Ivo Juránek

Biological importance of reactive oxygen species in relation to difficulties of treating pathologies involving oxidative stress by exogenous antioxidants

Findings about involvement of reactive oxygen species (ROS) not only in defense processes, but also in a number of pathologies, stimulated discussion about their role in etiopathogenesis of various diseases. Yet questions regarding the role of ROS in tissue injury, whether ROS may serve as a common cause of different disorders or whether their uncontrolled production is just a manifestation of the processes involved, remain unexplained. Dogmatically, increased ROS formation is considered to be responsible for development of the so-called free-radical diseases. The present review discusses importance of ROS in various biological processes, including origin of life, evolution, genome plasticity, maintaining homeostasis and organism protection. This may be a reason why no significant benefit was found when exogenous antioxidants were used to treat free-radical diseases, even though their causality was primarily attributed to ROS. Here, we postulate that ROS unlikely play a causal role in tissue damage, but may readily be involved in signaling processes and as such in mediating tissue healing rather than injuring. This concept is thus in a contradiction to traditional understanding of ROS as deleterious agents. Nonetheless, under conditions of failing autoregulation, ROS may attack integral cellular components, cause cell death and deteriorate the evolving injury.

Pro-oxidative effect of peroxynitrite regarding biological systems: a special focus on high-molar-mass hyaluronan degradation.

Current understanding on the role of peroxynitrite in etiology and pathogenesis of some human diseases, such as cardio-vascular diseases, stroke, cancer, inflammation, neurodegenerative disorders, diabetes mellitus and diabetic complications has recently led to intensive investigation of peroxynitrite involvement in physiology and pathophysiology. Mechanism of cytotoxic effects of peroxynitrite involve its reactions with lipids, DNA/RNA, proteins, and polysaccharides, thus triggering cellular responses ranging from subtle changes of cell functioning to severe oxidative damage of the affected macromolecules leading to necrosis or apoptosis. The present work is aimed at providing a brief overview of i) peroxynitrite biosynthesis and reaction pathways in vivo, ii) its synthetic preparation in vitro, and iii) to reveal its potential damaging role in vivo, on actions studied via monitoring in vitro hyaluronan degradation. The complex biochemical behavior of peroxynitrite is determined by a number of variables, such as chemistry of the reaction itself, depending mostly on the involvement of conformational structures of different energy states, concentration of the species involved, content of reactive intermediates and trace transition metal ions, contribution of carbon dioxide, presence of trace organics, and by the reaction kinetics. Recently, in vitro studies of oxidative cleavage of hyaluronan have, in fact, been the subject of growing interest. Here we also describe our experimental set-up for studying peroxynitrite-mediated degradation of hyaluronan, a system, which may be suitable for testing prospective pharmacological substances.

Influence of tiopronin, captopril and levamisole therapeutics on the oxidative degradation of hyaluronan

The ability to protect hyaluronic acid (HA) from oxidative degradation by cupric ions and ascorbate (production of (•)OH and peroxy-type radicals) during acute phase joint inflammation has been investigated using the following drugs: tiopronin, captopril, and levamisole. Radical scavenging activity, i.e. the propensity for donation of electrons was assessed for the drugs by ABTS and DPPH assays. The kinetics of HA degradation have been measured in the presence of each drug using rotational viscometry. The results of ABTS and DPPH assays show the highest radical scavenging activity for captopril, followed by tiopronin. For levamisole, no effect was observed. Captopril and tiopronin prevented HA degradation induced by (•)OH radicals in a similar manner, while tiopronin was more effective in scavenging peroxy-type radicals. On the other hand, levamisole was shown to be a pro-oxidant. Recovered HA fragments were characterized using FT-IR analysis, the incorporation of a sulphur atom from captopril and tiopronin but not from levamisole into the HA molecule was demonstrated.

Free-Radical Degradation of High-Molar-Mass Hyaluronan Induced by Ascorbate plus Cupric Ions: Evaluation of Antioxidative Effect of Cysteine- Derived Compounds

Based on our previous findings, the present study has focused on free‐radical‐mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissbergers system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine‐derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L‐glutathione, L‐cysteine, N‐acetyl‐L‐cysteine, and cysteamine, against free‐oxygen‐radical‐mediated degradation of high‐molar‐mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L‐glutathione exhibited the most significant protective and chain‐breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pKa, pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L‐glutathione, cysteamine, N‐acetyl‐L‐cysteine, and L‐cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion‐derived diseases.

Anthracycline-Dependent Cardiotoxicity and Extracellular Matrix Remodeling

The mechanisms of anthracycline-dependent cardiotoxicity have been studied widely, with the suggested principal mechanism of anthracycline damage being the generation of reactive oxygen species by iron-anthracycline complexes, leading to lipid peroxidation and membrane damage. An increasing number of researchers studying cardiovascular events associated with anthracycline-based chemotherapy are addressing cardiac extracellular matrix (ECM) remodeling. The heart is an efficient muscular pump, with the cardiomyocytes and intramural coronary vasculature of the heart tethered in an ECM consisting of a network of fibrillar, structural proteins, mostly collagens. Increasing evidence suggests that the ECM plays a complex and diverse role in the processes initiated by anthracycline-class drugs that lead to cardiac damage. This review discusses adverse myocardial remodeling induced by anthracyclines and focuses on their mechanisms of action.

Cerebral hypoxia–ischemia: Focus on the use of magnetic resonance imaging and spectroscopy in research on animals

Cerebral hypoxic-ischemic injury represents a serious health problem and is the third leading cause of mortality in developed countries. Early diagnosis of hypoxic-ischemic injury to the brain is inevitable for timely and efficient treatment. However, routinely applied cranial ultrasonography or computed tomography is often not sensitive enough to detect cerebral hypoxic-ischemic injury in its early stages. Therefore searching for a more effective diagnostic tool has been an intensive process in many laboratories within the last decades. Nowadays, magnetic resonance imaging (MRI) and spectroscopy (MRS) are the most promising non-invasive and non-destructive tools working in-real-time. These magnetic resonance-based techniques are progressively utilized in neurological and neonatology departments to confirm or refute cerebral hypoxic-ischemic injury in adults and neonates. The purpose of the present paper was therefore (i) to provide a brief overview on mechanisms of hypoxic-ischemic injury to the brain and (ii) to summarize main findings of both clinical reports and experimental studies, performed on various animal models of brain hypoxia-ischemia, with a particular focus on the monitoring of the evolving cerebral hypoxic-ischemic injury by means of in vivo MRI and MRS.

Aurothiomalate as Preventive and Chain-Breaking Antioxidant in Radical Degradation of High-Molar-Mass Hyaluronan

The potential anti‐ or pro‐oxidative effects of a disease‐modifying antirheumatic drug, aurothiomalate, to protect high‐molar‐mass hyaluronan against radical degradation were investigated along with L‐glutathione – tested in similar functions. Hyaluronan degradation was induced by the oxidative system CuII plus ascorbate known as the Weissbergers oxidative system. The time‐ and dose‐dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical‐scavenging capacity based on a decolorization 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L‐glutathione showed excellent scavenging of .OH and peroxyl‐type radicals, however, at the lowest concentration applied, its pro‐oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L‐glutathione, however, at the lowest concentration tested, no significant pro‐oxidant effect was observed.

Severe Hypoxia Inhibits Prostaglandin I2 Biosynthesis and Vasodilatory Responses Induced by Ionophore A23187 in the Isolated Rabbit Ear

The present study was designed to test the hypothesis that lack of oxygen in severely hypoxic tissue may inhibit arachidonic acid oxygenation and thereby result in an inhibition of eicosanoid synthesis. Hypoxia was induced in the isolated rabbit ear, and arachidonate metabolism and peripheral resistance of the preparation were monitored simultaneously. Severe hypoxia completely inhibited the biosynthesis of prostaglandin I2 induced by ionophore A23187 and converted the vasodilatory response observed under normoxia into vasoconstriction. The cyclooxygenase 1 inhibitor SC560 (1 µmol/l) effectively inhibited the normoxic prostaglandin I2 biosynthesis, while the cyclooxygenase 2 inhibitor DFU (1 µmol/l) did not. Neither SC560 nor DFU affected normoxic vasodilatory responses, indicating no involvement of prostanoids. The nitric oxide (synthase inhibitor Nω-nitro-L-arginine methyl ester (100 µmol/l) converted the vasodilation into vasoconstriction, similar to what was observed under hypoxia, suggesting that the hypoxia-mediated conversion might occur due to the inhibition of nitric oxide.

Hypoxia-induced inhibition of calcium channels in guinea-pig taenia caeci smooth muscle cells

1 The effects of hypoxia on whole‐cell current in single smooth muscle cells and on a high K+‐induced contraction of strips of the guinea‐pig taenia caeci were studied. 2 In physiological salt solution (PSS) and K+‐based pipette solution, hypoxia (PO2= 20 mmHg) reversibly inhibited both the inward Ca2+ current (ICa) and outward Ca2+‐activated K+ current (IK(Ca)) components of the whole‐cell current. 3 In PSS and Cs+‐based pipette solution, hypoxia reversibly suppressed ICa by 30 ± 5% at 0mV. 4 When Ba2+ was used as a charge carrier, the IBa was suppressed by hypoxia in a potential‐dependent manner, with the maximum of 40 ± 7% at +10mV. Alterations of concentrations of egta, GDBβS or ATP in the pipette solution did not change the inhibitory effects of hypoxia on ICa and IBa. 5 In PSS with 2 mm CaCl2 replaced by CoCl2, hypoxia did not affect the Ca2+ influx‐independent potassium current. 6 In cells voltage clamped at ‐20 mV hypoxia reversibly inhibited the spontaneous transient outward currents. 7 The response of high K+‐contracted taenia caeci to hypoxia was composed of an initial rapid relaxation followed by a small transient contraction and slow relaxation. The transient contraction was blocked by atropine (1–10 μm), while relaxations were unaffected by atropine and guanethidine (10 μm). 8 The results show that hypoxia reversibly inhibits ICa and secondarily suppresses IK(Ca) due to decreased Ca2+ influx through Ca2+ channels. 9 It is suggested that inhibition of ICa was responsible for the rapid relaxation, whereas transient contraction may have been due to release of acetylcholine from nerve terminals upon hypoxia.

Free-radical degradation of high-molecular-weight hyaluronan induced by ascorbate plus cupric ions. Testing of bucillamine and its SA981-metabolite as antioxidants

High-molecular-weight hyaluronan (HA) samples were exposed to free-radical chain-degradation reactions induced by ascorbate in the presence of Cu(II) ions - the so-called Weissbergers oxidative system. The concentrations of both reactants [ascorbate, Cu(II)] were comparable to those that may occur during an early stage of the acute phase of joint inflammation. The time-dependent changes of the viscosity of the HA solution in the absence of the substance tested were monitored by rotational viscometry. However, when the anti- or pro-oxidative effects of the antioxidants/drugs were investigated, their dose-dependency was also examined. Additionally, the anti-oxidative activities of these substances were screened by the well-established ABTS and DPPH decolorization assays. The actions of the disease-modifying anti-rheumatic drugs, namely bucillamine and D-penicillamine, were compared to those of L-cysteine and of SA981, the oxidized metabolite of bucillamine. The results indicated that bucillamine was the most efficient scavenger of hydroxyl- and/or peroxyl-type radicals, even at the lowest drug concentration. In contrast, SA981 demonstrated no scavenging activity against the aforementioned free radicals. D-Penicillamine and L-cysteine showed a dual effect, i.e. a pronounced anti-oxidative effect was, after a given time period, followed by a significant pro-oxidative effect.