Targeting of reactive isolevuglandins in mitochondrial dysfunction and inflammation.

Inflammation is a major cause of morbidity and mortality in Western societies. Despite use of multiple drugs, both chronic and acute inflammation still represent major health burdens. Inflammation produces highly reactive dicarbonyl lipid peroxidation products such as isolevuglandins which covalently modify and cross-link proteins via lysine residues. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation-induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria-targeted scavenger of isolevuglandins by conjugating 2-hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1 g/L) to lipopolysaccharide treated mice increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analyses supported mito2HOBA-mediated protection of renal cortex from cell injury. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols.

Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of "antioxidant" effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min) on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 µM) did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 µM) for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR) while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD)2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR spectra of Fe(MGD)2 supplemented mitochondrial lysates showed presence of both dinitrosyl iron complexes and nitrosothiols. We suggest that nitric oxide attenuates production of mitochondrial superoxide by post-translational modifications by nitrosylation of protein cysteine residues and formation of protein dinitrosyl iron complexes with thiol-containing ligands and, therefore, nitric oxide reduction in pathological conditions associated with endothelial dysfunction may increase mitochondrial oxidative stress.