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1.
Allergy ; 67(4): 560-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22229752

RESUMO

BACKGROUND: Chronic rhinosinusitis (CRS) is a frequent condition that is treated by functional endoscopic sinus surgery (FESS) when medical treatment fails. Endogenous as well as exogenous factors may be responsible for persisting symptoms after FESS. The role of occupational exposures on success of FESS has never been investigated. METHODS: In this case-control study, we tested the hypothesis that the outcome of FESS procedures is related to exposures at work. Questionnaires were sent to 890 patients who had undergone one or more FESS procedures and to 182 controls. Three independent experts assessed blindly the reported work exposures to inhaled agents. The relationship between exposure and the number of FESS procedures was analyzed. RESULTS: Relevant occupational exposure was reported by 25% of all responding patients undergoing FESS (n = 467) and 12% of controls (n = 69). The prevalence of occupational exposures increased linearly with the number of FESS procedures from 21% in those who had one FESS to 44% in those who had four or more FESS (χ(2)  = 12.74, P < 0.001). Logistic regression analysis with adjustments for potential confounders, including smoking, atopy, and asthma, confirmed that the odds ratio (OR) for reporting occupational exposures was significantly higher in those needing more than one FESS (OR = 1.64) or more than two FESS (OR = 1.97). These results were mainly driven by exposure to low molecular weight agents. CONCLUSION: Exposure at work appears to be a risk factor for the occurrence of CRS and for its recurrence or persistence, as evidenced by the need for revision surgery.


Assuntos
Exposição Ocupacional/efeitos adversos , Rinite/cirurgia , Sinusite/cirurgia , Estudos de Casos e Controles , Doença Crônica , Endoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Reoperação , Fatores de Risco , Inquéritos e Questionários , Resultado do Tratamento
2.
Eur Respir J ; 37(2): 299-309, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20530043

RESUMO

The aim of this study was to investigate the modulation of an asthmatic response by titanium dioxide (TiO2) or gold (Au) nanoparticles (NPs) in a murine model of diisocyanate-induced asthma. On days 1 and 8, BALB/c mice received 0.3% toluene diisocyanate (TDI) or the vehicle acetone-olive oil (AOO) on the dorsum of both ears (20 µL). On day 14, the mice were oropharyngeally dosed with 40 µL of a NP suspension (0.4 mg·mL⁻¹ (∼0.8 mg·kg⁻¹) TiO2 or Au). 1 day later (day 15), the mice received an oropharyngeal challenge with 0.01% TDI (20 µL). On day 16, airway hyperreactivity (AHR), bronchoalveolar lavage (BAL) cell and cytokine analysis, lung histology, and total serum immunoglobulin E were assessed. NP exposure in sensitised mice led to a two- (TiO2) or three-fold (Au) increase in AHR, and a three- (TiO2) or five-fold (Au) increase in BAL total cell counts, mainly comprising neutrophils and macrophages. The NPs taken up by BAL macrophages were identified by energy dispersive X-ray spectroscopy. Histological analysis revealed increased oedema, epithelial damage and inflammation. In conclusion, these results show that a low, intrapulmonary doses of TiO2 or Au NPs can aggravate pulmonary inflammation and AHR in a mouse model of diisocyanate-induced asthma.


Assuntos
Asma/induzido quimicamente , Asma/fisiopatologia , Ouro/efeitos adversos , Pulmão/fisiopatologia , Nanopartículas/efeitos adversos , Titânio/efeitos adversos , Tolueno 2,4-Di-Isocianato/toxicidade , Animais , Hiper-Reatividade Brônquica , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Eosinófilos , Imunoglobulina E/sangue , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/fisiopatologia
3.
Scand J Immunol ; 70(1): 25-33, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19522764

RESUMO

In a mouse model of chemical-induced asthma, we investigated the effects of multiple challenges, using toluene diisocyanate (TDI), a known cause of occupational asthma. On days 1 and 7, BALB/c mice received TDI or vehicle (acetone/olive oil). On days 10, 13 and 16 the mice received an intranasal instillation of TDI. Ventilatory function (Penh) was monitored by whole body plethysmography for 40 min after each challenge. Reactivity to methacholine was measured 22 h later. Pulmonary inflammation, TNF-alpha and MIP-2 levels were assessed 24 h after the last challenge by broncho-alveolar lavage (BAL). Other immunological parameters included total IgE, lymphocyte sub-populations in auricular and cervical lymph nodes, and IL-4, IFN-gamma and IL-13 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Early ventilatory function and airway reactivity increased in all groups that received a dermal application and one or multiple intranasal challenges of TDI. After multiple challenges, lung inflammation was characterized by neutrophils (approximately 15%), and eosinophils (approximately 4%), along with an increase in BAL MIP-2 and TNF-alpha levels. The auricular and cervical lymph node cells of all sensitized mice showed an increase in B cells, Th cells and an increased concentration of in vitro release of IL-4, IFN-gamma and IL-13 after stimulation with concanavalin A. Total serum IgE was elevated in dermally TDI-sensitized mice. This protocol including multiple challenges results in a model that resembles human asthma, indicating that responses found in the model using a single challenge could be a good first indication for the development of asthma.


Assuntos
Asma/induzido quimicamente , Asma/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Imunoglobulina E/sangue , Interferon gama/biossíntese , Interleucina-13/biossíntese , Interleucina-4 , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Função Respiratória , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossíntese
4.
Eur Respir J ; 32(5): 1184-94, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18653652

RESUMO

The current authors evaluated whether a system of co-cultures of relevant cells (pneumocytes (A549), macrophages (THP-1), mast cells (HMC-1) and endothelial cells (EAHY926)) would mimic the responses to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)) previously reported in vivo. The role of mast cells was considered of special interest. Single cultures, bicultures (A549 + HMC-1 in a 10:1 ratio; THP-1 + HMC-1 in a 2:1 ratio) and tricultures (A549 + THP-1 + HMC-1 in a 10:2:1 ratio) were exposed to urban PM(10) (24 h at 0, 10, 30 or 100 microg x cm(-2)). Additionally, EAHY926 cells were introduced in inserts above the tricultures. The released cytokines were evaluated with a fluorescence-activated cell sorter array system. THP-1 + HMC-1 bicultures and the tricultures released more granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-1beta, IL-8, IL-6, tumour necrosis factor-alpha and MIP-1alpha in response to PM(10) than the sum of the single cultures. Tricultures with EAHY926 released more G-CSF, MIP-1alpha, IL-8 and MIP-1beta than the EAHY926 single culture. The bicultures, tricultures and tricultures with EAHY926 provide results that are consistent with the local and systemic effects previously described for particulate matter effects, i.e. inflammation, endothelial dysfunction and bone marrow cell mobilisation. Mast cells seem to play a significant role in the co-culture responses.


Assuntos
Células Endoteliais/metabolismo , Macrófagos/metabolismo , Mastócitos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL4/metabolismo , Técnicas de Cocultura , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Modelos Biológicos , Tamanho da Partícula , Análise Serial de Proteínas , Fator de Necrose Tumoral alfa/metabolismo
5.
Toxicol In Vitro ; 21(7): 1215-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17629671

RESUMO

An in vitro model to study pulmonary translocation was created, using the human cell line Calu-3 and primary rat type II pneumocytes. Cells were seeded on permeable membranes with a 0.4 microm or 3 microm pore size, utilizing different culture conditions such as medium formulation and cell density. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. When seeded on inserts with 0.4 microm pore size, the Calu-3 cells and primary rat type II pneumocytes created high TEER values of 949+/-182 Omega cm(2) and 400+/-257 Omega cm(2), respectively. On membranes with 3 microm pores, Calu-3 cells achieved a high TEER value of 500+/-95 Omega cm(2). Our experiments indicate that the culture medium was more critical than the cell density, regarding the influence on TEER values. For both cell types a reduction of serum in the medium resulted in a decrease in TEER value. We established a good ('tight') monolayer of primary type II pneumocytes in Waymouth medium at a cell density of 0.9x10(6) cells/cm(2); the Calu-3 cells should be grown in DMEM medium containing Hepes at 0.75x10(6) cells/cm(2).


Assuntos
Técnicas de Cultura de Células , Permeabilidade da Membrana Celular , Mucosa Respiratória/metabolismo , Administração por Inalação , Animais , Transporte Biológico , Brônquios/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Impedância Elétrica , Células Epiteliais/metabolismo , Fluoresceína/farmacocinética , Humanos , Pulmão/metabolismo , Permeabilidade , Ratos , Mucosa Respiratória/citologia
6.
Drug Metab Dispos ; 29(10): 1349-54, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11560880

RESUMO

Because of the evidence for the involvement of xenobiotic bioactivation in pulmonary toxicity and carcinogenesis, it is important to improve our understanding of the xenobiotic-metabolizing enzymes in isolated and cultured specific pulmonary cell populations. Some phase I and phase II xenobiotic-metabolizing enzyme activities, reduced glutathione (GSH), and gamma-glutamyl transferase (gamma-GT) were studied in rat type II pneumocytes and alveolar macrophages cultured for up to 48 h and 3 h, respectively. In type II pneumocytes, 7-ethoxyresorufin activity was not detected. 7-Benzyloxyresorufin (BROD) and 7-pentoxyresorufin (PROD) O-dealkylation decreased at 24 h by 84 and 82%, respectively, and continued to decline over the next 24 h with no measurable PROD at 48 h. The activity of NADPH- and NADH-cytochrome c reductase at 48 h decreased by 31 and 67%, respectively. GST activity decreased by 25 and 42% at 24 and 48 h, respectively. A transient increase in DT-diaphorase activity was observed at 24 h (by 55%). GSH content and gamma-GT activity increased significantly with time in culture. In freshly isolated alveolar macrophages, BROD activity was the only cytochrome P450-dependent alkoxyresorufin-O-dealkylase activity measured. BROD activity decreased by 38% in 3-h-attached macrophages. There were no changes in NADPH- and NADH-cytochrome c reductase, GST, and DT-diaphorase. An increase of GSH (by 24%) was observed in attached macrophages. In conclusion, type II pneumocytes and to a lesser extent alveolar macrophages in primary cultures undergo changes in biotransformation-related enzyme activities and intracellular GSH level that may affect xenobiotic toxicity at different times in culture.


Assuntos
Glutationa Transferase/metabolismo , Pulmão/enzimologia , Macrófagos Alveolares/enzimologia , Xenobióticos/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Células Cultivadas , Pulmão/citologia , Masculino , Ratos , Ratos Wistar
7.
Environ Mol Mutagen ; 37(2): 155-63, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11246222

RESUMO

The possible genotoxicity of small particulate matter has been under investigation for the last 10 years. Diesel exhaust particles (DEP) are considered as "probably carcinogenic" (IARC group 2A) and a number of studies show genotoxic effects of urban particulate matter (UPM). Carbon black (CB) is carcinogenic in rats. In this study the cytotoxic and genotoxic potency of these three particle types was investigated by exposing human cells (A549 and THP-1 cell lines) in vitro to CB, DEP (SRM 1650, NIST), and UPM (SRM 1648, NIST) for 48 hr. Cytotoxicity was assessed using the Alamar Blue assay, whereas genotoxicity was assessed using the single-cell gel electrophoresis (comet assay). The particles were characterized with regard to their mean diameter in tissue culture medium (CB 100 nm, DEP 400 nm, UPM 2 microm), their total carbon content (CB 99%, DEP 85%, UPM 15%), and their acid-soluble metal composition (UPM >> CB approximately DEP). The concentrations ranged from 16 ng/ml to 16 microg/ml for cytotoxicity tests and from 16 ng/ml to 1.6 microg/ml for genotoxicity tests. In both assays, paraquat was used as a reference chemical. The CB, DEP, and UPM particles showed no significant cytotoxicity. However, all three particles were able to cause significant DNA damage, although to a different extent in the two cell lines. The genotoxicity of washed particles and dichloromethane extracts was also investigated. In THP-1 cells CB washed particles and DEP extracts caused significant DNA damage. This difference in effect may be related to differences in size, structure, and composition of the particles. These results suggest that CB, DEP, and UPM are able to cause DNA damage and, therefore, may contribute to the causation of lung cancer. More detailed studies on influence of size, structure, and composition of the particles are needed.


Assuntos
Poluentes Atmosféricos/toxicidade , Carbono/toxicidade , Monócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Emissões de Veículos/toxicidade , Poluentes Atmosféricos/química , Carbono/química , Linhagem Celular , Ensaio Cometa , Humanos , Mutagênicos/química , Tamanho da Partícula
8.
Am J Respir Cell Mol Biol ; 23(2): 146-53, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10919979

RESUMO

This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)-binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS). Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression. Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied. These cells produced LBP in response to interleukin (IL)-1beta, IL-6, and tumor necrosis factor- alpha, a response that was strongly enhanced by dexamethasone. In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation. The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7. Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7-derived LBP. Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS. Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells. The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.


Assuntos
Proteínas de Fase Aguda/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Glicoproteínas de Membrana , Mucosa Respiratória/metabolismo , Animais , Western Blotting , Células CHO , Proteínas de Transporte/genética , Linhagem Celular , Cricetinae , Citocinas/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia
9.
Biochem Pharmacol ; 59(11): 1467-75, 2000 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10751557

RESUMO

Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (

Assuntos
Acetaminofen/toxicidade , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Analgésicos não Narcóticos/farmacologia , Animais , Inibidores das Enzimas do Citocromo P-450 , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Técnicas In Vitro , Pulmão/citologia , Masculino , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar
10.
Free Radic Res ; 30(5): 371-81, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10342330

RESUMO

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([3H]-GSH, 0.1 microCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 microM) to inhibit gamma-glutamyltransferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 +/- 0.20 nmol/min/mg protein (37 degrees C) and 0.35 +/- 0.04 nmol/min/mg protein (4 degrees C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 +/- 0.30 nmol/min/mg protein (37 degrees C) and 0.32 +/- 0.06 nmol/min/mg protein (4 degrees C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 +/- 0.3 and 1.2 +/- 0.3 nmol/mg protein after 15 min at 37 degrees C, and 2.8 +/- 0.2 and 1.0 +/- 0.3 nmol/mg protein at 4 degrees C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37 degrees C and 4 degrees C was found. Pre-exposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by pre-exposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.


Assuntos
Glutationa/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Animais , Antimetabólitos/farmacologia , Transporte Biológico , Células Cultivadas , Cistina/farmacologia , Humanos , Isoxazóis/farmacologia , Pulmão/efeitos dos fármacos , Masculino , Ouabaína/farmacologia , Penicilamina/farmacologia , Ratos , Ratos Wistar , Compostos de Sulfidrila/farmacologia
11.
Pharmacol Toxicol ; 81(2): 74-80, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9298503

RESUMO

It has been demonstrated that hard metal dust, which consists of a mixture of cobalt and tungsten carbide, is more toxic toward mouse peritoneal and rat alveolar macrophages than pure cobalt (Co) or tungsten carbide (WC). The aim of this study was to investigate the toxic effects of Co and hard metal dust on alveolar epithelial type II cells (AT-II), and to compare these with alveolar macrophages. Freshly isolated rat and human AT-II and rat alveolar macrophages were exposed for 18 hr to particles of Co, WC or Co/WC. As an index for cell toxicity, release of lactate dehydrogenase was measured. For rat AT-II, TD50 values per 10(5) cells were 672 micrograms (95% C.I. = 264-1706 micrograms) for pure Co and 101 micrograms (95% C.I. = 59-172 micrograms) for Co in Co/WC mixture. For rat alveolar macrophages, TD50 values per 10(5) cells were 18 micrograms (95% C.I. = 15-24 micrograms) for pure Co and 5 micrograms (95% C.I. = 5-6 micrograms) for Co in Co/WC mixture. WC only caused an increase in lactate dehydrogenase at high concentrations. No toxicity was found in human AT-II for either Co, WC or Co/WC. These results indicate that 1) rat AT-II are less sensitive to Co than rat alveolar macrophages, 2) human AT are less sensitive to Co than rat AT-II, 3) the toxicity of Co is increased by the presence of WC.


Assuntos
Cobalto/toxicidade , Poeira/efeitos adversos , Macrófagos Alveolares/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Compostos de Tungstênio/toxicidade , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Macrófagos Alveolares/enzimologia , Masculino , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/enzimologia , Ratos , Ratos Wistar , Especificidade da Espécie , Compostos de Tungstênio/efeitos adversos
12.
Free Radic Biol Med ; 22(3): 525-34, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-8981045

RESUMO

The purpose of our study was to investigate the effect of oxidative stress or intracellular glutathione (GSH) depletion on gamma-glutamyltransferase (gamma-GT) activity in cultured type II pneumocytes. Twenty-four hours after isolation, primary cultures of rat type II pneumocytes were preincubated with one of four compounds: 15, 30, 60, 125, 250 microM L-buthionine-[SR]-sulfoximine (BSO) for 3 h; 100, 200, 400, 800 microM tertiary-butylhydroperoxide (t-BOOH) for 45 min; 10, 25, 50, 100 microM menadione for 15 min; 100, 1000 microM paraquat for 1 h. GSH levels, H2O2 and O2.- generation were measured immediately after the incubation, gamma-GT activity and GSH levels also up to 24 h or 48 h later. Exposure to BSO led to a persistent GSH depletion without increase in H2O2 or O2.- production, together with a dose and time-dependent increase (doubling) of gamma-GT activity with a nonsignificant increase in gamma-GT mRNA expression 24 h after exposure to BSO. Exposure to 100 microM menadione, which increased H2O2 production, decreased gamma-GT activity. t-BOOH or paraquat did not give rise to a measurable increase in H2O2 or O2.-. Paraquat did not affect initial GSH levels, but increased GSH and decreased gamma-GT activity 24 h later. t-BOOH (400 and 800 microM) initially decreased GSH, and tended to increase GSH 24 h later, 100 and 200 microM increased gamma-GT activity 24 h later, but 800 microM decreased it. Restoration of intracellular GSH levels by addition of GSH to the culture medium completely prevented the increase in gamma-GT activity by BSO, while the addition of catalase or DMTU had no effect. We conclude that at least two effects are operating upon gamma-GT activity: GSH depletion seems to increase gamma-GT activity, while exposure to compounds generating oxidative stress correlates with a decrease in gamma-GT activity.


Assuntos
Glutationa/metabolismo , Pulmão/enzimologia , gama-Glutamiltransferase/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Peróxido de Hidrogênio/metabolismo , Pulmão/efeitos dos fármacos , Masculino , Estresse Oxidativo , Paraquat/administração & dosagem , Paraquat/farmacologia , Peróxidos/administração & dosagem , Peróxidos/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Vitamina K/administração & dosagem , Vitamina K/farmacologia , terc-Butil Hidroperóxido
13.
Br J Cancer ; 73(1): 96-100, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8554991

RESUMO

Type II pneumocytes and Clara cells, both epithelial cells that possess an active uptake system for polyamines, have been identified as possible precursor cells of at least some types of lung tumours. In this study we have investigated whether human pulmonary tumours exhibit putrescine uptake. Lung slices from both tumoral tissue and non-tumoral tissue, obtained from patients undergoing surgery for lung cancer, were incubated with radiolabelled putrescine at both 37 degrees C and 4 degrees C. The accumulation of putrescine was evaluated by its apparent kinetic parameters, in the presence or absence of cystamine, and by autoradiography. The investigated tumoral tissue (six squamous carcinomas and five adenocarcinomas) did not show accumulation of putrescine above that attributable to simple diffusion, except for one adenocarcinoma. In this specimen autoradiography showed that the accumulation was not specifically associated with any particular cell type, but that practically every cell accumulated putrescine. We conclude that human pulmonary tumours do not accumulate polyamines in a manner similar to normal pulmonary epithelial cells.


Assuntos
Neoplasias Pulmonares/metabolismo , Putrescina/farmacocinética , Adenocarcinoma/metabolismo , Idoso , Autorradiografia , Radioisótopos de Carbono , Carcinoma de Células Escamosas/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Valores de Referência , Trítio
14.
Am J Physiol ; 269(5 Pt 1): L681-9, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7491989

RESUMO

Putrescine is accumulated in the lungs of various species by an active uptake system that also mediates the uptake of cystamine and paraquat. We have characterized this uptake in both lung slices and type II pneumocytes isolated from hamsters by trypsin digestion, differential adherence on plastic, and centrifugation on a discontinuous Percoll gradient. The accumulation of [14C]putrescine in lung slices was shown to be temperature and energy dependent, and to obey saturation kinetics, with mean calculated values of apparent Michaelis constant (Km) 29.4 microM and maximum rate of uptake (Vmax) 637 nmol.g-1.h-1. In the presence of cystamine or paraquat, the putrescine uptake was reduced in a manner compatible with competitive inhibition. The calculated inhibitor constants (Ki) were 16 and 1,017-1,328 microM for the inhibition by cystamine and paraquat, respectively. The cellular localization of [3H]putrescine in lung slices after incubation with 2.5 microM putrescine was determined by light-microscopic autoradiography. Labeling was present in type II and possibly also in type I pneumocytes of the alveolar epithelium but not in macrophages, endothelium, or cells of the interstitium. Two days after their isolation, cultured type II pneumocytes exhibited an uptake of putrescine that had kinetic characteristics similar to those of slices (Km of 23 microM and Vmax of 3.06 mumol.g protein-1.h-1) and was also competitively inhibited by paraquat (Ki of 222-350 microM paraquat). Our data demonstrate the presence of an active uptake system for putrescine in both lung slices and cultured type II pneumocytes.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Pulmão/metabolismo , Putrescina/farmacocinética , Animais , Autorradiografia , Células Cultivadas , Cricetinae , Cistamina/farmacologia , Feminino , Técnicas In Vitro , Pulmão/citologia , Pulmão/efeitos dos fármacos , Masculino , Mesocricetus , Paraquat/farmacocinética , Paraquat/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Putrescina/antagonistas & inibidores
15.
Biochem Pharmacol ; 50(7): 981-9, 1995 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-7575683

RESUMO

Putrescine uptake in type II pneumocytes is a carrier-mediated active process. Our hypothesis was that oligoamines might be taken up into the cell at least in part by gamma-glutamyltransferase (gamma-GT). This was investigated in rat type II pneumocytes 24 hr after their isolation. Preexposure to 125 microM L-buthionine-[SR]-sulfoximine (BSO) or 100 microM diethylmaleate (DEM), both of which affect intracellular glutathione (GSH) only, were found to decrease GSH by 85% (p < 0.05) and 62%, respectively (p < 0.05), without change in [3H]-putrescine uptake. Preexposure to 20 microM N-ethylmaleimide (NEM), which affects intra- and extracellular GSH, decreased intracellular GSH by 79% (p = 0.015) and putrescine uptake by 39% (p = 0.03). Selective extracellular GSH depletion by 10 microM copper-o-phenanthroline complex (CuP) led to a decrease in putrescine uptake of 41% (p = 0.001), while intracellular GSH remained unchanged. Specific inhibition of gamma-GT by 5-20 mM serine-borate or 5 mM acivicin gave similar degrees of putrescine uptake inhibition (39.5% and 40.5%). The kinetic properties of the putrescine uptake system in the presence of acivicin and serine-borate indicated that the Vmax decreased by 25%, while Km remained unchanged. In experiments with pure gamma-GT, the oligoamines putrescine, spermidine and spermine, and cystamine proved to be acceptor substrates for gamma-GT, all having similar efficiencies (Vmax/Km); methylglyoxal-bis-(guanyl-hydrazone) and paraquat were not accepted. As extracellular GSH is required for gamma-GT, and because its extracellular depletion inhibits putrescine uptake as much as specific inhibition of gamma-GT, we suggest that 30-40% of the putrescine uptake in type II pneumocytes occurs by gamma-GT and that, therefore, at least two systems are involved in the uptake of putrescine.


Assuntos
Pulmão/metabolismo , Putrescina/metabolismo , gama-Glutamiltransferase/metabolismo , Animais , Boratos/farmacologia , Separação Celular , Sobrevivência Celular , Células Cultivadas , Cobre , Regulação para Baixo , Glutationa/metabolismo , Glutationa/farmacologia , Isoxazóis/farmacologia , Cinética , Masculino , Compostos Organometálicos/farmacologia , Fenantrolinas/farmacologia , Ratos , Ratos Wistar , Serina/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores
16.
Biochem Pharmacol ; 48(3): 517-24, 1994 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-8068038

RESUMO

Paraquat is accumulated into the lungs of various species by an active uptake system which also appears to mediate the uptake of endogenous polyamines, such as putrescine. The accumulation of putrescine in the human lung has been previously shown to be mainly located in the type II cells. In the present study, we have studied the mutually competitive inhibition of putrescine and paraquat in human lung slices and the inhibition of putrescine by paraquat or cystamine in isolated human type II pneumocytes. Peripheral lung tissue taken from patients undergoing pneumectomy or lobectomy was used. The initial steps of the cell isolation procedure differed from the literature in that the tissue was first sliced in 0.7 mm thick slices, which were washed in phosphate buffered saline without calcium and magnesium (PBS-), followed by incubations with trypsin. The type II cells were purified and isolated by differential adherence on plastic followed by Percoll gradient centrifugation. Uptake was determined 48 hr after cell isolation. The accumulation of radiolabelled putrescine showed saturation kinetics, with the following apparent kinetic parameters: Km 6.7 and 6.2-7.6 microM and Vmax 2.7 and 3.0-3.4 mumol/g prot/hr for slices and isolated cells, respectively. In the presence of paraquat, putrescine uptake was reduced, in both systems, in a manner compatible with competitive inhibition, with calculated inhibition constants (Ki) of 549-614 and 659-895 microM paraquat for slices and isolated cells, respectively. The accumulation of putrescine in isolated human pneumocytes was strongly reduced in the presence of cystamine, with calculated Ki of 3.7 microM cystamine. These data indicate that putrescine, paraquat and cystamine accumulate in the human lung by the same uptake system, but that the affinities for the three substrates differ. The presence of an uptake system for putrescine in cultured human pulmonary type II is probably useful as a functional viability test.


Assuntos
Pulmão/metabolismo , Paraquat/farmacologia , Putrescina/metabolismo , Ligação Competitiva , Células Cultivadas , Cistamina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética
17.
Thorax ; 48(12): 1235-41, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8303630

RESUMO

BACKGROUND: The polyamines (putrescine, spermidine, and spermine) are involved in cellular growth, proliferation, and differentiation. In the lungs of various species, polyamines are accumulated by an active uptake system which also mediates the uptake of cystamine and paraquat. In the rat lung putrescine uptake has been shown to be cell-specific, occurring predominantly in the alveolar epithelium. The aim of this study was to characterise the uptake of putrescine in human lung. METHODS: Lung tissue was obtained from 31 patients undergoing surgery for lung cancer. Slices (0.7 mm thick) from non-tumour containing lung parenchyma were incubated for 15-60 minutes in Krebs-Ringer phosphate buffer with various concentrations of putrescine (2.5 to 80 mumol/l) containing 0.1 microCi [1,4-14C]-putrescine. Uptake was assessed from tissue radioactivity. For autoradiographic imaging, slices were incubated for 30 minutes with 2.5 mumol/l putrescine containing 2.5 mCi [1,4n-3H]-putrescine. RESULTS: The accumulation of [14C]-putrescine into slices was time-dependent and energy-dependent, and obeyed saturation kinetics, with mean calculated values for Vmax (maximal rate of uptake) of 414 nmol/g/hour and for Km (medium concentration at which the rate of uptake is half Vmax) of 7.2 mumol/l, with a large interindividual variation. Competitive inhibition was observed on incubation with cystamine, which appears to have a high affinity for the uptake system since its calculated Ki (concentration of inhibitor at which the Km is doubled) was 3.2 mumol/l. Ultrastructural autoradiography showed labelling over both type I and type II cells of the alveolar epithelium, but not over the endothelium or any cells of the interstitium. Alveolar macrophages were also devoid of label. CONCLUSIONS: These results show that the human lung possesses an active uptake system for putrescine, and probably also cystamine, which is located in both cell types of the alveolar epithelium. These findings may be used to develop tests for the assessment of the alveolar epithelium.


Assuntos
Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Putrescina/farmacocinética , Autorradiografia , Cistamina/metabolismo , Metabolismo Energético , Humanos , Cinética , Pulmão/patologia , Neoplasias Pulmonares/patologia , Fatores de Tempo
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