TNFR1/phox interaction and TNFR1 mitochondrial translocation Thwart silica-induced pulmonary fibrosis.

Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.

Covalent modifications of hemoglobin by nitrite anion: formation kinetics and properties of nitrihemoglobin.

The green nitrihemoglobin (α(2)ß(2) tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A(0) under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion, and oxygen concentrations: -d[HbNO(2)]/dt = [k(1) + k(2)(K(a)[HNO(2)])[O(2)](1/2)][HbNO(2)], where k(1) = (2.4 ± 0.9) × 10(-4) s(-1), k(2) = (1 ± 0.2) × 10(5) M(-5/2) s(-1), and K(a) is the acid dissociation constant for nitrous acid (4.5 × 10(-4) M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Because nonphotochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A(0) and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.

The resistance of electron-transport chain Fe-S clusters to oxidative damage during the reaction of peroxynitrite with mitochondrial complex II and rat-heart pericardium.

The effects of peroxynitrite and nitric oxide on the iron-sulfur clusters in complex II (succinate dehydrogenase) isolated from bovine heart have been studied primarily by EPR spectroscopy and no measurable damage to the constitutive 2Fe-2S, 3Fe-4S, or 4Fe-4S clusters was observed. The enzyme can be repeatedly oxidized with a slight excess of peroxynitrite and then quantitatively re-reduced with succinate. When added in large excess, peroxynitrite reacted with at least one tyrosine in each subunit of complex II to form 3-nitrotyrosines, but activity was barely compromised. Examination of rat-heart pericardium subjected to conditions leading to peroxynitrite production showed a small inhibition of complex II (16%) and a greater inhibition of aconitase (77%). In addition, experiments performed with excesses of sodium citrate and sodium succinate on rat-heart pericardium indicated that the "g approximately 2.01" EPR signal observed immediately following the beginning of conditions modeling oxidative/nitrosative stress, could be a consequence of both reversible oxidation of the constitutive 3Fe-4S cluster in complex II and degradation of the 4Fe-4S cluster in aconitase. However, the net signal envelope, which becomes apparent in less than 1min following the start of oxidative/nitrosative conditions, is dominated by the component arising from complex II. Taking into account the findings of a previous study concerning complexes I and III (L.L. Pearce, A.J. Kanai, M.W. Epperly, J. Peterson, Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors, Nitric Oxide 13 (2005) 254-263) it is now apparent that, with the exception of the cofactor in aconitase, mammalian (mitochondrial) iron-sulfur clusters are surprisingly resistant to degradation stemming from oxidative/nitrosative stress.

LC/MS characterization of rotenone induced cardiolipin oxidation in human lymphocytes: implications for mitochondrial dysfunction associated with Parkinson's disease.

SCOPE: Rotenone is a toxicant believed to contribute to the development of Parkinson's disease. METHODS AND RESULTS: Using human peripheral blood lymphocytes we demonstrated that exposure to rotenone resulted in disruption of electron transport accompanied by the production of reactive oxygen species, development of apoptosis and elevation of peroxidase activity of mitochondria. Employing LC/MS-based lipidomics/oxidative lipidomics we characterized molecular species of cardiolipin (CL) and its oxidation/hydrolysis products formed early in apoptosis and associated with the rotenone-induced mitochondrial dysfunction. CONCLUSION: The major oxidized CL species - tetra-linoleoyl-CL - underwent oxidation to yield epoxy-C18:2 and dihydroxy-C18:2 derivatives predominantly localized in sn-1 and sn-2 positions, respectively. In addition, accumulation of mono-lyso-CL species and oxygenated free C18:2 were detected in rotenone-treated lymphocytes. These oxidation/hydrolysis products may be useful for the development of new biomarkers of mitochondrial dysfunction.

Adsorption of surfactant lipids by single-walled carbon nanotubes in mouse lung upon pharyngeal aspiration.

The pulmonary route represents one of the most important portals of entry for nanoparticles into the body. However, the in vivo interactions of nanoparticles with biomolecules of the lung have not been sufficiently studied. Here, using an established mouse model of pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs), we recovered SWCNTs from the bronchoalveolar lavage fluid (BALf), purified them from possible contamination with lung cells, and examined the composition of phospholipids adsorbed on SWCNTs by liquid chromatography mass spectrometry (LC-MS) analysis. We found that SWCNTs selectively adsorbed two types of the most abundant surfactant phospholipids: phosphatidylcholines (PC) and phosphatidylglycerols (PG). Molecular speciation of these phospholipids was also consistent with pulmonary surfactant. Quantitation of adsorbed lipids by LC-MS along with the structural assessments of phospholipid binding by atomic force microscopy and molecular modeling indicated that the phospholipids (∼108 molecules per SWCNT) formed an uninterrupted "coating" whereby the hydrophobic alkyl chains of the phospholipids were adsorbed onto the SWCNT with the polar head groups pointed away from the SWCNT into the aqueous phase. In addition, the presence of surfactant proteins A, B, and D on SWCNTs was determined by LC-MS. Finally, we demonstrated that the presence of this surfactant coating markedly enhanced the in vitro uptake of SWCNTs by macrophages. Taken together, this is the first demonstration of the in vivo adsorption of the surfactant lipids and proteins on SWCNTs in a physiologically relevant animal model.

Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity.

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.