The neuropathological sequelae of carbon monoxide (CO) poisoning cannot be explained by hypoxic stress alone. CO poisoning also causes adduct formation between myelin basic protein (MBP) and malonylaldehyde, a reactive product of lipid peroxidation, resulting in an immunological cascade. MBP loses its normal cationic characteristics, and antibody recognition of MBP is altered. Immunohistochemical evidence of degraded MBP occurs in brain over days, along with influx of macrophages and CD-4 lymphocytes. Lymphocytes from CO-poisoned rats subsequently exhibit an autoreactive proliferative response to MBP, and there is a significant increase in the number of activated microglia in brain. Rats rendered immunologically tolerant to MBP before CO poisoning exhibit acute biochemical changes in MBP but no lymphocyte proliferative response or brain microglial activation. CO poisoning causes a decrement in learning that is not observed in immunologically tolerant rats. These results demonstrate that delayed CO-mediated neuropathology is linked to an adaptive immunological response to chemically modified MBP. myelin basic protein ͉ malonylaldehyde ͉ lymphocyte activation ͉ CD-40 ͉ microglia

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in immunocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantify nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L -nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.

Prior studies have shown that exposure to carbon monoxide (CO) will elevate the steady-state concentration of nitric oxide ( ⅐ NO) in several cell types and body organs and that some toxic effects of CO are directed toward endothelial cells. Studies reported in this paper were conducted with bovine pulmonary artery endothelial cells exposed to 10 to 100 ppm CO to achieve concentrations between 11 and 110 nM in air-saturated buffer. Exposure to 11 nM CO increased synthesis of manganous superoxide dismutase and conferred resistance against the lethal effects of 110 nM CO. At concentrations of 88 nM CO or more, exposures for 1 h or longer caused cell death that became apparent 18 h after the exposure ceased. Caspase-1 was activated in response to CO, and cell death was inhibited by a caspase-1 inhibitor. Alteration of proteolytic pathways by CO was indicated by the presence of ubiquitincontaining intracellular inclusion bodies. Morphological changes and caspase activation indicated that cell death was an apoptotic process. Cells exposed to 110 nM CO had higher concentrations of manganous superoxide dismutase and heme oxygenase-1 but no changes in glutathione peroxidase, glucose-6-phosphate dehydrogenase, thiols, or catalase. Elevated levels of antioxidant enzymes and apoptosis were inhibited by the nitric oxide synthase inhibitor, S-isopropylisothiourea, and the peroxynitrite scavenger, selenomethionine. These results show that biochemical effects of CO occur at environmentally relevant concentrations, that apoptotic cell death follows exposure to relatively high concentrations of CO, and that these actions of CO are mediated by nitric oxide. C arbon monoxide (CO) is a ubiquitous environmental pollutant. The National Ambient Air Quality Standards in the United States for CO have been set at 35 ppm for a 1-h average exposure, and 9 ppm for an 8-h average exposure. Concentrations of CO found in urban environments have been correlated with hospital admissions, mortality, and morbidity caused by cardiovascular and pulmonary diseases (1-8). Average CO concentrations have been found to be 1-9 ppm, but there are many occupational settings where exposures exceed these levels (9-17). The carboxyhemoglobin values associated with levels of CO typically found in the environment are so low that a direct hypoxic stress is doubtful and compensatory responses are sufficient to maintain tissue oxygenation (18-21). Therefore, the pathophysiological basis for toxic effects of low concentrations of CO is not clear.The goal of the current investigation was to evaluate the mechanism for cell death and manifestations of oxidative stress in cultured endothelial cells exposed to CO. Studies in humans have suggested that CO exposures will cause vascular and perivascular abnormalities (22-25); these suggestions increase interest regarding the effects of CO on endothelial cells. Our previous work with experimental animals has shown that CO has a number of effects on the vasculature. Nitrotyrosine is a major product when peroxynitrite reacts w...

Animal and clinical investigations have reported that exposure to hyperbaric O(2) improved the outcome of some reperfusion injuries. Animal studies have suggested that this may be due to an inhibition of leukocyte adherence to injured endothelium. This investigation tested the hypothesis that exposure to hyperbaric O(2) would inhibit beta2-integrin-dependent adherence of human neutrophils. Subjects were exposed to O(2) at partial pressures of up to 3 atmospheres absolute (ATA; 1 ATA = 0.1 MPa) for 45 min, and neutrophil binding to nylon columns and to fibrinogen-coated surfaces was measured. Exposure to O(2) at 2.8 or 3.0 ATA inhibited beta2-integrin-dependent neutrophil adherence but had no effect on the cell-surface expression of beta2-integrins, respiratory burst in response to phorbol ester, or non-beta2-integrin-dependent adherence to plastic plates coated with a fibronectin-like protein. beta2-Integrin adherence was restored by incubating blood with 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and hyperbaric O(2) inhibited synthesis of cGMP by neutrophils stimulated with N-formyl-Met-Leu-Phe (FMLP). In studies of cell fractions, the activity of membrane guanylate cyclase was found to be increased by incubation with FMLP as well as by atrial natriuretic peptide (ANP) plus ATP. Hyperbaric O(2) had no effect on the basal activity of soluble or membrane-bound guanylate cyclase. However, hyperbaric O(2) inhibited the function of both the extracellular binding domain of membrane guanylate cyclase as well as intracellular catalytic activity. There are approximately 7,300 membrane guanylate cyclase molecules per cell, based on binding studies with ANP, with a dissociation constant of approximately 450 pM. Hyperbaric O(2) inhibits the function of human neutrophil beta2-integrins by a process linked to impaired synthesis of cGMP.

Peroxiredoxin 6 (Prdx6) and cytosolic GSH peroxidase (GPx1) both GSH-dependent peroxidases, were compared for effects of their "knock-out" on lung injury and lipid peroxidation in: a) mice exposed to 0.85 or 1.0 ata O 2 , b) isolated perfused mouse lungs exposed to 5 mM tert-butyl hydroperoxide (t-BOOH) or 1 mM paraquat, and c) primary mouse pulmonary microvascular endothelial cells exposed to 50 μM t-BOOH. Derangements in GPx1 null were similar or slightly greater than wild type for all parameters in the different models of oxidant stress, while Prdx6 null showed markedly increased effects. GSH peroxidase activity with phosphatidylcholine hydroperoxide as substrate in GPx1 null lung homogenate was decreased only slightly vs. wild type while activity in Prdx6 null lungs was decreased by ~ 95%, indicating that Prdx6 is the major enzyme for reduction of oxidized lung phospholipids. Expression levels of oxidant related genes measured with a PCR-based gene array indicated no significant differences between the Prdx6 and the GPx1 null except for the target genes. Thus, Prdx6 null mice are significantly more sensitive to oxidant stress as compared to GPx1 null, suggesting that scavenging of phospholipid hydroperoxides by Prdx6 plays a major role in lung antioxidant defense.