RESUMO
We assessed the safety and efficacy of combined intravenous and aerosolized antioxidant administration to attenuate chlorine gas-induced airway alterations when administered after exposure. Adult male Sprague-Dawley rats were exposed to air or 400 parts per million (ppm) chlorine (a concentration likely to be encountered in the vicinity of industrial accidents) in environmental chambers for 30 minutes, and returned to room air, and they then received a single intravenous injection of ascorbic acid and deferoxamine or saline. At 1 hour and 15 hours after chlorine exposure, the rats were treated with aerosolized ascorbate and deferoxamine or vehicle. Lung antioxidant profiles, plasma ascorbate concentrations, airway morphology, and airway reactivity were evaluated at 24 hours and 7 days after chlorine exposure. At 24 hours after exposure, chlorine-exposed rats had significantly lower pulmonary ascorbate and reduced glutathione concentrations. Treatment with antioxidants restored depleted ascorbate in lungs and plasma. At 7 days after exposure, in chlorine-exposed, vehicle-treated rats, the thickness of the proximal airways was 60% greater than in control rats, with twice the amount of mucosubstances. Airway resistance in response to methacholine challenge was also significantly elevated. Combined treatment with intravenous and aerosolized antioxidants restored airway morphology, the amount of airway mucosubstances, and airway reactivity to control levels by 7 days after chlorine exposure. Our results demonstrate for the first time, to the best of our knowledge, that severe injury to major airways in rats exposed to chlorine, as characterized by epithelial hyperplasia, mucus accumulation, and airway hyperreactivity, can be reversed in a safe and efficacious manner by the post-exposure administration of ascorbate and deferoxamine.
Assuntos
Antioxidantes/uso terapêutico , Brônquios/patologia , Cloro/toxicidade , Traqueia/patologia , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/metabolismo , Brônquios/efeitos dos fármacos , Testes de Provocação Brônquica , Cloro/administração & dosagem , Glutationa/metabolismo , Hiperplasia/prevenção & controle , Exposição por Inalação , Pulmão/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Traqueia/efeitos dos fármacosRESUMO
Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (â¼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Cloro/toxicidade , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Substâncias para a Guerra Química/toxicidade , Citocinas/metabolismo , Endotélio Vascular/efeitos dos fármacos , Imunofluorescência , Exposição por Inalação , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Vasodilatação/efeitos dos fármacosRESUMO
Chlorine (Cl(2)) gas exposure poses an environmental and occupational hazard that frequently results in acute lung injury. There is no effective treatment. We assessed the efficacy of antioxidants, administered after exposure, in decreasing mortality and lung injury in C57BL/6 mice exposed to 600 ppm of Cl(2) for 45 minutes and returned to room air. Ascorbate and deferoxamine were administered intramuscularly every 12 hours and by nose-only inhalation every 24 hours for 3 days starting after 1 hour after exposure. Control mice were exposed to Cl(2) and treated with vehicle (saline or water). Mortality was reduced fourfold in the treatment group compared with the control group (22 versus 78%; P = 0.007). Surviving animals in the treatment group had significantly lower protein concentrations, cell counts, and epithelial cells in their bronchoalveolar lavage (BAL). Lung tissue ascorbate correlated inversely with BAL protein as well as with the number of neutrophils and epithelial cells. In addition, lipid peroxidation was reduced threefold in the BAL of mice treated with ascorbate and deferoxamine when compared with the control group. Administration of ascorbate and deferoxamine reduces mortality and decreases lung injury through reduction of alveolar-capillary permeability, inflammation, and epithelial sloughing and lipid peroxidation.
Assuntos
Lesão Pulmonar Aguda/mortalidade , Lesão Pulmonar Aguda/prevenção & controle , Ácido Ascórbico/uso terapêutico , Cloro/toxicidade , Desferroxamina/uso terapêutico , Lesão Pulmonar Aguda/patologia , Animais , Antioxidantes/uso terapêutico , Substâncias para a Guerra Química/toxicidade , Cromatografia Líquida de Alta Pressão , Exposição por Inalação , Injeções Intramusculares , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/mortalidade , Pneumonia/patologia , Pneumonia/prevenção & controle , Sideróforos/uso terapêutico , Taxa de SobrevidaRESUMO
We investigated the mechanisms by which chlorine (Cl(2)) and its reactive byproducts inhibit Na(+)-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na(+) channels (ENaC) by measuring AFC in mice exposed to Cl(2) (0-500 ppm for 30 min) and Na(+) and amiloride-sensitive currents (I(Na) and I(amil), respectively) across Xenopus oocytes expressing human alpha-, beta-, and gamma-ENaC incubated with HOCl (1-2000 microm). Both Cl(2) and HOCl-derived products decreased AFC in mice and whole cell and single channel I(Na) in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I(Na) in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I(Na) by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the gamma-ENaC subunit restored the ability of HOCl to inhibit I(Na). Finally, I(Na) of oocytes expressing wild type alpha- and gamma-ENaC and a mutant form of beta ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.
Assuntos
Cloraminas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ácido Hipocloroso/metabolismo , Pulmão/metabolismo , Canais de Sódio/metabolismo , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , XenopusRESUMO
Nitrite (NO(2)(-)) has been shown to limit injury to the heart, liver, and kidneys in various models of ischemia-reperfusion injury. Potential protective effects of systemic NO(2)(-) in limiting lung injury or enhancing repair have not been documented. We assessed the efficacy and mechanisms by which postexposure intraperitoneal injections of NO(2)(-) mitigate chlorine (Cl(2))-induced lung injury in rats. Rats were exposed to Cl(2) (400 ppm) for 30 min and returned to room air. NO(2)(-) (1 mg/kg) or saline was administered intraperitoneally at 10 min and 2, 4, and 6 h after exposure. Rats were killed at 6 or 24 h. Injury to airway and alveolar epithelia was assessed by quantitative morphology, protein concentrations, number of cells in bronchoalveolar lavage (BAL), and wet-to-dry lung weight ratio. Lipid peroxidation was assessed by measurement of lung F(2)-isoprostanes. Rats developed severe, but transient, hypoxemia. A significant increase of protein concentration, neutrophil numbers, airway epithelia in the BAL, and lung wet-to-dry weight ratio was evident at 6 h after Cl(2) exposure. Quantitative morphology revealed extensive lung injury in the upper airways. Airway epithelial cells stained positive for terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL), but not caspase-3. Administration of NO(2)(-) resulted in lower BAL protein levels, significant reduction in the intensity of the TUNEL-positive cells, and normal lung wet-to-dry weight ratios. F(2)-isoprostane levels increased at 6 and 24 h after Cl(2) exposure in NO(2)(-)- and saline-injected rats. This is the first demonstration that systemic NO(2)(-) administration mitigates airway and epithelial injury.
Assuntos
Exposição por Inalação , Lesão Pulmonar/patologia , Lesão Pulmonar/prevenção & controle , Nitrito de Sódio/administração & dosagem , Nitrito de Sódio/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Cloro , F2-Isoprostanos/metabolismo , Marcação In Situ das Extremidades Cortadas , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/fisiopatologia , Masculino , Dióxido de Nitrogênio/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Respiração/efeitos dos fármacosRESUMO
Industrial and transport accidents, accidental releases during recreational swimming pool water treatment, household accidents due to mixing bleach with acidic cleaners, and, in recent years, usage of chlorine during war and in acts of terror, all contribute to the general and elevated state of alert with regard to chlorine gas. We here describe chemical and physical properties of Cl(2) that are relevant to its chemical reactivity with biological molecules, including water-soluble small-molecular-weight antioxidants, amino acid residues in proteins, and amino-phospholipids such as phosphatidylethanolamine and phosphatidylserine that are present in the lining fluid layers covering the airways and alveolar spaces. We further conduct a Cl(2) penetration analysis to assess how far Cl(2) can penetrate the surface of the lung before it reacts with water or biological substrate molecules. Our results strongly suggest that Cl(2) will predominantly react directly with biological molecules in the lung epithelial lining fluid, such as low-molecular-weight antioxidants, and that the hydrolysis of Cl(2) to HOCl (and HCl) can be important only when these biological molecules have been depleted by direct chemical reaction with Cl(2). The results from this theoretical analysis are then used for the assessment of the potential benefits of adjuvant antioxidant therapy in the mitigation of lung injury due to inhalation of Cl(2) and are compared with recent experimental results.
Assuntos
Cloro/toxicidade , Pulmão/efeitos dos fármacos , Animais , Antioxidantes/uso terapêutico , Líquidos Corporais/efeitos dos fármacos , Líquidos Corporais/metabolismo , Cloro/química , Cloro/metabolismo , Cloro/farmacocinética , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Hidrólise , Pulmão/metabolismo , Lesão Pulmonar/tratamento farmacológico , TermodinâmicaRESUMO
S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.
Assuntos
Etanol/farmacologia , Hepatopatias Alcoólicas/prevenção & controle , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteoma/efeitos dos fármacos , S-Adenosilmetionina/farmacologia , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Masculino , Mitocôndrias Hepáticas/química , Proteínas Mitocondriais/análise , Consumo de Oxigênio/efeitos dos fármacos , Proteômica , Ratos , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.
Assuntos
Eritrócitos/efeitos dos fármacos , Alho/química , Sulfeto de Hidrogênio/farmacologia , Acetilcisteína/farmacologia , Cromatografia Líquida de Alta Pressão , Eletroquímica , Eritrócitos/metabolismo , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Humanos , Sulfeto de Hidrogênio/sangueRESUMO
Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.
Assuntos
Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Pirazóis/farmacologia , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Humanos , Camundongos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivo activity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivo activity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivo LDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivo within 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo.
Assuntos
Quimioterapia Combinada/métodos , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias/imunologia , Animais , Humanos , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
Solvent "lens" effects for the reaction kinetics of NO(2) can be evaluated on the basis of published Henry's law constants for nitrogen dioxide in various solvents. Water-to-organic solvent partition coefficients were derived from Henry's law constants and used to assess the tendencies of NO(2) toward fleeing the aqueous environments and concentrating in biological hydrophobic media. It is concluded, based only on the estimated aqueous medium-to-cell membrane partition coefficient for NO(2), that such tendencies will be relatively small, and that they may account for an acceleration of chemical reactions in biological hydrophobic media with reaction kinetics that are first order on NO(2) by a factor of approximately 3+/-1. Thus, kinetic effects due to mass action will be relatively small but it is also important to recognize that because NO(2) will tend to dissolve in cell membranes, reactions with cell membrane components will not be hindered by lack of physical solubility at these loci. In comparison to other gases, nitrogen dioxide is less hydrophobic than NO, O(2) and N(2).
Assuntos
Modelos Químicos , Dióxido de Nitrogênio/química , Algoritmos , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Cinética , Dióxido de Nitrogênio/metabolismo , Compostos Orgânicos/química , Solventes/química , Termodinâmica , Água/químicaRESUMO
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , L-Lactato Desidrogenase/antagonistas & inibidores , Sarcoma de Ewing/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Sarcoma de Ewing/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Populations of Drosophila melanogaster face significant mortality risks from parasitoid wasps that use species-specific strategies to locate and survive in hosts. We tested the hypothesis that parasitoids with different strategies select for alternative host defense characteristics and in doing so contribute to the maintenance of fitness variation and produce trade-offs among traits. We characterized defense traits of Drosophila when exposed to parasitoids with different host searching behaviors (Aphaereta sp. and Leptopilina boulardi). We used host larvae with different natural alleles of the gene Dopa decarboxylase (Ddc), a gene controlling the production of dopamine and known to influence the immune response against parasitoids. Previous population genetic analyses indicate that our focal alleles are maintained by balancing selection. Genotypes exhibited a trade-off between the immune response against Aphaereta sp. and the ability to avoid parasitism by L. boulardi. We also identified a trade-off between the ability to avoid parasitism by L. boulardi and larval competitive ability as indicated by differences in foraging and feeding behavior. Genotypes differed in dopamine levels potentially explaining variation in these traits. Our results highlight the potential role of parasitoid biodiversity on host fitness variation and implicate Ddc as an antagonistic pleiotropic locus influencing larval fitness traits.
Assuntos
Dopa Descarboxilase/genética , Drosophila melanogaster/genética , Comportamento Alimentar , Interações Hospedeiro-Parasita/genética , Vespas/fisiologia , Animais , Dopamina/metabolismo , Drosophila melanogaster/imunologia , Drosophila melanogaster/parasitologia , Feminino , Genótipo , Interações Hospedeiro-Parasita/imunologia , Larva/parasitologia , Especificidade da EspécieRESUMO
Tissues are exposed to exogenous and endogenous nitrogen dioxide ((·)NO(2)), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas-phase (·)NO(2) exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to ()NO(2) covered by +/- DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low[DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with (·)NO(2) compared to ascorbate, assessed via (·)NO(2) reactive absorption and aqueous-phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk-phase conditions, and inhibited 3-NT generated by active myeloperoxidase "bound" to RCM. Per the above and kinetic analyses suggesting preferential DFO versus (·)NO(2) reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents (·)NO(2) addition, and thus nitration, and potentially influences biochemical functionalities.
Assuntos
Desferroxamina/farmacologia , Sequestradores de Radicais Livres/farmacologia , Proteínas/metabolismo , Tirosina/análogos & derivados , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Desferroxamina/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Sequestradores de Radicais Livres/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Dióxido de Nitrogênio/química , Dióxido de Nitrogênio/farmacologia , Oxidantes Fotoquímicos/química , Oxidantes Fotoquímicos/farmacologia , Peroxidase/metabolismo , Proteínas/química , Sideróforos/química , Sideróforos/farmacologia , Tirosina/química , Tirosina/metabolismoRESUMO
BACKGROUND: Chlorine (Cl(2))-induced lung injury is a serious public health threat that may result from industrial and household accidents. Post-Cl(2) administration of aerosolized ascorbate in rodents decreased lung injury and mortality. However, the extent to which aerosolized ascorbate augments depleted ascorbate stores in distal lung compartments has not been assessed. METHODS: We exposed rats to Cl(2) (300 ppm for 30 min) and returned them to room air. Within 15-30 min postexposure, rats breathed aerosolized ascorbate and desferal or vehicle (mean particle size 3.3 µm) through a nose-only exposure system for 60 min and were euthanized. We measured the concentrations of reduced ascorbate in the bronchoalveolar lavage (BAL), plasma, and lung tissues with high-pressure liquid chromatography, protein plasma concentration in the BAL, and the volume of the epithelia lining fluid (ELF). RESULTS: Cl(2)-exposed rats that breathed aerosolized vehicle had lower values of ascorbate in their BAL, ELF, and lung tissues compared to air-breathing rats. Delivery of aerosolized ascorbate increased reduced ascorbate in BAL, ELF, lung tissues, and plasma of both Cl(2) and air-exposed rats without causing lung injury. Based on mean diameter of aerosolized particles and airway sizes we calculated that approximately 5% and 1% of inhaled ascorbate was deposited in distal lung regions of air and Cl(2)-exposed rats, respectively. Significantly higher ascorbate levels were present in the BAL of Cl(2)-exposed rats when aerosol delivery was initiated 1 h post-Cl(2). CONCLUSIONS: Aerosol administration is an effective, safe, and noninvasive method for the delivery of low molecular weight antioxidants to the lungs of Cl(2)-exposed individuals for the purpose of decreasing morbidity and mortality. Delivery is most effective when initiated 1 h postexposure when the effects of Cl(2) on minute ventilation subside.
Assuntos
Ácido Ascórbico/administração & dosagem , Cloro/toxicidade , Sistemas de Liberação de Medicamentos , Lesão Pulmonar/tratamento farmacológico , Administração por Inalação , Aerossóis , Animais , Antioxidantes/administração & dosagem , Antioxidantes/efeitos adversos , Antioxidantes/farmacocinética , Ácido Ascórbico/efeitos adversos , Ácido Ascórbico/farmacocinética , Líquido da Lavagem Broncoalveolar/química , Cromatografia Líquida de Alta Pressão , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/patologia , Masculino , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição TecidualRESUMO
Cl(2) gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl(2) exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1 mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30 min after Cl(2) exposure. Exposure of rats to Cl(2) gas (400 ppm, 30 min) significantly increased ALI and caused RAS 6-24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl(2)-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl(2)-exposed rats. Both ip and im nitrite improved airway histology compared to Cl(2) gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl(2) gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl(2) exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl(2)-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl(2) gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Pulmão/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Nitrito de Sódio/farmacologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Cloro , Exposição por Inalação , Injeções Intramusculares , Injeções Intraperitoneais , Pulmão/imunologia , Pulmão/patologia , Masculino , Cloreto de Metacolina/administração & dosagem , Neutrófilos/imunologia , Neutrófilos/patologia , Ratos , Ratos Sprague-Dawley , Nitrito de Sódio/uso terapêuticoRESUMO
Inhalation experiments using laboratory animals are performed under controlled conditions to assess the toxicity of and to investigate interventional strategies to ameliorate injury resulting from oxidant gas exposures. A variety of dynamic inhalation exposure systems that use whole-body or nose-only exposure chambers have been developed for rodents. In a whole-body exposure chamber, the animals are immersed in the test atmosphere, whereas in nose-only or head-only exposure systems, exposures are localized primarily to the head and/or nasal regions. There are advantages and disadvantages with both types of exposure approaches. Considerations such as animal number, exposure duration, end points of study, and availability of test material should influence the selection of a particular exposure system.
Assuntos
Câmaras de Exposição Atmosférica , Cloro/toxicidade , Gases/toxicidade , Modelos Animais , Nariz , Animais , Desenho de Equipamento , Exposição por InalaçãoRESUMO
Chlorine (Cl(2)) is a reactive oxidant gas used extensively in industrial processes. Exposure of both humans and animals to high concentrations of Cl(2) results in acute lung injury, which may resolve spontaneously or progress to acute respiratory failure. Injury to airway and alveolar epithelium may result from chemical reactions of Cl(2), from HOCl (the hydrolysis product of Cl(2)), and/or from the various reaction products, such as chloramines, that are formed from the reactions of these chlorinating species with biological molecules. Subsequent reactions may initiate self-propagating reactions and induce the production of inflammatory mediators compounding injury to pulmonary surfactant, ion channels, and components of lung epithelial and airway cells. Low-molecular-weight antioxidants, such as ascorbate, glutathione, and urate, present in the lung epithelial lining fluid and tissue, remove Cl(2) and HOCl and thus decrease injury to critical target biological targets. However, levels of lung antioxidants of animals exposed to Cl(2) in concentrations likely to be encountered in the vicinity of industrial accidents decrease rapidly and irreversibly. Our measurements show that prophylactic administration of a mixture containing ascorbate and desferal N-acetyl-cysteine, a precursor of reduced glutathione, prevents Cl(2)-induced injury to the alveolar epithelium of rats exposed to Cl(2). The clinical challenge is to deliver sufficient quantities of antioxidants noninvasively, after Cl(2) exposure, to decrease morbidity and mortality.
Assuntos
Acetilcisteína/farmacologia , Ácido Ascórbico/farmacologia , Cloro/toxicidade , Gases/toxicidade , Pneumopatias/diagnóstico , Pneumopatias/prevenção & controle , Pulmão/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar/química , Cloro/química , Gases/química , Exposição por Inalação , Modelos Animais , Coelhos , RatosRESUMO
Chlorine (Cl(2)) is a highly reactive oxidant gas used extensively in a number of industrial processes. Exposure to high concentrations of Cl(2) results in acute lung injury that may either resolve spontaneously or progress to acute respiratory failure. Presently, the pathophysiological sequelae associated with Cl(2)-induced acute lung injury in conscious animals, as well as the cellular and biochemical mechanisms involved, have not been elucidated. We exposed conscious Sprague-Dawley rats to Cl(2) gas (184 or 400 ppm) for 30 min in environmental chambers and then returned them to room air. At 1 h after exposure, rats showed evidence of arterial hypoxemia, respiratory acidosis, increased levels of albumin, IgG, and IgM in bronchoalveolar lavage fluid (BALF), increased BALF surfactant surface tension, and significant histological injury to airway and alveolar epithelia. These changes were more pronounced in the 400-ppm-exposed rats. Concomitant decreases of ascorbate (AA) and reduced glutathione (GSH) were also detected in both BALF and lung tissues. In contrast, heart tissue AA and GSH content remained unchanged. These abnormalities persisted 24 h after exposure in rats exposed to 400 ppm Cl(2). Rats injected systemically with a mixture of AA, deferoxamine, and N-acetyl-L-cysteine before exposure to 184 ppm Cl(2) had normal levels of AA, lower levels of BALF albumin and normal arterial Po(2) and Pco(2) values. These findings suggest that Cl(2) inhalation damages both airway and alveolar epithelial tissues and that resulting effects were ameliorated by prophylactic administration of low-molecular-weight antioxidants.
Assuntos
Antioxidantes/farmacologia , Pneumopatias/induzido quimicamente , Pneumopatias/prevenção & controle , Acidose Respiratória/complicações , Ar , Animais , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Líquido da Lavagem Broncoalveolar , Cloro , Amarelo de Eosina-(YS) , Epitélio/efeitos dos fármacos , Epitélio/patologia , Hematoxilina , Hipóxia/complicações , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Masculino , Peso Molecular , Pressão Parcial , Fosfolipídeos/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Surfactantes Pulmonares/metabolismo , Ratos , Ratos Sprague-Dawley , Tensão Superficial/efeitos dos fármacosRESUMO
We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (SOD) and both ground-state, triplet oxygen ((3)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(3)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.