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Bortolami, Silvia (2009) H2O2 release by isoloated mitochondria and a new approach for the evaluation of H2O2 sources in intact cells. [Tesi di dottorato]

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Abstract (inglese)

Reactive Oxygen Species (ROS), considered for many years as the unwanted toxic by-products of living systems in an aerobic environment, are now recognised as mediators of physiological functions. For H2O2, the more stable among ROS, a role as second messenger is suggested also for its permeability across membranes. H2O2 can be produced directly by several oxidases or after dismutation of superoxide (O2.-) originating from the membrane NAD(P)H oxidases and from mitochondrial electron transport chain.
In the first part of the thesis are summarised some evidences obtained in brain and heart mitochondrial suspensions, of how Complex I can be responsible of most of the mitochondrial O2.-/H2O2 release. The measure of H2O2 release was performed with the sensitive fluorescent method Amplex red/HRP. H2O2 release is low during the oxidation of the NAD linked substrates but high during succinate oxidation, a condition considered not physiological in the scientific literature being attributed to the high succinate concentration used. In this work the succinate dependent H2O2 production was measured also at submillimolar succinate concentrations, more consistent with physiological conditions. Moreover long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of the succinate dependent H2O2 release, showing that it can be modulated by the presence of these important metabolites. It was also shown that the inhibitory action of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA.
These data pose the question of how and in which conditions the mitochondrial ETC can give rise to a sustained H2O2 production in intact cells. In fact the majority of the evidences of the mitochondrial involvement comes from subcellular preparations. The aim of my work was then to extend the work done in mitochondria to intact viable cells
We soon realized that the use of “specific” fluorescent probes, often used for the H2O2 measurements, were unreliable as also largely debated in literature. We tried to overcome the general problem of the inadequacy of the H2O2 measurements in intact cells by transiently expressing HRP in two mammalian cell lines, C2C12 and SHSY-5Y. HRP is a well known specific H2O2 reagent when coupled to fluorescent probe and this detection system is widely accepted for H2O2 measurements in subcellular preparations.
HRP expression largely enhance the sensitivity and specificity of the detection of H2O2 by the cell ROS sensitive fluorescent probes DCFH2 and DHR. In HRP expressing cells we are able to measure with fluorescence microscopy, but also directly at a fluorescent plate reader, the H2O2 production derived from a constitutive MAO activity for the first time in intact cells.
The viability and metabolic response confirmed that HRP transfected cells behave as the corresponding wild type. The specificity of the measurements (obtained with exogenous H2O2 delivery and for competition with endogenous H2O2 removal systems), and the limits and reliability of this procedure largely confirm that HRP transfected cells are suitable for studies involving H2O2 in cell physiology.
In the last part of the research we also obtained preliminary results, confirmed in isolated mitochondria in parallel, that the method allows measurements of the mitochondrial H2O2 release. We give evidence that the dynamic H2O2 measurements performed following the rate of oxidation of DHR is increased by addition of rotenone. The rise in cells is expected since the prevailing NAD dependent substrates should induce an higher H2O2 release in the presence of the Complex I inhibitor Rotenone as largely reported in mitochondria. The oxidation rate is also increased, again as in mitochondria, when a permeant form of succinate is exogenously added, and the increase is inhibited by DPI, which inhibits the oxidation of both NAD dependent substrates and succinate. Comparative analysis of the maximal rate of H2O2 production by succinate and by the MAO activity in HRP transfected cells shows that the ETC contribution to H2O2 production in the cells is far lower than the maximal production following the addition of the MAO substrate tyramine. Our data in cells are in agreements with our data in isolated mitochondria and as also suggested by several authors.
On the whole HRP transfected cells appear a suitable model to study different experimental conditions mimicking physiological or pathological conditions where ROS are thought to be involved.

Abstract (italiano)

L’ H2O2 è un metabolita accertato nelle cellule viventi. A lungo considerata una forma attivata dell’ossigeno particolarmente dannosa, recentemente è stata rivalutata come mediatore fondamentale della funzionalità cellulare per i suoi molteplici ruoli in situazioni sia fisiologiche che patologiche. Oltre a riconoscerne un ruolo importante nei meccanismi di difesa contro patogeni viene ampiamente suggerita come molecola messaggero in una serie di comunicazioni intra e intercellulari grazie alla sua caratteristiche di stabilità e diffusibilità uniche fra i derivati reattivi dell’ossigeno (ROS). Può essere prodotta direttamente da alcune ossidasi o in seguito a dismutazione del superossido prodotto principalmente dalle NADPH ossidasi di membrana e dalla catena respiratoria mitocondriale.
Nella catena respiratoria mitocondriale il superossido (O2•-), dismutato ad H2O2 dalla superossido dismutasi Mn-dipendente, viene prodotto soprattutto a livello del complesso I e risente del potenziale e dello stato redox di vari componenti la catena respiratoria. Tale produzione è bassa in presenza di substrati NAD dipendenti (nel nostro caso glutammato più malato), e alta, e rotenone sensibile, in presenza di succinato. Nella prima parte della tesi si evidenzia come la produzione di O2.- in mitocondri isolati possa avvenire a concentrazioni submillimolari di succinato, anche in presenza di substrati NAD dipendenti, condizioni ritenute simili a quelle fisiologiche. Inoltre la presenza di acil-CoA a lunga catena inibisce la produzione di H2O2 dipendente da succinato, in un modo indipendente dalla loro ossidazione. I mitocondri appaiono quindi una delle più probabili fonti di produzione fisiologica modulabile di H2O2 intra-cellulare.
La critica più pesante a studi fatti su preparazioni subcellulari è la loro effettiva congruità al modello cellulare o tissutale. Obiettivo del lavoro era quindi estendere i dati ottenuti in mitocondri isolati a un modello più complesso: la cellula intera. Il grosso limite allo studio è determinato dalla particolare esiguità della produzione di H2O2 in condizioni fisiologiche e dalla scarsa affidabilità dei metodi di misura intracellulari. Infatti, le principali sonde fluorescenti, come la diidrorodamina (DHR) o la diclorofluoresceina (DCFH) utilizzate per la detection dell’H2O2 in cellula intera non sono affidabili, come ampiamente discusso in letteratura. Il basso segnale e la mancanza di specificità di queste sonde, a nostro avviso, è dovuta soprattutto alla carenza di uno catalizzatore per la reazione sonda/H2O2. Abbiamo pensato quindi di esprimere all’interno delle cellule l’enzima Horseradish Peroxidase (HRP), ampiamente utilizzato per la misura dell’ H2O2 in esperimenti in vitro accoppiato a sonde spettroscopiche.
L’espressione dell’HRP all’interno delle cellule aumenta visibilmente la sensibilità delle sonde e la specificità della reazione con l’H2O2. Infatti è stato possibile misurare, per la prima volta in cellula intera, la produzione di H2O2 derivata dall’attività Mono-Ammino Ossidasica (MAO) come incremento di velocità della ossidazione della DHR o DCFH2 indotta da Tiramina, substrato della MAO. L’integrità cellulare e la risposta metabolica non vengono modificate significativamente dalla trasfezione con HRP. Inoltre la specificità della misura (verificata con l’aggiunta esogena di H2O2 e per competizione con i sistemi di rimozione endogeni) conferma che le cellule trasfettate con HRP sono un buon modello per lo studio del coinvolgimento dell’H2O2 nella fisiologia cellulare. Nell’ultima parte della ricerca vengono presentati risultati preliminari che indicano come il metodo sia sufficientemente sensibile alla misura della quota di H2O2 di origine mitocondriale. Infatti si evidenzia una maggiore velocità di ossidazione della DHR in presenza di rotenone, simile all’incremento ottenuto nei mitocondri isolati in presenza di Glutammato/Malato. Inoltre si evidenzia un aumento dell’ossidazione della DHR in presenza del dietil-succinato, una forma permeabile di succinato. L’aumentata ossidazione viene inibita da difenilene iodonio, che si dimostra essere anche un potente inibitore dell’ossidazione e quindi della produzione di H2O2 sia dei substrati NAD dipendenti che del succinato in mitocondri isolati. Inoltre, come già ipotizzato in letteratura, mostriamo che la produzione di H2O2 dipendente dalla MAO è largamente superiore a quella mitocondriale indotta da alte concentrazioni di dietil-succinato in cellula.
Complessivamente il modello di cellule trasfettate con HRP sembra essere sufficientemente sensibile per misurare il contributo alla produzione di H2O2 di varie fonti intracellulari e quindi tale da poter essere utilizzato in varie condizioni fisiologiche o patologiche.

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Tipo di EPrint:Tesi di dottorato
Relatore:Alexandre, Adolfo
Correlatore:Cavallini, Lucia
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOCHIMICA E BIOTECNOLOGIE > BIOCHIMICA E BIOFISICA
Data di deposito della tesi:29 Gennaio 2009
Anno di Pubblicazione:29 Gennaio 2009
Parole chiave (italiano / inglese):Reactive oxygen species, intracellular H2O2 detection, mitochondrial H2O2 release, MAO, HRP, transfection, cell signalling
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Chimica Biologica
Codice ID:1647
Depositato il:29 Gen 2009
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