4-hydroxy-2-nonenal and peroxynitric-acid

Oxidative modification of lipoproteins plays an important role in atherogenesis. We investigated a variety of different oxidatively modified epitopes (malondialdehyde (MDA)-2, hydroxynonenal (HNE)-7, peroxynitrite, hypochlorite, EO-6) in parallel and compared normal vessel wall, early and advanced atherosclerotic lesions in WHHL rabbits. Early atherosclerotic lesions showed abundant intracellular staining in macrophages for all ox-epitopes, apo B and apo E; advanced lesions showed a more prominent peri- and extracellular staining for ox-epitopes, which tended to colocalize more with apo B than apo E. Hypochlorite-modified epitopes showed intense staining in all types of lesions, followed by MDA-2. Early and advanced atherosclerotic lesions differed significantly in that early stages revealed abundant cellular positivity for EO-6 and weak staining for HNE-7 modified proteins whereas the opposite was observed in advanced lesions. Nuclear factor-kappa B (NF-kappa B) was nearly exclusively detected in macrophages with no difference between early and advanced lesions. We conclude that hypochlorite-modified epitopes are abundantly present at all stages of atherogenesis. EO-6 might be a marker for early, HNE-7 a marker for advanced lesions. Colocalization of ox-epitopes with apolipoproteins further supports that oxidation of lipoproteins is one of the key mechanisms in atherogenesis. Chronic stable expression and activation of NF-kappa B could be a useful target for therapeutic interventions.

Topics: Aldehydes; Animals; Arteriosclerosis; Epitopes; Hypochlorous Acid; Immunohistochemistry; Malondialdehyde; Nitrates; Oxidation-Reduction; Rabbits

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Topics: Aldehydes; Animals; Apoptosis; Cell Nucleus; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Intracellular Membranes; Ion Channels; Jurkat Cells; Lipid Peroxidation; Membrane Proteins; Mice; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nitrates; Nitric Oxide; Oxidants; Permeability; Proteins; Proteolipids; Proto-Oncogene Proteins c-bcl-2

Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

Topics: Aldehydes; Animals; Blood Urea Nitrogen; Creatinine; Free Radicals; Glutathione; Kidney; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Proteins; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tyrosine

To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1.. Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay.. Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P: < 0. 01).. Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.

Topics: Aldehydes; Animals; Cell Death; Cyclin D1; Fluorescent Antibody Technique, Indirect; Free Radical Scavengers; Lipid Peroxidation; Lipid Peroxides; Male; Nitrates; Propidium; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Retina; Retinal Diseases; Thiobarbituric Acid Reactive Substances; Thioredoxins

Peroxynitrite may be generated in and around muscles in several pathophysiological conditions (e.g., sepsis) and may induce muscle dysfunction in these disease states. The effect of peroxynitrite on muscle force generation has not been directly assessed. The purpose of the present study was to assess the effects of peroxynitrite administration on diaphragmatic force-generating capacity in 1) intact diaphragm muscle fiber bundles (to model the effects produced by exposure of muscles to extracellular peroxynitrite) and 2) single skinned diaphragm muscle fibers (to model the effects of intracellular peroxynitrite on contractile protein function) by examining the effects of both peroxynitrite and a peroxynitrite-generating solution, 3-morpholinosydnonimine, on force vs. pCa characteristics. In intact diaphragm preparations, peroxynitrite reduced diaphragm force generation and increased muscle levels of 4-hydroxynonenal (an index of lipid peroxidation). In skinned fibers, both peroxynitrite and 3-morpholinosydnonimine reduced maximum calcium-activated force. These data indicate that peroxynitrite is capable of producing significant diaphragmatic contractile dysfunction. We speculate that peroxynitrite-mediated alterations may be responsible for much of the muscle dysfunction seen in pathophysiological conditions such as sepsis.

Topics: Aldehydes; Animals; Calcium; Diaphragm; Kinetics; Lipid Peroxidation; Molsidomine; Muscle Contraction; Nitrates; Nitric Oxide Donors; Rats; Thiobarbituric Acid Reactive Substances

Few studies have been conducted focusing on a potential role of reactive oxygen species in tumor cell metabolism. Here we studied human colorectal adenocarcinomas and adenomas to determine whether oxidative stress is imposed on cancer cells in vivo and used specific antibodies against 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE)-modified proteins, and 3-nitro-L-tyrosine (3-NT) to determine whether there is an association between oxidative stress and cellular proliferation. Higher levels of oxidative modifications in DNA and proteins were observed in carcinoma cells, but not in adenoma cells, than in the corresponding nontumorous epithelial cells by immunohistochemistry as well as high-performance liquid chromatography (HPLC)-based 8-OHdG determination. The fraction of proliferating cell nuclear antigen-positive cells was proportionally associated in adenocarcinomas with the staining intensities of 8-OHdG and 3-NT. Furthermore, Western blot analysis of the proteins extracted from carcinoma cells revealed several specific proteins modified by HNE or peroxynitrite. Thus we concluded that colorectal carcinoma, but not adenoma cells, are exposed to more oxidative stress than their corresponding nontumorous epithelial cells, regardless of clinical stage and histology, and further that the oxidative stress in carcinoma cells might stimulate cellular proliferation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenocarcinoma; Adenoma; Adult; Aged; Aldehydes; Case-Control Studies; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Deoxyguanosine; Epithelial Cells; Female; Humans; Immunohistochemistry; Male; Middle Aged; Nitrates; Oxidants; Oxidative Stress; Reactive Oxygen Species; Time Factors

Topics: Aldehydes; Alzheimer Disease; Analysis of Variance; Biomarkers; DNA Damage; Glycation End Products, Advanced; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Histocytochemistry; Humans; Iron; Membrane Proteins; Neurodegenerative Diseases; Neurofibrillary Tangles; Neurons; Nitrates; Oxidation-Reduction; Oxidative Stress; Oxygen; Phenylhydrazines; Plaque, Amyloid; Tyrosine