levels of selenium, zinc and copper compared with M. cepalus. In antarctic fishes, in the place of Q10, the coenzyme Q form generally observed in temperature fishes, only the homolog coenzyme Q9 was found. This particular finding has been related to the difference in the crystallization temperature existing between CoQ10 and CoQ9. Moreover, the high levels of zinc and selenium, observed in C. hamatus, correlate very well with a recent report on the presence, in the liver of this fish, of a zinc-binding protein that, in addition, is characterized by an amino acid sequence closely similar to that of a selenium-containing protein isolated from ovine heart. All together, the results indicate, for the antarctic species, a high level of antioxidant defenses with respect to the mediterranean fish and may be considered as a stimulating basis for further studies on the oxidative metabolism of Antarctic organisms.

Two-equivalent oxidation of metmyoglobin (MbIII) by hydrogen peroxide (H2O2) yields an oxoferryl moiety (MbIV) plus a protein radical which presumably originates from the conversion of tyrosines to tyrosyl radicals (-MbIV). In the absence of electron donors, MbIII oxidation is followed by (i) heme degradation or (ii) tyrosyl radical-dependent reactions, such as irreversible dimerization or covalent binding of the heme group to the apoprotein. Moreover, the oxidizing equivalents of H2O2-activated MbIII promote the peroxidative decomposition of polyunsaturated fatty acids. In this study, water-soluble short chain coenzyme Q analogs (CoQ1H2 and CoQ2H2) were found to reduce the oxoferryl moiety, preventing heme degradation and regenerating MbIII and, more slowly, MbIIO2. CoQ1H2 and CoQ2H2 were also found to reduce tyrosyl radicals generated by UV irradiation of tyrosine solutions. Accordingly, CoQ1H2 and CoQ2H2 effectively prevented tyrosyl radical-dependent reactions such as the dimerization of sperm whale myoglobin and heme-apoprotein covalent binding in horse heart myoglobin. By competing for the oxidizing equivalents of hypervalent myoglobin, CoQ1H2 and CoQ2H2 also prevented the peroxidation of arachidonic acid. Collectively, these studies suggest that the proposed function of coenzyme Q as a naturally occurring antioxidant might well relate to its ability of reducing H2O2-activated myoglobin. Coenzyme Q should therefore mitigate cardiac or muscular dysfunctions that are caused by an abnormal generation of H2O2.

Plasma coenzyme Q10 (CoQ10) is currently assayed in our laboratory for its well-known diagnostic meaning; in fact plasma CoQ10 levels are inversely related to metabolic demand. Definite levels of CoQ10 are also found in white and red blood cell components, as well as in platelets. Plasma and erythrocyte CoQ10 has a well assessed antioxidant role, which was demonstrated through a series of experiments. Erythrocytes previously enriched with exogenous CoQ10 were found more resistant to a hemolysis induced by a free radical initiator. Several enzymatic activities of erythrocyte ghosts were also protected by different side chain CoQ homologues, both when reduced and, although at a lesser extent, in the oxidized state. CoQ was not effective in preventing metal-catalyzed oxidation of erythrocyte membrane enzymes, and this effect is likely to be due to lack of interaction of CoQ with the metal target. Moreover CoQ was able to protect isolated enzymes and erythrocyte membrane bound enzymes from the inactivating effect of free radicals generated by water sonolysis or radiolysis. As far as plasma lipoproteins are concerned it is well known that LDL isolated from healthy volunteers supplemented with CoQ10 are more resistant to peroxidation induced by an azoinitiator. We started to systematically investigate CoQ10 and vitamin E levels in isolated human LDL and HDL. Both CoQ10 and vitamin E concentrations, referred to protein, were found higher in LDL than in HDL. Susceptibility to exogenously applied peroxidation did not correlate with the endogeneous content of the two antioxidants, possibly on the basis of different lipid content of these lipoproteins.

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.

Desferrioxamine (DFO) nearly doubles alkaline phosphatase oxidative inactivation by the ascorbate system. The effect is dependent on ascorbate and desferrioxamine concentrations, exhibiting in both cases a saturation mechanism. Conversion of desferrioxamine to ferrioxamine abolishes the prooxidant action. Desferrioxamine also increases ascorbate-dependent oxygen consumption and nitroblue tetrazolium reduction. Superoxide dismutase, which blocks the desferrioxamine enhancing effect on enzyme inactivation, markedly slows down nitroblue tetrazolium reduction as well as oxygen consumption by ascorbate plus desferrioxamine, while it fails to protect against the ascorbate system alone. Therefore, in the presence of desferrioxamine, the metal-catalyzed ascorbate autooxidation becomes superoxide-dependent and thus inhibitable by superoxide dismutase. Catalase, peroxidase, and ascorbate oxidase protect alkaline phosphatase from inactivation by both ascorbate and ascorbate-desferrioxamine systems. Hemin shields the enzyme from ascorbate plus DFO attack but not from ascorbate alone. In air-saturated solution, desferrioxamine seems to mediate one electron transfer from ascorbate to oxygen, generating superoxide anions, which can either trigger a Fenton reaction or produce desferal nitroxide radicals. In the absence of oxygen, ascorbate alone is ineffective, but the ascorbate plus desferrioxamine system still inactivates the enzyme; catalase, peroxidase, and ascorbate oxidase, but not superoxide dismutase, afford protection.

Clinical use of the anticancer anthracyclines doxorubicin (DOX) and daunorubicin (DNR) is limited by development of cardiotoxicity upon chronic administration. Secondary alcohol metabolites, formed after two-equivalent reduction of a carbonyl group in the side chain of DOX or DNR, have been implicated as potential mediators of chronic cardiotoxicity. In the present study we characterized how human heart converted DOX or DNR to their alcohol metabolites DOXol or DNRol. Experiments were carried out using post-mortem myocardial samples obtained by ethically-acceptable procedures, and results showed that DOXol and DNRol were formed by flavin-independent cytoplasmic reductases which shared common features like pH-dependence and requirement for NADPH, but not NADH, as a source of reducing equivalents. However, studies performed with inhibitors exhibiting absolute or mixed specificity toward best known cytoplasmic reductases revealed that DOX and DNR were metabolized to DOXol or DNRol through the action of distinct enzymes. Whereas DOX was converted to DOXol by aldehyde-type reductase(s) belonging to the superfamily of aldo-keto reductases, DNR was converted to DNRol by carbonyl reductase(s) belonging to the superfamily of short-chain dehydrogenase/reductases. This pattern changed in cardiac cytosol derived from rabbit, a laboratory animal often exploited to reproduce cardiotoxicity induced by anthracyclines and to develop protectants for use in cancer patients. In fact, only carbonyl reductases were involved in metabolizing DOX and DNR in rabbit cardiac cytosol, although with different K(m) and V(max). Collectively, these results demonstrate that human myocardium convert DOX and DNR to DOXol or DNRol by virtue of different reductases, an information which may be of value to prevent alcohol metabolite formation during the course of anthracycline-based anticancer therapy. These results also raise caution on the preclinical value of animal models of anthracycline cardiotoxicity, as they demonstrate that the metabolic routes leading to DOXol in a laboratory animal may not be the same as those occurring in patients.

Bilirubin (BR), the final product of heme catabolism, plays a crucial role in the defense against reactive oxygen species in various cell types. In this study, we addressed the hypothesis that BR can act as a physiological scavenger of nitric oxide (NO), a gaseous mediator involved in many cellular functions and able to trigger the formation of reactive nitrogen species with pro-oxidant activity. We found that S -nitrosocysteine (SNOC) and S -nitrosoglutathione (GSNO), which have a half-life of 0.52±0.07 hr and 38±5 hr and release NO at a constant rate of 1.42±0.2 hr −1 and 0.018±0.002 hr −1 , respectively, were able to decrease BR half-life in a concentration-dependent manner under physiological conditions. This effect appears to be dependent on NO formation as l -cysteine and GSH did not affect BR consumption and nitrite was four to five times less efficient than SNOC in reducing BR half-life. Oxyhemoglobin, a well-known scavenger of NO, protected BR from SNOC-mediated degradation. In addition, the reaction between SNOC/GSNO and BR modified the absorption spectrum of the bile pigment showing a gradual increase in the absorbance at 316 nm. This change in the BR spectrum indicates that the bile pigment could be a target for N -nitrosation reactions, since it resembles the modifications occurred when other molecules such as di-peptides and uric acid are nitrosated. Taken together, these data suggest that BR should not be considered only as an endogenous antioxidant but also as a molecule with the potential ability to counteract intracellular nitrosative stress reactions.

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.

Homocysteine is a non-protein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H2O2, enhanced platelet activity and increased production of macrophage-derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine-generated H2O2 has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol-dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol–hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at µmol/L concentrations (10–100 µmol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol–hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H2O2-mediated neutrophil activity is not supported and, probably, the common opinion that views the H2O2 generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered. Copyright © 2000 John Wiley & Sons, Ltd.

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.

Idebenone [2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone] is a synthetic analogue of coenzyme Q that is currently employed in the treatment of vascular and degenerative diseases of the central nervous system. There is some evidence to suggest that idebenone might function as an antioxidant; however, it has not been demonstrated whether this function pertains to the quinone or hydroquinone form of idebenone. Here we demonstrate that idebenone can scavenge a variety of free radical species, including organic radicals such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and diphenylpicrylhydrazyl, peroxyl and tyrosyl radicals, and peroxynitrite. Idebenone can also redox couple with hypervalent species of Mb or Hb, thus preventing lipid peroxidation promoted by these species. Likewise, idebenone inhibits microsomal lipid peroxidation induced by ADP-iron complexes or organic hydroperoxides. In so doing, idebenone prevents the destruction of cytochrome P450, which otherwise would accompany lipid peroxidation. Irrespective of the experimental system under investigation, idebenone functions by virtue of the electron-donating properties of the hydroquinone form. Redox coupling of this hydroquinone with free radicals generates the quinone compound, which per se lacks antioxidant activity. In many experiments, the antioxidant effects of idebenone become appreciable at approximately 2 microM, which is well in the range of plasma levels attainable in patients given oral doses of this drug. Moreover, comparative experiments have shown that the antioxidant efficiency of idebenone varies from no less than 50% to slightly more than 100% of that of vitamin E or Trolox. We would therefore propose that the neuroprotective effects of idebenone can be attributed, at least in part, to its ability to function as an antioxidant, involving redox cycling between hydroquinone and quinone.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20 degrees C. However, on going from 20 degrees C to 37 degrees C, the difference in O2 affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.

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.

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (MbIV and/or HbIV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (MbIV and/or HbIV) back to met and oxy forms (Mb and/or HbIIO2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.

Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.

Tryptophanyl fluorescence quenching by ascorbate has been studied in monomeric and tetrameric forms of melittin. The strong efficiency of ascorbate quenching is inconsistent with a collisional type of mechanism. Moreover, from absorption spectra alteration, temperature dependence of the quenching, lack of diffusion control and the unchanged fluorescence lifetime, it is demonstrated that the mechanism of the process is static (formation of a complex) and not dynamic. Thermodynamic parameters suggest that polar interactions mostly contribute to the formation of the complex (negative ΔH value) but that some hydrophobic interactions also occur in the stabilization of the complex. The bell-shaped relationship of the association constants vs. pH indicates that monoprotonated melittin is quenched more easily and that the active species is the reduced ascorbate monoanion. It can be inferred from the pK values (about 6.8 and 8.0) that the involved ionizable residues are probably Lys-21 and Lys-23. Polarization experiments reveal that ascorbate induces a large phosphate-like effect on melittin self-association, increasing the degree of structural organization of the peptide.

Alkaline phosphatase (EC 3.1.3.1), a zinc metallo-enzyme, is irreversibly inhibited by ascorbate, probably through a modification of active site environment (Martorana et al., 1986; Miggiano et al., 1984). A metal substituted form was prepared from bovine kidney alkaline phosphatase purchased from Calbiochem (La Jolla, CA, U.S.A.) and further purified as described (Martorana et al., 1986). Cobalt can replace zinc yielding a partially active phosphohydrolase, which lacks phosphotransferase activity (Ensinger et al., 1978) and presents a relevant visible absorption (Simpson and Vallee, 1968).

Moderate concentrations of guanidinium chloride induce both instantaneous and time-dependent modifications of the catalytic and optical properties of intestinal alkaline phosphatase, which undergoes consecutive conformational transitions at about 0.05 M, 0.25 M and 1.0 M denaturant. A paradoxical activation is observed up to 1.0 M-guanidine, with a maximum at 0.25 M- and a mid-point around 0.5 M-guanidine. Difference absorbance and fluorescence spectra imply a change in the state of ionization of the protein residues, with variation in molecular size suggested by light-scattering. Random-coil formation is indicated by a lower fluorescence yield, a more polar environment of the aromatic residues and another separate tryptophan emission. Iodide quenching confirms the alterations of conformation. Deprotonation favours the loss of the intramolecular constraints and the enhancement of the structure disruption by guanidine.

Ascorbic acid, isoascorbic acid and dehydroascorbic acid inhibit bovine kidney alkaline phosphatase activity. Ascorbic acid free radicals seem not to be involved. Dialysis does not make the inactivation reversible. A competitive mechanism can be inferred from experiments with phosphate and substrates, which block the activity decay. The influence of temperature, pH, other inhibitors and tertiary structure modifications on the inactivation process is also investigated.

Urea inhibits the activity of alkaline phosphatase during the reaction course. The inactivation is progressively stronger for the placental, intestinal and renal subforms. Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin and enzyme concentration on the inactivation mechanism is evaluated. In all experimental conditions the process follows pseudofirst-order kinetics and the inactivation profiles are distinct and typical for each enzymatic subform. With a simple graphical analysis, a single inactivation curve in controlled experimental conditions, allows the identification of each isoenzyme from the slope and the calculation of the respective fractional amount from the intercept of the time-activity plot.

Ascorbic acid is found strikingly to decrease the activity of bovine kidney alkaline phosphatase in vitro. The inhibition of alkaline phosphatase is a function of ascorbic acid concentration and is time and temperature dependent. The presence of the substrate protects the enzyme against the inhibitory action of the vitamin.

Alkaline phosphatase is inactivated by mixed function oxidation systems. OH. radicals, generated via an ascorbate-modified Haber-Weiss cycle or a Fenton-type reaction, seem to be responsible for the protein oxidative damage. Experiments with hydroxyl radical scavengers, enzyme substrates, products, and metal cofactors suggest that a "site-specific" radical attack takes place at or near the active center. Vitamin E fails to protect alkaline phosphatase; uric acid, instead, is particularly effective in shielding the protein against covalent modifications.

The occurrence of alkaline phosphatase (AP) activity was examined in several human endometrial adenocarcinomas. Catalytic activities were detectable only in 10 out of 15 tumors, with no apparent correlation between elevated AP and histological type. The apparent molecular weight of the enzyme after partial purification was about 140,000 daltons. Kinetic activity, thermodynamic properties and the pH dependence of the activity were in the ranges reported for other subforms. Several other physicochemical properties were also investigated and compared with those displayed by enzymes obtained from normal human tissues. The inhibition studies show that the enzyme shares several properties with the placental form, particularly in resistance to zinc chloride and EDTA action. On the other hand, in sensitivity to uncompetitive inhibitors and to urea and ascorbic acid, it is closer to other non-Regan heat-sensitive forms. The results support the view that a polymorphism in the expression of AP in neoplastic tissues can occur. A wider spectrum of physicochemical properties is clearly needed to define better the characteristics of oncodevelopmental enzymes.

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

A procedure for the selective fractionation of the bone and liver alkaline phosphatase activity in tissue extracts and human sera is proposed. Optimized conditions of the assay are: urea 3.7 mol/l in 0.5 mol/l DEA buffer, pH 9.8; 0.5 mmol/l MgCl2; 10.0 mmol/l p-nitrophenyl phosphate. The sample is diluted 1:20 in the reagent solution and the activity is recorded for 10 min at 37 degrees C. By means of a computerized or manual graphic analysis, based on 'peeling-off' the exponentials, the two differently urea-sensitive subforms are identified and the slow-(liver) and the fast-decaying (bone) activities are easily discriminated and their respective values calculated. Interference due to the intestinal isoenzyme can be also accounted for. The analytical variability is very satisfactory (within run CV = 7.5 and 4.5% for osseous and hepatic form, respectively; day-to-day CV less than 10% for both). The lower limits of detection are about 10 U/l and the serum or plasma reference values together with the influence on the assay of hemoglobin and protein content are also investigated.

Aromatic residues of bovine kidney alkaline phosphatase appear to be involved in the interaction with ascorbate, as shown by the strong quenching of intrinsic fluorescence and absorption. Difference u.v.-absorption spectra clearly indicate that conformational changes also occur. The pH value at which the greatest fluorescence deactivation is found is close to that necessary for optimal catalytic activity and for maximal inhibition by ascorbate. A protective effect against ascorbate is afforded by Pi. Time profiles of inactivation on one side and of absorbance and emission quenching on the other display opposite behaviours. Attempts to reverse the effects by the use of KOH fail to restore enzyme activity or to modify the spectral effects of ascorbate. The protein alterations are related, directly or indirectly, to the enzyme active centre and can be probably ascribed to the redox and chelating properties of ascorbate.

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.