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1.
Antioxidants (Basel) ; 11(7)2022 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-35883743

RESUMO

The innate immune response to infection results in inflammation and oxidative damage, creating a paradox where most anti-inflammatory and antioxidant therapies can further suppress an already inadequate immune response. We have previously reported the beneficial effects of the exogenous supplementation of innate immunity with small pseudohalide thiocyanate (-SCN) in a mouse model of a cystic fibrosis (CF) lung infection and inflammation. The object of this study was to evaluate the use of -SCN as a counter anion for cationic manganese porphyrin (MnP) catalytic antioxidants, which could increase the parent compound's antioxidant spectrum against hypohalous acids while supplementing innate immunity. The antioxidant activities of the parent compound were examined, as its chloride salt was compared with the -SCN-anion exchanged compound, (MnP(SCN) versus MnP(Cl)). We measured the superoxide dismutase activity spectrophotometrically and performed hydrogen peroxide scavenging using oxygen and hydrogen peroxide electrodes. Peroxidase activity was measured using an amplex red assay. The inhibition of lipid peroxidation was assessed using a thiobarbituric acid reactive species (TBARS) assay. The effects of the MnP compounds on macrophage phagocytosis were assessed by flow cytometry. The abilities of the MnP(Cl) formulations to protect human bronchiolar epithelial cells against hypochlorite (HOCl) and glycine chloramine versus their MnP(SCN) formulations were assessed using a cell viability assay. We found that anions exchanging out the chloride for -SCN improved the cellular bioavailability but did not adversely affect the cell viability or phagocytosis and that they switched hydrogen-peroxide scavenging from a dismutation reaction to a peroxidase reaction. In addition, the -SCN formulations improved the ability of MnPs to protect human bronchiolar epithelial cells against hypochlorous acid (HOCl) and glycine chloramine toxicity. These novel types of antioxidants may be more beneficial in treating lung disease that is associated with chronic infections or acute infectious exacerbations.

2.
Toxicol Sci ; 182(2): 297-309, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34051097

RESUMO

Upon returning from deployment to Afghanistan, a substantial number of U.S. military personnel report deployment-related lung disease (DRLD) symptoms, including those consistent with an asthma-like airways disease. DRLD is thought to be caused by prolonged inhalation of toxic desert particulate matter, which can persist in the postdeployment setting such as exposure to common household allergens. The goal of this study was to define the transcriptomic responses of lung leukocytes of mice exposed to Afghanistan desert particulate matter (APM) and house dust mite (HDM). C57BL/6 mice (n = 15/group) were exposed to filtered air or aerosolized APM for 12 days, followed by intranasal PBS or HDM allergen challenges for 24 h. Bronchoalveolar lavage (BAL) cells were collected for single-cell RNA sequencing (scRNAseq), and assessment of inflammation and airway hyper-responsiveness. Unsupervised clustering of BAL cell scRNAseq data revealed a unique monocyte population induced only by both APM and allergen treatments. This population of monocytes is characterized by the expression of genes involved in allergic asthma, including Alox15. We validated Alox15 expression in monocytes via immunostaining of lung tissue. APM pre-exposure, followed by the HDM challenge, led to significantly increased total respiratory system resistance compared with filtered air controls. Using this mouse model to mimic DRLD, we demonstrated that inhalation of airborne PM during deployment may prime airways to be more responsive to allergen exposure after returning home, which may be linked to dysregulated immune responses such as induction of a unique lung monocyte population.


Assuntos
Alérgenos , Material Particulado , Afeganistão , Alérgenos/toxicidade , Animais , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Monócitos , Material Particulado/toxicidade , Análise de Sequência de RNA
3.
Toxicol Appl Pharmacol ; 404: 115186, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777237

RESUMO

Increased symptoms of asthma-like respiratory illnesses have been reported in soldiers returning from tours of duty in Afghanistan. Inhalation of desert particulate matter (PM) may contribute to this deployment-related lung disease (DRLD), but little is known about disease mechanisms. The IL-33 signaling pathway, including its receptor ST2, has been implicated in the pathogenesis of lung diseases including asthma, but its role in PM-mediated airway dysfunction has not been studied. The goal of this study was to investigate whether IL-33/ST2 signaling contributes to airway dysfunction in preclinical models of lung exposure to Afghanistan PM (APM). Wild-type (WT) and ST2 knockout (KO) mice on the BALB/C background were oropharyngeally instilled with a single dose of saline or 50 µg of APM in saline. Airway hyperresponsiveness (AHR) and inflammation were assessed after 24 h. In WT mice, a single APM exposure induced AHR and neutrophilic inflammation. Unlike the WT mice, ST2 KO mice that lack the receptor for IL-33 did not demonstrate AHR although airway neutrophilic inflammation was comparable to the WT mice. Oropharyngeal delivery of a soluble ST2 decoy receptor in APM-exposed WT mice significantly blocked AHR. Additional data in mouse tracheal epithelial cell and lung macrophage cultures demonstrated a role of APM-induced IL-33/ST2 signaling in suppression of regulator of G protein signaling 2 (RGS2), a gene known to protect against bronchoconstriction. We present for the first time that APM may increase AHR, one of the features of asthma, in part through the IL-33/ST2/RGS2 pathway.


Assuntos
Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Pneumopatias/induzido quimicamente , Material Particulado/toxicidade , Afeganistão , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/genética , Macrófagos/efeitos dos fármacos , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Tamanho da Partícula , Alvéolos Pulmonares/citologia , Transdução de Sinais/efeitos dos fármacos
4.
J Inflamm Res ; 13: 175-185, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368126

RESUMO

PURPOSE: Electronic cigarettes (e-cigs) are relatively new devices that allow the user to inhale a heated and aerosolized solution. At present, little is known about their health effects in the human lung, particularly in the small airways (<2 mm in diameter), a key site of airway obstruction and destruction in chronic obstructive pulmonary disease and other acute and chronic lung conditions. The aim of this study was to investigate the effect of e-cigarettes on human distal airway inflammation and remodeling. METHODS: We isolated primary small airway epithelial cells from donor lungs without known lung disease. Small airway epithelial cells were cultured at air-liquid interface and exposed to 15 puffs vapor obtained by heating a commercially available e-cigarette solution (e-vapor) with or without nicotine. After 24 hrs of e-vapor exposure, basolateral and apical media as well as cell lysates were collected to measure the pleiotropic cytokine interleukin 6 (IL6) and MUC5AC, one of the major components in mucus. RESULTS: Unlike the nicotine-containing e-vapor, nicotine-free e-vapor significantly increased the amount of IL6, which was coupled with increased levels of intracellular MUC5AC protein. Importantly, a neutralizing IL6 antibody (vs an IgG isotype control) significantly inhibited the production of MUC5AC induced by nicotine-free e-vapor. CONCLUSION: Our results suggest that human small airway epithelial cells exposed to nicotine-free e-vapor increase the inflammatory response and mucin production, which may contribute to distal lung airflow limitation and airway obstruction.

5.
Free Radic Biol Med ; 152: 91-99, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32142878

RESUMO

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by severe bacterial infections, excessive neutrophilic inflammation and oxidative stress. The neutrophil enzyme myeloperoxidase (MPO), which produces hypochlorous acid, is associated with worse disease outcomes. Therefore, pharmacological inhibition of MPO in the airways has therapeutic potential. We investigated whether treating mice with an MPO inhibitor during pulmonary infection decreases oxidative stress and improves infection outcomes in mice with CF-like lung inflammation without impacting on bacterial clearance. METHODS: Transgenic ß-epithelial sodium channel (ßENaC)-overexpressing mice (n = 10) were infected with Burkholderia multivorans and treated twice daily with the MPO inhibitor AZM198 (125 µmol/kg) or vehicle administered by oral gavage for two days. Bodyweight was recorded daily. MPO activity, markers of oxidative stress, inflammatory cytokines and leukocytes numbers were measured in bronchoalveolar lavage fluid (BALF). Bacterial burden was determined in lung tissue homogenates. RESULTS: During the course of infection, mice treated with AZM198 lost less weight than vehicle-treated mice (p < 0.01). MPO activity and glutathione sulfonamide, a hypochlorous acid-specific glutathione oxidation product, were significantly lower in BALF from AZM198-treated mice (p < 0.05). The inflammatory cytokines CXCL1 and TNF-α in BALF and bacterial burden in the lung were not significantly different between treated and control mice. CONCLUSIONS: Orally administered AZM198 inhibits MPO activity in epithelial lining fluid. Blocking hypochlorous acid production in epithelial lining fluid during pulmonary infections through inhibition of MPO improves morbidity in mice with CF-like lung inflammation without interfering with clearance of bacteria. Pharmacological inhibition of MPO is an approach to limit destructive oxidative stress in cystic fibrosis lung disease in humans.


Assuntos
Fibrose Cística , Pneumonia , Animais , Líquido da Lavagem Broncoalveolar , Burkholderia , Fibrose Cística/tratamento farmacológico , Inflamação , Pulmão/metabolismo , Camundongos , Morbidade , Estresse Oxidativo , Peroxidase/metabolismo , Pneumonia/tratamento farmacológico
6.
Free Radic Biol Med ; 146: 324-332, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31740228

RESUMO

A hallmark of cystic fibrosis (CF) lung pathology is an increased susceptibility to pulmonary infections. Thiocyanate (-SCN) is an endogenous component of the innate immunity's peroxidase system that converts -SCN to the antimicrobial agent hypothiocyanite (HOSCN). We have previously shown that the host thioredoxin reductase (TrxR), but not the pathogen's TrxR, can selectively detoxify HOSCN thereby decreasing inflammation and oxidative stress. We tested whether the -SCN analog selenocyanate (-SeCN) shares these properties against several clinical CF bacterial isolates. We examined oxidant production from a lactoperoxidase (LPO) system using -SeCN as a potential substrate. The LPO system generated an oxidant similar in nature to HOSCN and consistent with being HOSeCN. The rate of oxidant generation using -SeCN was significantly less than seen for -SCN. An LPO system was used to generate HOSCN or HOSeCN and compared for antimicrobial activity during in situ exposure of clinical CF isolates of P. aeruginosa (PA), B. cepacia complex (BCC), and methicillin-resistant S. aureus (MRSA) obtained from CF sputum samples. Bacterial viability was assessed by colony forming units. Selective detoxification of HOSeCN was determined by comparing its metabolism by mammalian thioredoxin reductase (TrxR) to bacterial TrxR following the consumption of NADPH. We also assessed potential toxicity of equivalent HOSeCN generation, which demonstrated in situ antimicrobial activity, in human bronchial epithelial cells with a cell viability assay. The -SeCN/HOSeCN system was much more potent than -SCN/HOSCN system at killing PA, BCC and MRSA isolates. The -SeCN/HOSeCN system was more effective at killing -SCN/HOSCN resistant isolates. Mammalian TrxR selectively detoxified HOSeCN whereas the bacterial TrxR enzyme showed little activity. Human bronchial epithelial cells exposed to equivalent flux of HOSeCN that killed several CF pathogens showed no decrease in viability. -SeCN may be an effective therapeutic for the treatment of CF lung pathogens that are difficult to treat with current antibiotics.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Pró-Fármacos , Animais , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Cianatos , Humanos , Compostos de Selênio , Tiocianatos
7.
Toxicol Sci ; 154(2): 341-353, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27605419

RESUMO

Sulfur mustard (bis 2-chloroethyl ethyl sulfide, SM) is a powerful bi-functional vesicating chemical warfare agent. SM tissue injury is partially mediated by the overproduction of reactive oxygen species resulting in oxidative stress. We hypothesized that using a catalytic antioxidant (AEOL 10150) to alleviate oxidative stress and secondary inflammation following exposure to SM would attenuate the toxic effects of SM inhalation. Adult male rats were intubated and exposed to SM (1.4 mg/kg), a dose that produces an LD50 at approximately 24 h. Rats were randomized and treated via subcutaneous injection with either sterile PBS or AEOL 10150 (5 mg/kg, sc, every 4 h) beginning 1 h post-SM exposure. Rats were euthanized between 6 and 48 h after exposure to SM and survival and markers of injury were determined. Catalytic antioxidant treatment improved survival after SM inhalation in a dose-dependent manner, up to 52% over SM PBS at 48 h post-exposure. This improvement was sustained for at least 72 h after SM exposure when treatments were stopped after 48 h. Non-invasive monitoring throughout the duration of the studies also revealed blood oxygen saturations were improved by 10% and clinical scores were reduced by 57% after SM exposure in the catalytic antioxidant treatment group. Tissue analysis showed catalytic antioxidant therapy was able to decrease airway cast formation by 69% at 48 h post-exposure. To investigate antioxidant induced changes at the peak of injury, several biomarkers of oxidative stress and inflammation were evaluated at 24 h post-exposure. AEOL 10150 attenuated SM-mediated lung lipid oxidation, nitrosative stress and many proinflammatory cytokines. The findings indicate that catalytic antioxidants may be useful medical countermeasure against inhaled SM exposure.


Assuntos
Antídotos/farmacologia , Antioxidantes/farmacologia , Substâncias para a Guerra Química/toxicidade , Lesão Pulmonar/prevenção & controle , Pulmão/efeitos dos fármacos , Metaloporfirinas/farmacologia , Gás de Mostarda/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/prevenção & controle , Animais , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Mediadores da Inflamação/metabolismo , Exposição por Inalação , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
8.
Toxicol Sci ; 147(2): 466-74, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26149495

RESUMO

The study objective was to assess age-related changes in glutathione (GSH) adaptive response to cigarette smoke (CS) exposure. Older cigarette smokers show a decline (67%) in lung epithelial lining fluid (ELF) GSH and a 1.8-fold decreased GSH adaptive response to cigarette smoking with a concomitant elevation (47%) of exhaled nitric oxide compared with younger smokers. In order to isolate the changes in tissue GSH from other age-related effects, pharmacological inhibition of the rate limiting step in GSH synthesis was employed to examine the lung's response to CS exposure in young mice. The γ-glutamylcysteine ligase inhibitor L-buthionine-sulfoximine (BSO) was administered in the drinking water (20 mM) to decrease by half the in vivo GSH levels to those found in aged mice and humans. Mice were then exposed to CS (3 h/day) for 5 or 15 days. Biochemical analysis of the ELF and lung tissue revealed an inhibition of the CS-induced GSH adaptive response by BSO with a concurrent increase in mixed protein-GSH disulfides indicating increased cysteine oxidation. The prevention of the GSH adaptive response led to an increase in pro-inflammatory cytokines present in the lung. Airspace enlargement is a hallmark of lung emphysema and was observed in mice treated with BSO and exposed to CS for as little as 15 days, whereas these types of changes normally take up to 6 months in this model. BSO treatment potentiated both lung elastase and matrix metalloproteinase activity in the CS group. These data suggest that age-related decline in the GSH adaptive response can markedly accelerate many of the factors thought to drive CS-induced emphysema.


Assuntos
Glutationa/deficiência , Inflamação/induzido quimicamente , Sistema Respiratório/efeitos dos fármacos , Fumar/efeitos adversos , Adolescente , Adulto , Fatores Etários , Idoso , Animais , Líquido da Lavagem Broncoalveolar/química , Citocinas/análise , Glutationa/análise , Glutationa/metabolismo , Glutationa/fisiologia , Humanos , Inflamação/fisiopatologia , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Masculino , Metaloproteinases da Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Sistema Respiratório/fisiopatologia , Adulto Jovem
9.
Am J Respir Cell Mol Biol ; 53(2): 193-205, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25490247

RESUMO

Thiocyanate (SCN) is used by the innate immune system, but less is known about its impact on inflammation and oxidative stress. Granulocytes oxidize SCN to evolve the bactericidal hypothiocyanous acid, which we previously demonstrated is metabolized by mammalian, but not bacterial, thioredoxin reductase (TrxR). There is also evidence that SCN is dysregulated in cystic fibrosis (CF), a disease marked by chronic infection and airway inflammation. To investigate antiinflammatory effects of SCN, we administered nebulized SCN or saline to ß epithelial sodium channel (ßENaC) mice, a phenotypic CF model. SCN significantly decreased airway neutrophil infiltrate and restored the redox ratio of glutathione in lung tissue and airway epithelial lining fluid to levels comparable to wild type. Furthermore, in Pseudomonas aeruginosa-infected ßENaC and wild-type mice, SCN decreased inflammation, proinflammatory cytokines, and bacterial load. SCN also decreased airway neutrophil chemokine keratinocyte chemoattractant (also known as C-X-C motif chemokine ligand 1) and glutathione sulfonamide, a biomarker of granulocyte oxidative activity, in uninfected ßENaC mice. Lung tissue TrxR activity and expression increased in inflamed lung tissue, providing in vivo evidence for the link between hypothiocyanous acid metabolism by TrxR and the promotion of selective biocide of pathogens. SCN treatment both suppressed inflammation and improved host defense, suggesting that nebulized SCN may have important therapeutic utility in diseases of both chronic airway inflammation and persistent bacterial infection, such as CF.


Assuntos
Antibacterianos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Fibrose Cística/tratamento farmacológico , Tiocianatos/administração & dosagem , Administração por Inalação , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Linhagem Celular , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Pulmão/enzimologia , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/enzimologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Tiocianatos/farmacologia , Tiorredoxina Dissulfeto Redutase/metabolismo
10.
J Allergy Clin Immunol ; 131(4): 1041-7, 1047.e1-3, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23154082

RESUMO

BACKGROUND: Asthma in obese adults is typically more severe and less responsive to glucocorticoids than asthma in nonobese adults. OBJECTIVE: We sought to determine whether the clearance of apoptotic inflammatory cells (efferocytosis) by airway macrophages was associated with altered inflammation and reduced glucocorticoid sensitivity in obese asthmatic patients. METHODS: We investigated the relationship of efferocytosis by airway (induced sputum) macrophages and blood monocytes to markers of monocyte programming, in vitro glucocorticoid response, and systemic oxidative stress in a cohort of adults with persistent asthma. RESULTS: Efferocytosis by airway macrophages was assessed in obese (n=14) and nonobese (n=19) asthmatic patients. Efferocytosis by macrophages was 40% lower in obese than nonobese subjects, with a mean efferocytic index of 1.77 (SD, 1.07) versus 3.00 (SD, 1.25; P<.01). A similar reduction of efferocytic function was observed in blood monocytes of obese participants. In these monocytes there was also a relative decrease in expression of markers of alternative (M2) programming associated with efferocytosis, including peroxisome proliferator-activated receptor δ and CX3 chemokine receptor 1. Macrophage efferocytic index was significantly correlated with dexamethasone-induced mitogen-activated protein kinase phosphatase 1 expression (ρ=0.46, P<.02) and baseline glucocorticoid receptor α expression (ρ=0.44, P<.02) in PBMCs. Plasma 4-hydroxynonenal levels were increased in obese asthmatic patients at 0.33 ng/mL (SD, 0.15 ng/mL) versus 0.16 ng/mL (SD, 0.08 ng/mL) in nonobese patients (P=.006) and was inversely correlated with macrophage efferocytic index (ρ=-0.67, P=.02). CONCLUSIONS: Asthma in obese adults is associated with impaired macrophage/monocyte efferocytosis. Impairment of this anti-inflammatory process is associated with altered monocyte/macrophage programming, reduced glucocorticoid responsiveness, and systemic oxidative stress.


Assuntos
Asma/patologia , Macrófagos/patologia , Monócitos/patologia , Obesidade/patologia , Adulto , Aldeídos/sangue , Apoptose , Asma/complicações , Asma/tratamento farmacológico , Asma/imunologia , Biomarcadores/metabolismo , Estudos de Coortes , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/imunologia , Feminino , Expressão Gênica , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Inflamação/imunologia , Inflamação/patologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/imunologia , Estresse Oxidativo , PPAR delta/genética , PPAR delta/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/imunologia , Escarro/citologia
11.
Free Radic Biol Med ; 52(7): 1201-6, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22266045

RESUMO

One of the most abundant antioxidants in the lung is glutathione (GSH), a low-molecular-weight thiol, which functions to attenuate both oxidative stress and inflammation. GSH is concentrated in the epithelial lining fluid (ELF) of the lung and can be elevated in response to the increased oxidant burden from cigarette smoke (CS). However, the transporter(s) responsible for the increase in ELF GSH with cigarette smoke is not known. Three candidate apical GSH transporters in the lung are CFTR, BCRP, and MRP2, but their potential roles in ELF GSH transport in response to CS have not been investigated. In vitro, the inhibition of CFTR, BCRP, or MRP2 resulted in decreased GSH efflux in response to cigarette smoke extract. In vivo, mice deficient in CFTR, BCRP, or MRP2 were exposed to either air or acute CS. CFTR-deficient mice had reduced basal and CS-induced GSH in the ELF, whereas BCRP or MRP2 deficiency had no effect on ELF GSH basal or CS-exposed levels. Furthermore, BCRP or MRP2 deficiency had little effect on lung tissue GSH. These data indicate that CFTR is predominantly involved in maintaining basal ELF GSH and increasing ELF GSH in response to CS.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Quimiocinas CC/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Pulmão/efeitos dos fármacos , Proteínas Inflamatórias de Macrófagos/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Fumar/efeitos adversos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Brônquios/citologia , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glutationa/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos
12.
Toxicol Appl Pharmacol ; 258(1): 1-9, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21856323

RESUMO

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5-25 µM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5-30 µM) and DOX (0.025-3.0 µM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC(50) values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Flavonoides/farmacologia , Glutationa/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/patologia , Células Tumorais Cultivadas
13.
Respir Res ; 12: 133, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21982222

RESUMO

BACKGROUND: Smoking tobacco is a leading cause of chronic obstructive pulmonary disease (COPD), but although the majority of COPD cases can be directly related to smoking, only a quarter of smokers actually develop the disease. A potential reason for the disparity between smoking and COPD may involve an individual's ability to mount a protective adaptive response to cigarette smoke (CS). Glutathione (GSH) is highly concentrated in the lung epithelial lining fluid (ELF) and protects against many inhaled oxidants. The changes in GSH that occur with CS are not well investigated; therefore the GSH adaptive response that occurs with a commonly utilized CS exposure was examined in mice. METHODS: Mice were exposed to CS for 5 h after which they were rested in filtered air for up to 16 h. GSH levels were measured in the ELF, bronchoalveolar lavage cells, plasma, and tissues. GSH synthesis was assessed by measuring γ-glutamylcysteine ligase (GCL) activity in lung and liver tissue. RESULTS: GSH levels in the ELF, plasma, and liver were decreased by as much as 50% during the 5 h CS exposure period whereas the lung GSH levels were unchanged. Next, the time course of rebound in GSH levels after the CS exposure was examined. CS exposure initially decreased ELF GSH levels by 50% but within 2 h GSH levels rebound to about 3 times basal levels and peaked at 16 h with a 6-fold increase and over repeat exposures were maintained at a 3-fold elevation for up to 2 months. Similar changes were observed in tissue GCL activity which is the rate limiting step in GSH synthesis. Furthermore, elevation in ELF GSH levels was not arbitrary since the CS induced GSH adaptive response after a 3d exposure period prevented GSH levels from dropping below basal levels. CONCLUSIONS: CS exposures evoke a powerful GSH adaptive response in the lung and systemically. These data suggests there may be a sensor that sets the ELF GSH adaptive response to prevent GSH levels from dipping below basal levels. Factors that disrupt GSH adaptive responses may contribute to the pathophysiology of COPD.


Assuntos
Adaptação Fisiológica , Glutationa/metabolismo , Exposição por Inalação , Pulmão/metabolismo , Fumar/metabolismo , Adaptação Fisiológica/imunologia , Animais , Linhagem Celular , Glutationa/fisiologia , Humanos , Exposição por Inalação/efeitos adversos , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiopatologia , Fumar/efeitos adversos
14.
PLoS One ; 6(10): e25704, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21991336

RESUMO

A number of inflammatory lung diseases have abnormally low glutathione (GSH) levels in the airway fluids. Lung macrophages are common mediators of inflammation, make up the majority of cells that are found in the airway epithelial lining fluid (ELF), and are commonly elevated in many lung diseases. Several animal models with altered ELF GSH levels are associated with similar alterations in the intracellular GSH levels of bronchoalveolar lavage (BAL) cells. The possible mechanisms and outcomes for this association between ELF GSH levels and intracellular BAL cell GSH are unknown. To investigate these issues, macrophages were grown in media supplemented with 500 µM GSH. GSH supplementation resulted in a 2-3 fold increase in macrophage intracellular GSH levels. The increase in macrophage intracellular GSH levels was associated with a significant reduction in NF-κB nuclear translocation and tumor necrosis factor α (TNFα) release upon LPS stimulation. Furthermore, co-treatment of macrophages with GSH and inhibitors of GSH breakdown or synthesis did not block GSH accumulation. In contrast, treatment with cytochalasin D, an inhibitor of actin dependent endocytosis, and amiloride, an inhibitor of macropinocytosis blocked, at least in part, GSH uptake. Furthermore, using two cigarette smoke exposure paradigms that result in two different GSH levels in the ELF and thus in the BAL cells resulted in modulation of cytokine release when stimulated with LPS ex vivo. These data suggest that macrophages are able to utilize extracellular GSH which can then modulate inflammatory signaling in response to proinflammatory stimuli. This data also suggests the lung can modulate inflammatory responses triggered by proinflammatory stimuli by altering ELF GSH levels and may help explain the dysregulated inflammation associated with lung diseases that have low ELF GSH levels.


Assuntos
Espaço Extracelular/metabolismo , Glutationa/metabolismo , Ativação de Macrófagos , Macrófagos Alveolares/citologia , Macrófagos Alveolares/metabolismo , Pinocitose , Fator de Necrose Tumoral alfa/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Corantes Fluorescentes/metabolismo , Glutationa/administração & dosagem , Mediadores da Inflamação/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Pinocitose/efeitos dos fármacos , Fumar/efeitos adversos
15.
Respir Res ; 11: 119, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20799947

RESUMO

BACKGROUND: Cystic fibrosis is a debilitating lung disease due to mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) and is associated with chronic infections resulting in elevated myeloperoxidase activity and generation of hypochlorous acid (HOCl). CFTR mutations lead to decreased levels of glutathione (GSH) and thiocyanate (SCN) in the epithelial lining fluid (ELF). Hypertonic saline is used to improve lung function however the mechanism is uncertain. METHODS: In the present study, the effect of GSH and SCN on HOCl-mediated cell injury and their changes in the ELF after hypertonic saline nebulization in wild type (WT) and CFTR KO mice was examined. CFTR sufficient and deficient lung cells were assessed for GSH, SCN and corresponding sensitivity towards HOCl-mediated injury, in vitro. RESULTS: CFTR (-) cells had lower extracellular levels of both GSH and SCN and were more sensitive to HOCl-mediated injury. In vivo, hypertonic saline increased ELF GSH in the WT and to a lesser extent in the CFTR KO mice but only SCN in the WT ELF. Finally, potential protective effects of GSH and SCN at concentrations found in the ELF against HOCl toxicity were examined in vitro. CONCLUSIONS: While the concentrations of GSH and SCN associated with the WT ELF protect against HOCl toxicity, those found in the CFTR KO mice were less sufficient to inhibit cell injury. These data suggests that CFTR has important roles in exporting GSH and SCN which are protective against oxidants and that hypertonic saline treatment may have beneficial effects by increasing their levels in the lung.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/prevenção & controle , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Glutationa/metabolismo , Mucosa Respiratória/metabolismo , Solução Salina Hipertônica/uso terapêutico , Compostos de Sulfidrila/fisiologia , Tiocianatos/metabolismo , Lesão Pulmonar Aguda/genética , Animais , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Knockout , Camundongos Transgênicos , Oxirredução/efeitos dos fármacos , Substâncias Protetoras/uso terapêutico , Mucosa Respiratória/efeitos dos fármacos
16.
Am J Respir Crit Care Med ; 182(9): 1114-22, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20622027

RESUMO

RATIONALE: Cigarette smoke (CS) is the leading cause of chronic obstructive pulmonary disease, accounting for more than 90% of cases. The prevalence of chronic obstructive pulmonary disease is much higher in the elderly, suggesting an age dependency. A prominent defense against the oxidant burden caused by CS is the glutathione (GSH) adaptive response in the lung epithelial lining fluid (ELF) and tissue. However, as one ages the ability to maintain GSH levels declines. OBJECTIVES: Examine the effect of aging on the GSH adaptive response to CS and resulting lung sensitization to inflammation and oxidation. METHODS: Both young (2 mo old) and aged (8, 13, 19, and 26 mo old) mice were used to study the effects of age on the GSH adaptive response after an acute exposure to CS. MEASUREMENTS AND MAIN RESULTS: Young mice had a robust sixfold increase in ELF GSH after a single exposure to CS. The GSH response to CS decreased as a function of age and diminishes in the older mice to only a twofold increase over air controls. As a consequence, levels of CS-induced tumor necrosis factor-α and nitric oxide synthase, markers of inflammation, and 8-hydroxy-2-deoxyguanosine, a marker of DNA oxidation, were elevated in the aged mice compared with the young mice. Additionally, depletion of ELF GSH with buthionine sulfoximine in young mice recapitulated changes in ELF tumor necrosis factor-α as seen in old mice. CONCLUSIONS: These data suggest that the age-related maladaptive response to CS sensitizes the lung to both inflammation and oxidation potentially contributing to the development of CS-induced chronic obstructive pulmonary disease.


Assuntos
Envelhecimento/fisiologia , Glutationa/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/fisiopatologia , Adaptação Fisiológica/fisiologia , Fatores Etários , Animais , Líquido da Lavagem Broncoalveolar/química , Progressão da Doença , Feminino , Glutationa/análise , Imuno-Histoquímica , Pulmão/química , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos , Estresse Oxidativo/fisiologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Nicotiana
17.
Free Radic Biol Med ; 48(9): 1188-96, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20138141

RESUMO

Sulfur mustard (bis-2-(chloroethyl) sulfide; SM) is a highly reactive vesicating and alkylating chemical warfare agent. A SM analog, 2-chloroethyl ethyl sulfide (CEES), has been utilized to elucidate mechanisms of toxicity and as a screen for therapeutics. Previous studies with SM and CEES have demonstrated a role for oxidative stress as well as decreased injury with antioxidant treatment. We tested whether posttreatment with the metalloporphyrin catalytic antioxidant AEOL 10150 would improve outcome in CEES-induced lung injury. Anesthetized rats inhaled 5% CEES for 15 min via a nose-only inhalation system. At 1 and 9 h after CEES exposure, rats were given AEOL 10150 (5 mg/kg, sc). At 18 h post-CEES exposure BALF lactate dehydrogenase activity, protein, IgM, red blood cells, and neutrophils were elevated but were decreased by AEOL 10150 treatment. Lung myeloperoxidase activity was increased after CEES inhalation and was ameliorated by AEOL 10150. The lung oxidative stress markers 8-OHdG and 4-HNE were elevated after CEES exposure and significantly decreased by AEOL 10150 treatment. These findings demonstrate that CEES inhalation increased lung injury, inflammation, and oxidative stress, and AEOL 10150 was an effective rescue agent. Further investigation utilizing catalytic antioxidants as treatment for SM inhalation injury is warranted.


Assuntos
Antioxidantes/farmacologia , Substâncias para a Guerra Química/toxicidade , Metaloporfirinas/farmacologia , Gás de Mostarda/análogos & derivados , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Inflamação/tratamento farmacológico , Exposição por Inalação , L-Lactato Desidrogenase/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Gás de Mostarda/administração & dosagem , Gás de Mostarda/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
18.
Free Radic Biol Med ; 47(6): 722-30, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19520157

RESUMO

Electrophilic cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)), initiate redox-based cell signaling responses including increased intracellular glutathione (GSH) synthesis. We investigated whether cyPGs facilitated GSH efflux and if members of the ATP-binding cassette (ABC) protein family mediated the efflux. Four human cell lines were treated with 1-6 microM cyPGs for 48 h. Media and cells were harvested for GSH measurements using HPLC-EC. CyPG treatment increased extracellular GSH levels two- to threefold over controls in HN4 and C38 cells and five- to sixfold in SAEC and MDA 1586 cells and was dependent on increased GSH synthesis. Our studies show that prostaglandin D(2) and its metabolites, prostaglandin J(2) and 15dPGJ(2), specifically induce GSH efflux compared to other eicosanoids. These higher extracellular GSH levels were associated with protection from tert-butylhydroperoxide. Superarray analysis of ABC transporters suggested only ABCG2 expression had a positive relationship in the four cell types compared with extracellular GSH increases after cyPG treatment. The ABCG2 substrate Hoechst 33342 inhibited extracellular GSH increase after 15dPGJ(2) treatment. We report for the first time that ABCG2 may play a role in GSH efflux in response to cyPG treatment and may link inflammatory signaling with antioxidant adaptive responses.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/biossíntese , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Prostaglandina D2/análogos & derivados , Mucosa Respiratória/efeitos dos fármacos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Citoproteção/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glutationa/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Prostaglandina D2/farmacologia , Prostaglandina D2/uso terapêutico , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Especificidade por Substrato , terc-Butil Hidroperóxido/metabolismo
19.
Am J Physiol Lung Cell Mol Physiol ; 292(6): L1590-7, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17369290

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glutationa/farmacocinética , Pulmão/metabolismo , Administração Oral , Animais , Líquido da Lavagem Broncoalveolar/citologia , Glutationa/sangue , Absorção Intestinal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Knockout
20.
Free Radic Biol Med ; 41(9): 1459-69, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17023273

RESUMO

Aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in epithelial cells and stromal keratocytes of mammalian cornea and is believed to play an important role in cellular defense. To explore a potential protective role against oxidative damage, a rabbit corneal fibroblastic cell line (TRK43) was stably transfected with the human ALDH3A1 and subjected to oxidative stress induced by H(2)O(2), mitomycin C (MMC), or etoposide (VP-16). ALDH3A1-transfected cells were more resistant to H(2)O(2,) MMC, and VP-16 compared to the vector-transfected cells. All treatments induced apoptosis only in vector-transfected cells, which was associated with increased levels of 4-hydroxy-2-nonenal (4-HNE)-adducted proteins. Treatment with H(2)O(2) resulted in a rise in reduced glutathione (GSH) levels in all groups but was more pronounced in the ALDH3A1-expressing cells. Treatment with the DNA-damaging agents led to GSH depletion in control groups, although the depletion was significantly less in ALDH3A1-expressing cells. Increased carbonylation of ALDH3A1 but not significant decline in enzymatic activity was observed after all treatments. In conclusion, our results suggest that ALDH3A1 may act to protect corneal cells against cellular oxidative damage by metabolizing toxic lipid peroxidation products (e.g., 4-HNE), maintaining cellular GSH levels and redox balance, and operating as an antioxidant.


Assuntos
Aldeído Desidrogenase/metabolismo , Antioxidantes/farmacologia , Córnea/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Aldeídos/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Córnea/citologia , Córnea/metabolismo , Dano ao DNA/efeitos dos fármacos , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Mitomicina/farmacologia , Estresse Oxidativo , Coelhos , Células Estromais/metabolismo , Transfecção
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