RIUNIONI SCIENTIFICHE SULLA RICERCA BIOMEDICA DELL'OSPEDALE PEDIATRICO BAMBINO GESU' E DELL'UNIVERSITA' ROMA TRE
Martedì 28 Maggio 2013 "Stress ossidativo e patologie correlate"
Università Roma Tre, Dipartimento di Scienze, Aula 2 (Piano terra), Viale Marconi, 446 Roma
Ore 14.30 - 19.00 NATURAL ANTIOXIDANTS. ARE THEY AN ANSWER TO "OXIDATIVE STRESS" OF MODERN LIFE?
Daniela Tofani, Department of Sciences, University Roma Tre, c/o via della vasca navale 79, 00146 Roma Oxidative stress is a destabilization of cell redox balance caused by an excess of reactive radical species (ROS and RNS). Such free radicals formation increases in living organisms as the life time increases as well and this is connected to many diseases and aging pathologies. Natural antioxidants, normally introduced by diet, are able to trap ROS and RNS and, therefore, constitute a fundamental aid to the redox balance. At the end of the last century, many research groups focused their attention on this class of compounds. The good results obtained grew up the simplistic idea that maximizing antioxidant concentrations could result in an everlasting youth. Today many myths have been dispelled and scientific approach to antioxidants is more prudent. Nevertheless, research continues in order to get both a better understanding of structure/antioxidant activity relationships and the physiological role of the bioactive compounds. In order to find a conscious way to use natural antioxidants, the most important antioxidant classes and their synthetic derivatives will be presented, together with their mechanism of action, beneficial health effects and drawbacks. MECHANISMS OF FREE RADICAL SUPPRESSION BY ANTIOXIDANTS
Francesco Caruso1,2, Miriam Rossi2, Jens Z. Pedersen3, Sandra Incerpi4
1 CNR, Istituto di Chimica Biomolecolare, presso Univ Roma "La Sapienza" Roma (email@example.com),
2Vassar College, Department of Chemistry, Poughkeepsie, New York, USA, 3Department of Biology, University Tor Vergata, Roma,4Department of Sciences, University Roma Tre, Viale Marconi, 446 Roma, Italy Our group combines a series of techniques in order to improve the activity of antioxidant compounds in their action against free radicals. Our goal is to ameliorate the decline in health conditions due to aging, and more generally, degenerative diseases in humans. In this interdisciplinary approach we include the following: 1)
Perform synthesis and chemical characterization of potential antioxidants, including determination of
molecular structure using X-ray crystal diffraction; 2)
We study the reactivity of free radicals, both naturally occurring (superoxide, hydroxyl) and from
models (DPPH, galvinoxyl), using cyclovoltammetry and spectroscopic techniques as UV-visible and electron spin resonance (EPR). Since the reactivity of a naturally occurring free radical is very high, classical chemical tools give poor characterization. The use of less reactive models (DPPH, etc.) allows more control reactivity, and help describing antioxidant scavenging features. 3)
Theoretical methods are extremely useful for fast reactions and so we also use Density Functional
Theory (DFT) to analyze potential path reactions between our scavengers and free radicals. 4)
We also study docking interactions on potential targets (proteins, enzymes, DNA) of free radicals.
Our general approach is based on the study of a series of homogeneous antioxidants (for instance the stilbene group, which includes resveratrol, its metabolites and non-naturally occurring related derivatives). We try to figure out the structure-activity relationship, select the best antioxidants and apply them on cells that are subjected to free radical attack from peroxides. In vivo model are also studied through collaborations and our students; for instance we have a recently accepted manuscript which includes the techniques described above plus an in vivo study of diabetes performed on rats. PROTECTION OF CELLS AGAINST OXIDATIVE STRESS BY NANOMOLAR LEVELS OF HYDROXYFLAVONES Emanuele Lombardo1, Cristian Sabellico1,Valentina Balducci1, Paolo De Vito2, Stefano Leone1, Ilaria Proietti Silvestri3, Giuliana Righi3, Paolo Luly2, Luciano Saso4, Paolo Bovicelli3, Jens Z. Pedersen2, Sandra Incerpi1
1Department of Sciences, University Roma Tre, 2 Department of Biology, University Tor Vergata, Rome, Rome;
3Department of Chemistry, Sapienza University of Rome, Rome, 4 Institute of Biomolecular Chemistry, National Research Council (CNR), Rome,4Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University of Rome, Rome, Italy Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals. OXIDATIVE STRESS IN PARKINSON'S DISEASE: EFFECTS OF PHARMACOLOGICAL THERAPY. Colamartino M.1, Padua L.2, Leone S.1, Testa A.3 and Cozzi R.1
1 Dipartimento di Biologia, Università "Roma TRE", 2 Fondazione "Don Carlo Gnocchi", Roma, 3 Sezione di Tossicologia e Scienze Biomediche, ENEA-Casaccia, Roma Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorder. It is characterized by the depletion of dopamine (DA) in the dopaminergic neurons of the striatum of the brain. The substantia nigra pars compacta of PD patients is in a state of oxidative stress. Furthermore, there is evidence of oxidative damage to DNA, lipids and proteins. During DA metabolism, hydrogen peroxide (H2O2) is generated: H2O2 could undergo autoxidation in the presence of high levels of iron, giving rise to highly toxic hydroxyl radicals. For these reasons oxidative stress, resulting from the imbalance between ROS formation and anti- oxidant defenses, is thought to play a pivotal role in the pathogenesis of the disease. L-Dopa, in association with carbidopa (an amino acid decarboxylase inhibitor) is the "gold" standard drug for the treatment of PD; it is a natural dopamine precursor that can cross the blood brain barrier to reach the brain and to be converted into dopamine, it replenishes the lack of DA in the striatum and increases the DA content of the brain. It has been postulated that the chronic administration of L-Dopa would exacerbate the production and the accumulation of ROS leading neurons to death. We utilized catecholaminergic human neuroblastoma cells in vitro to study DNA damage and ROS production after L-Dopa and carbidopa treatments. Our data lead us to assert that L-Dopa
may have a protective effect on dopaminergic cells toward the production of ROS and their toxic effects on DNA. Furthermore in combined treatments (+H2O2) carbidopa is effective in reducing DNA damage caused by reactive oxygen intermediates both alone and in combination with L-Dopa.
Ore 16.30 Coffee - Break A NEURONAL MODEL OF NEURODEGENERATIVE DISEASE: OXIDATIVE STRESS IN THE AXONAL DAMAGE.
Emanuela Piermarinia, Barbara Carlettia, Giulia Tozzia, Lorena Travaglinia, Anna Pastoreb, Alessandra Torracoa, Enrico Bertinia and Fiorella Piemontea
aUnit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, bLaboratory of Metabolomics and Proteomics, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. Neurons are highly susceptible to oxidative stress and oxidation of cytoskeletal proteins is considered one of the first steps of neurodegeneration, thus potentially inducing axonal damage. However, few neuronal cell models have been so far developed in order to study the oxidative stress-mediated neurodegeneration. Here, we describe a neuronal model of Friedreich's Ataxia (FRDA), a hereditary neurodegenerative disease characterized by a reduced synthesis of the mitochondrial iron chaperon protein frataxin as a result of a large GAA triplet- repeat expansion within the first intron of the frataxin gene. To generate this neuronal cell model, we silenced the frataxin gene using a shRNA lentiviral vector. This approach led to a reduction of frataxin levels to 30-60% of normal levels, and determined several biochemical and morphological modifications that are highly suggestive of some aspects of the axonal pathological profile of FRDA. Frataxin-deficient neurons showed a specific inhibition of mitochondrial Complex I activity and glutathione imbalance, revealing an oxidative stress condition. These biochemical hallmarks were associated with the inhibition of cell proliferation and with morphological changes at the axonal compartment. Interestingly, the in vivo treatment of silenced cells with the permeable form of reduced glutathione revealed a partial rescue of cell proliferation. Our findings indicate that motor neurons are a suitable model to study the effect of oxidative stress on neurodegeneration, and support glutathione as a beneficial therapeutic target for FRDA. GLUTATHIONE AND PROTEIN GLUTATHIONYLATION IN NON-ALCOHOLIC FATTY LIVER DISEASE
1Anna Alisi, 2Anna Pastore, 1Nadia Panera, 1Sara Ceccarelli, 1Daniela Gnani, 1Cristiano De Stefanis, 3Fiorella Piemonte, 1Valerio Nobili.
1Hepato-Metabolic Disease Unit and Liver Research Unit, 2Laboratory of Metabolomic and Proteomics, 3Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, "Bambino Gesù" Children's Hospital, IRCCS, Rome, Italy Non-alcoholic fatty liver disease (NAFLD) ranges from simple fatty liver to non-alcoholic steatohepatitis (NASH), which often progresses into advanced fibrosis and cirrhosis. NAFLD/NASH has a complex etiology resulting from the interplay of multiple risk factors; however all molecular pathogenetic mechanisms have not yet been discovered. Along with human data several in vivo and in vitro models demonstrated that oxidative stress may solve part of the mystery around molecular pathogenesis of NAFLD, and it might explain most of the second-level events resulting in the appearance of NASH. Interestingly, an impaired glutathione (GSH) metabolism, as a marker of redox homeostasis, correlates with a higher intake of saturated fat and a lower intake of carbohydrates in NASH subjects. Moreover, it is relevant that the ability of GSH to form mixed disulfides with protein sulfhydryl groups, a process known as protein glutathionylation, may be altered during hepatic damage. Here, we report our data regarding the role of GSH in pediatric NAFLD and in experimental models that mimic the disease. Furthermore, we highlight how glutathionylation may control, either positively or negatively, the activity of some regulatory, structural, and metabolic proteins that are crucial in NAFLD pathogenesis.
OXIDATIVE STRESS IN THE PATHOGENESIS OF X-LINKED ADRENOLEUKODYSTROPHY
Sara Petrilloa, Anna Pastoreb, Giulia Tozzia, Marco Cappac, Enrico Bertinia and Fiorella Piemontea aUnit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, bLaboratory of Metabolomics and Proteomics, cUnit of Endocrinology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder of X-linked inheritance caused by a mutation in the ABCD1 gene which determines an accumulation of long-chain fatty acids (VLCFA) in plasma and tissues. Recent evidence shows that oxidative stress may be a hallmark in the pathogenesis of X-ALD and glutathione plays an important role in the defence against free radicals. In this study we have analyzed glutathione homeostasis in lymphocytes of 14 patients with X-ALD and evaluated the balance between oxidized and reduced forms of glutathione, in order to define the role of this crucial redox marker in this condition. All glutathione forms have been found abnormal in the blood of X-ALD patients, including a profound decrease of GSH associated with high levels of the oxidized forms (GSSG + GS-Pro). The decrease of GSH levels may be a consequence of the VLCFA-induced oxidative stress, but may play itself an active role in X-ALD pathogenesis. Indeed, key enzymes of glycolysis and tricarboxylic acid cycle are sensitive to oxidation and represent additional targets for GSSG-driven oxidative damage in adrenoleukodystrophy. Our results additionally confirmed by the decrease of plasma thiols and the high level of carbonyls, strongly support a role for oxidative stress in the pathophysiology of X-ALD and strengthen the importance of the balance among glutathione forms as a hallmark and a potential biomarker of the disease. Also, our findings underscore the pertinence of using antioxidants aiming at replenishing GSH levels, such as N-acetyl-L-cysteine, for therapeutic purposes in this disease EFFECT OF EPI-743 ON THE CLINICAL COURSE OF LEIGH SYNDROME
Michela Catteruccia1, Diego Martinelli2, Fiorella Piemonte1, Anna Pastore2, Giulia Tozzi1, Giuseppe Pontrelli3, Susanna Livadiotti3, Carlo Dionisi-Vici2, Enrico Bertini1
1Unit for Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, IRCCS, Rome
2 Division of Metabolism, Bambino Gesù Children's Hospital, IRCCS, Rome
3 Clinical Trial Center, Bambino Gesù Children's Hospital, IRCCS, Rome Background: Genetically defined Leigh syndrome is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. No treatment is available. EPI-743 is a novel small molecule developed for the treatment of Leigh syndrome and other inherited mitochondrial diseases. In compassionate use cases and in an FDA Expanded Access protocol, children with Leigh syndrome treated with EPI-743 demonstrated objective signs of neurologic and neuromuscular improvement. To confirm these initial findings, a phase 2A open label trial of EPI-743 for children with genetically-confirmed Leigh syndrome was conducted and herein we report the results. Methods: A single arm clinical trial was performed in children with genetically defined Leigh syndrome. Subjects were treated for 6 months with EPI-743 three times daily and all were eligible for a treatment extension phase. The primary objective of the trial was to arrest disease progression as assessed by neuromuscular and quality of life metrics. Results were compared to the reported natural history of the disease. Results: Ten consecutive children, ages 1-13 years, were enrolled; they possessed seven different genetic defects. All children exhibited reversal of disease progression regardless of genetic determinant or disease severity. The primary endpoints-Newcastle Pediatric Mitochondrial Disease Scale, the Gross Motor Function Measure, and PedsQL Neuromuscular Module-demonstrated statistically significant improvement (p<0.05). In addition, all children had an improvement of one class on the Movement Disorder-Childhood Rating Scale. No significant drug-related adverse events were recorded. Conclusions: In comparison to the natural history of Leigh syndrome, EPI-743 improves clinical outcomes in children with genetically confirmed Leigh syndrome.
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