H2O2 release by isoloated mitochondria and a new approach for the evaluation of H2O2 sources in intact cells
Year: 2009
Type: dissertation
Abstract: 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.
Author: Silvia Bortolami
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W179404301