RESUMO
Inhaled 'NO2 is absorbed by a free radical-dependent reaction mechanism that localizes the initial oxidative events to the extracellular space of the pulmonary surface lining layer (SLL). Because 'NO2 per se is eliminated upon absorption, most likely the SLL-derived reaction products are critical to the genesis of 'NO2-induced lung injury. We utilized analysis of the rate of 'NO2 disappearance from the gas phase to determine the preferential absorption substrates within rat SLL. SLL was obtained via bronchoalveolar lavage and was used either as the cell-free composite or after constituent manipulation [(i) dialysis, treatment with (ii) N-ethylmaleimide, (iii) ascorbate oxidase, (iv) uricase, or (v) combined ii + iii]. Specific SLL constituents were studied in pure chemical systems. Exposures were conducted under conditions where 'NO2 is the limiting reagent and disappears with first-order kinetics ([NO2]0 < or = 10 ppm). Reduced glutathione and ascorbate were the principle rat SLL absorption substrates. Nonsulfhydryl amino acids and dipalmitoyl phosphatidylcholine exhibited negligible absorption activity. Whereas uric acid and vitamins A and E displayed rapid absorption kinetics, their low SLL concentrations preclude appreciable direct interaction. Unsaturated fatty acids may account for < or = 20% of absorption. The results suggest that water soluble, low molecular weight antioxidants are the preferential substrates driving 'NO2 absorption. Consequently, their free radicals, produced as a consequence of 'NO2 exposure, may participate in initiating the 'NO2-induced cascade, which results in epithelial injury.
Assuntos
Dióxido de Nitrogênio/farmacocinética , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Administração por Inalação , Adsorção , Aminoácidos/metabolismo , Animais , Antioxidantes/metabolismo , Ascorbato Oxidase/farmacologia , Ácido Ascórbico/metabolismo , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar , Etilmaleimida/farmacologia , Radicais Livres/administração & dosagem , Radicais Livres/farmacocinética , Glutationa/metabolismo , Cinética , Pulmão/metabolismo , Masculino , Matemática , Modelos Teóricos , Dióxido de Nitrogênio/administração & dosagem , Ratos , Ratos Sprague-Dawley , Urato Oxidase/farmacologia , Ácido Úrico/metabolismoRESUMO
With the use of an isolated rat lung model, we investigated pulmonary air space absorption kinetics of the reactive gas NO2 in an effort to determine the contributory role of chemical reaction(s) vs. physical solubility. Unperfused lungs were employed, because vascular perfusion had no effect on acute (0- to 60-min) NO2 absorption rates. We additionally found the following: 1) Uptake was proportional to exposure rates (2-14 micrograms NO2/min; 10-63 ppm; 37 degrees C) but saturated with exposures greater than or equal to 14 micrograms NO2/min. 2) Uptake was temperature (22-48 degrees C) dependent but, regardless of temperature, attained apparent saturation at 10.6 micrograms NO2/min. 3) Lung surface area (SA) was altered by increasing functional residual capacity (FRC). Expanded SA (8 ml FRC) and temperature (48 degrees C) both raised fractional uptakes (greater than or equal to 0.81) relative to 4 ml FRC, 37 degrees C (0.67). Uptake rates normalized per unit estimated SA revealed no independent effect of FRC on fractional uptake. However, temperature produced a profound effect (48 degrees C = 0.93; 4 and 8 ml FRC = 0.54). 4) Arrhenius plots (ln k' vs. 1/T), which utilized derived reactive uptake coefficients (k'), showed linearity (r2 = 0.94) and yielded an activation energy of 7,536 kcal.g-1.mol-1 and Q10 of 1.43, all consistent with a reaction-mediated process. These findings, particularly the effects of temperature, suggest that acute NO2 uptake in pulmonary air spaces is, in part, rate limited by chemical reaction of NO2 with epithelial surface constituents rather than by direct physical solubility.
Assuntos
Barreira Alveolocapilar/fisiologia , Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Poluentes Atmosféricos , Animais , Técnicas In Vitro , RatosRESUMO
Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.
Assuntos
Espaço Extracelular/metabolismo , Óxido Nitroso/farmacocinética , Troca Gasosa Pulmonar/fisiologia , Absorção , Animais , Líquido da Lavagem Broncoalveolar/metabolismo , Epitélio/metabolismo , Glutationa/metabolismo , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
In a previous study (J. Appl. Physiol. 68: 594-603, 1990) in isolated rat lungs, we suggested that the rate of pulmonary air space absorption of inhaled NO2 is limited, in part, by chemical reaction(s) rather than by physical solubility. Because the initial site of primary absorption interactions involves the epithelial lining fluid (ELF), we investigated whether ELF-NO2 interactions could account for pulmonary NO2 reactive absorption. Rat ELF, obtained by bronchoalveolar lavage (BAL), was compared with a model chemical system (reduced glutathione, GSH). In vitro exposures (NO2-air) used constant gas flow and planar gas-liquid interfaces. 1) Solvent pH notably altered NO2 uptake by GSH but to a lesser extent by BAL. 2) Uptake displayed [GSH]-dependent saturation. [ELF] in BAL was augmented by sequential lavage (lavagate reuse) of multiple lungs. Uptake was proportional to [ELF] but did not saturate under these exposure conditions. 3) The uptake rate exhibited [NO2] dependence. However, relative to increasing [NO2], fractional uptakes decreased for BAL and 1 mM GSH but not for 10 mM GSH. 4) Altered convective gas flow produced nonlinear increments in uptake (10 mM GSH) and substantial decrements in fractional uptake. 5) Arrhenius plots [ln(r) vs. 1/T, where r is reaction rate and T is absolute temperature (degree K)] for BAL and 1 mM GSH yielded respective activation energies of 4,952 and 4,149 kcal.g-1.mol-1 and degree of change in the rate of NO2 uptake per 10 degrees C (Q10) of 1.32 and 1.25. These results imply that the rate of NO2 uptake into rat ELF, like intact lung, is limited, in part, by chemical reaction(s).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Líquido da Lavagem Broncoalveolar/metabolismo , Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Absorção , Animais , Epitélio/metabolismo , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Solubilidade , TemperaturaRESUMO
We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO2 interfacial transfer into ELF exhibits first-order kinetics with respect to NO2, attains [NO2]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510, 1991). We have extended these observations by evaluating the kinetics of NO2 gas phase disappearance in isolated ventilating rat lungs. Transient exposures (2-3/lung at 25 degrees C) employed rebreathing (NO2-air) from a non-compliant continuously stirred closed chamber. We observed that 1) NO2 uptake rate is independent of exposure period, 2) NO2 gas phase disappearance exhibited first-order kinetics [initial rate (r*) saturation occurred when [NO2] > 11 ppm], 3) the mean effective rate constant (k*) for NO2 gas phase disappearance ([NO2] < or = 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 +/- 5 ml/min, 4) with [NO2] < or = 11 ppm, k* and r* were proportional to tidal volume, and 5) NO2 fractional uptakes were constant across [NO2] (< or = 11 ppm) and tidal volumes but exceeded quasi-steady-state observations. Preliminary data indicate that this divergence may be related to the inspired PCO2. These results suggest that NO2 reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Absorção , Animais , Epitélio/metabolismo , Capacidade Residual Funcional , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Volume de Ventilação Pulmonar/fisiologiaRESUMO
Nitrogen dioxide (NO2) is a ubiquitous, pollutant gas that produces a broad range of pathological and physiological effects on the lung. Absorption of inhaled NO2 is coupled to near-interfacial reactions between the solute gas and constituents of the airway and alveolar epithelial lining fluid. Although alveolar surfactant imparts limited resistance to respiratory gas exchange compared with that contributed by either the pulmonary membrane or uptake in red blood cells, resistance to NO2 flux could have a significant effect on NO2 absorption kinetics. To investigate the effect of interfacial surfactant on NO2 absorption, we designed an apparatus permitting exposure of variably compressed monolayers. Our results suggest that compressed monolayers enriched in 1,2-dipalmitoyl-sn-3-glycero-phosphocholine present significant resistance to NO2 absorption even at surface tensions greater than those achieved in vivo. However, monolayers composed of pure unsaturated phospholipids failed to alter NO2 absorption significantly when compressed, in spite of similar reductions in surface tension. The results demonstrate that phospholipid monolayers appreciably limit NO2 absorption and further that monolayer-induced resistance to NO2 flux is related to physicochemical properties of the film itself rather than alterations within the aqueous and gas phases. On the basis of these findings, we propose that pulmonary surfactant may influence the intrapulmonary gas phase distribution of inhaled NO2.
Assuntos
Membranas Artificiais , Dióxido de Nitrogênio/química , Fosfolipídeos/química , 1,2-Dipalmitoilfosfatidilcolina , Algoritmos , Animais , Líquido da Lavagem Broncoalveolar , Indicadores e Reagentes , Masculino , Surfactantes Pulmonares/química , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Tensão SuperficialRESUMO
Although NO2-induced cytotoxic responses have been well characterized, the specific mechanisms responsible for initiating toxicity remain equivocal. The inhomogeneous distribution of epithelial injury suggests that differential interactions between NO2 and the lung surfaces may contribute to the extent of regional responses. Consequently, we have initiated studies to characterize the mechanisms which govern NO2 absorption and the initiation of the toxic cascade. Due to limitations in whole animal models, we have utilized numerous in vitro exposure models. Herein we examine our recent investigations. In brief synopsis: NO2 uptake is governed by reaction between inhaled NO2 and constituents of the pulmonary surface lining layer (SLL). The predominant reaction pathway involves hydrogen abstraction producing HNO2 and an organic radical. NO2 uptake is first-order with respect to NO2 ([NO2] < 10 ppm), is aqueous substrate-dependent, and is saturable. Conditions at the gas/liquid interface proper modulate the rate of transfer into the aqueous phase. Most likely, NO2 does not diffuse unreacted through the SLL. Absorption is proportional to inspired dose. The clearance efficiency may be modulated by ventilation frequency and the effective surface area of the exposed air space surfaces. We propose that the profile and concentration of SLL constituents mediate both the dosimetry and the extent of epithelial responses. Due to differential lining layer conditions, the relationship between absorbed dose and response may be complex and may exhibit anatomic and host variability.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Absorção , Animais , Técnicas In Vitro , Modelos Biológicos , RatosRESUMO
The effects of nitrogen dioxide (NO2) exposure on primary cultured monolayers of rat type II pneumocytes were investigated as a function of the isolation and culture conditions. Monolayers were cultured in Eagle's minimum essential medium (MEM) and in MEM supplemented with Ham's F-12; in some experiments, the initial cell suspension was also replated after 3 h. Both supplementation of the basal medium and replating increased the sensitivity of the monolayers to NO2, as measured by reduction in dome formation of plastic dishes 24 h post-exposure. These findings suggest that comparisons of in vitro toxicologic observations may be complicated by the effects of specific experimental conditions.
Assuntos
Dióxido de Nitrogênio/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Animais , Células Cultivadas , Meios de Cultura , Epitélio/efeitos dos fármacos , Masculino , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
This study analyzed the effects of nitrogen dioxide (NO2) on alveolar epithelial permeability and transport properties. Primary cultured monolayers of rat Type II pneumocytes, cultured on both nonporous and porous surfaces, were used as models of isolated alveolar epithelium for in vitro exposure to nitrogen dioxide. The effects of nitrogen dioxide exposure for monolayers cultured on nonporous substrata were monitored by observing the changes in the net volume of fluid under the monolayer; for cells cultured on porous substrata, alterations in tissue bioelectric properties were noted. As a first step, primary cultured monolayers of rat Type II pneumocytes plated on nonporous plastic Petri dishes were used to investigate the effects of nitrogen dioxide on alveolar epithelial barrier properties. Such monolayers form fluid filled domes that are thought to result from active solute transport from medium to substratum, with water following passively. We used dome formation as a transport marker. Five-day-old cultures were directly exposed to 30 ppm NO2 in 5 percent CO2 in air at 25 degrees C, by cyclically tilting culture plates from side to side, so that both halves of the monolayer were exposed during each cycle. Exposures consisted of 10 cycles of four minutes each (two minutes per side), for a cell exposure time of 20 minutes. Control plates were simultaneously exposed to 5 percent CO2 in air under identical conditions. One day after the exposure, nitrogen dioxide-exposed monolayers exhibited significant decreases in dome density and individual dome volume, compared to the controls. By 48 hours post-exposure, differences between nitrogen dioxide-exposed and control monolayers were less, but remained significant. These results showed that short-term sublethal exposures to nitrogen dioxide produce a decrease in dome formation in Type II alveolar epithelial cell monolayers. This finding is most likely due to a decrease in the active transepithelial sodium transport rate, or an increase in the permeability of cell membranes or tight junctions, or both. Addition of vitamin E-containing liposomes to the culture media 24 hours pre-exposure did not affect the nitrogen dioxide-induced decrease in dome formation, indicating that under these circumstances no protective effect was provided by the antioxidant.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Dióxido de Nitrogênio/efeitos adversos , Alvéolos Pulmonares/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Alvéolos Pulmonares/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
While the pulmonary toxicity of NO2 is clearly established, the mechanism by which it is removed from inspired air is poorly understood. Uptake is most likely dependent on chemical reaction since, despite limited per se gaseous NO2 aqueous solubility, uptake proceeds rapidly without ready saturation. We utilized an isolated perfused rat lung model to characterize the effect of dose rate on uptake and transformation. Dose rate was varied via alterations in inspired concentration, tidal volume, and ventilation frequency. Dose equaled the total amount inhaled, uptake the amount removed from inspired air, and transformation the amount of NO2- that accumulated in the perfusate. We found a linear proportionality between both inspired concentration (4-20 ppm) and minute ventilation (45-130 ml/min) and uptake. Fractional uptakes (65%) were similar for all groups. Regression of combined concentration and minute ventilation data yielded a linear relationship between total inspired dose (25-330 micrograms NO2) and both uptake (r2 = 0.99) and transformation (r2 = 0.98). Testing of the functional descriptions resulted in measured uptakes and transformation that fell within a few percentage points of those predicted. We conclude that in acutely exposed isolated lungs (1) NO2 uptake is dependent on total inhaled dose rather than on the variables which serve to affect dose rate, (2) transformation is related to both total inspired dose and uptake, and (3) uptake is more accurately described using a regression equation rather than by use of fractional uptakes.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Animais , Biotransformação , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Ozônio/farmacocinética , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Nitrogen dioxide (NO2) is a relatively insoluble, reactive gas that, on inhalation, generates a diverse array of pulmonary toxic effects. Its uptake and transformation in isolated lungs have been shown to be proportional to inspired dose and associated with significant accumulations of the nitrite ion. However, not all absorbed NO2 is directly detectable as soluble nitrite. To further characterize its uptake and chemical disposition, we determined the chemical fate of 15NO2-nitrogen in isolated perfused (red cell-free) rat lungs that were exposed to 20 ppm 15NO2 for 60 min. Total excess 15N (relative to unexposed controls) was determined by isotope ratio mass spectrometry and total nitrogen analysis. Excess 15N was detected in whole lungs and in soluble and insoluble fractions but not in the total lipid pool. Perfusate excess 15N and nitrate correlated and accounted for all absorbed NO2 not detectable in tissue fractions. Exogenously instilled [15N]nitrite distributed within lung tissue, bound to insoluble elements, and diffused to the vascular space similar to NO2-nitrogen. Instilled [15N]nitrate did not distribute or bind like NO2-nitrogen or nitrite. Dialysis (1000 molecular weight cutoff) of cytosol, membranes, and perfusate removed excess 15N and nitrite derived from NO2, nitrite, or nitrate sources. We conclude that in isolated lungs, inhaled NO2 (1) undergoes rapid uptake and transformation in sites accessible to the pulmonary circulation; (2) does not form stable addition products with lipids; and (3) forms small-molecular-weight soluble reaction product(s) that behave similarly to nitrite, most likely indicating predominant univalent reduction of NO2 via initial hydrogen abstraction and subsequent HNO2 dissociation.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Nitrogênio/metabolismo , Administração por Inalação , Animais , Radicais Livres , Metabolismo dos Lipídeos , Masculino , Nitritos/metabolismo , Oxirredução , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Nitrogen dioxide (NO2) is an environmental oxidant that causes acute lung injury. Absorption of this aqueous insoluble gas into the epithelial lining fluid (ELF) that covers air space surfaces is, in part, governed by reactions with ELF constituents. Consequently, NO2 absorption is coupled to its chemical elimination and the formation of ELF-derived products. To investigate mechanisms of acute epithelial injury, we developed a model encompassing the spatial arrangements of the lung surface wherein oxidation of cell membranes immobilized below a chemically defined aqueous compartment was assessed after NO2 exposures. Because aqueous-phase unsaturated fatty acids displayed minimal NO2 absorptive activity, these studies focused on glutathione (GSH) and ascorbic acid (AH2) as the primary NO2 absorption substrates. Results demonstrated that membrane oxidation required both gasphase NO2 and aqueous-phase GSH and/or AH2. Membrane oxidation was antioxidant concentration and exposure duration dependent. Furthermore, studies indicated that GSH- and AH2-mediated NO2 absorption lead to the production of the reactive oxygen species (ROS) O-2. and H2O2 but not to .OH and that Fe-O2 complexes likely served as the initiating oxidant. Similar results were also observed in combined systems (GSH + AH2) and in isolated rat ELF. These results suggest that the exposure-induced prooxidant activities of ELF antioxidants generate extracellular ROS that likely contribute to NO2-induced cellular injury.
Assuntos
Antioxidantes/metabolismo , Pulmão/fisiologia , Dióxido de Nitrogênio/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Ácido Ascórbico/metabolismo , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Eritrócitos/fisiologia , Glutationa/metabolismo , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Modelos Biológicos , Dióxido de Nitrogênio/farmacocinética , Fosfatidilcolinas , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
An isolated rat lung preparation was ventilated with NO2 while being perfused with a medium containing morpholine. After 60 min of ventilation-perfusion, N-nitrosomorpholine was detected in both lung tissue and perfusate.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacologia , Nitrosaminas/metabolismo , Animais , Pulmão/efeitos dos fármacos , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
The fate of inhaled NO2 was studied with isolated perfused rat lungs. The isolated lungs were exposed to 5 ppm NO2 for 90 min at a ventilation rate of 34 ml/min. The NO2 exposure had no adverse effects on the lungs as judged from their weights, glucose uptake, or lactate production compared to control lungs. Isolated lungs absorbed 36% of ventilated NO2, which was detected in perfusate and lung tissue as NO2- but not NO3-. The NO2- concentration in perfusate increased linearly with time, and after 90 min of ventilation with NO2 and perfusion with erythrocyte-free medium the NO2- accumulation was 6.36 +/- 0.39 micrograms. If perfusate contained 10% erythrocytes, the ventilated NO2 product was mostly NO3- in perfusate but NO2- in lung tissue. Protein solutions absorbed NO2 more effectively than simple salt solutions, but they all yielded mainly NO2- unless erythrocytes were present, when the product was mostly NO3-. The results indicate that absorbed NO2 in the lung is converted predominantly to NO2-, but after its diffusion into the vascular space it is oxidized to NO3- by interactions with erythrocytes.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/metabolismo , Absorção , Animais , Eritrócitos/metabolismo , Glucose/metabolismo , Técnicas In Vitro , Lactatos/metabolismo , Pulmão/patologia , Masculino , Nitratos/metabolismo , Nitritos/metabolismo , Perfusão , RatosRESUMO
Isolated rat lung (IPL) studies have suggested that the pulmonary uptake of inhaled nitrogen dioxide (NO2) is governed via a chemical reaction-dependent process which results in NO2-derived reaction products diffusing into the vascular space. Experimental results indicated that substantial proportions of this reactive absorption occur in distal sites. However, gas phase deposition in proximal locations cannot be ruled out due to the lack of information on bronchial perfusion in rat IPL preparations. Consequently, we evaluated the presence of pulmonary-to-bronchial anastomotic perfusate flow in control and NO2-exposed (10.3 ppm) rat IPL. Monastral blue (MB) was used as a vascular marker and was infused into the pulmonary artery catheter either for recirculation at time zero or as an end-experiment (60 min) bolus. In addition, MB was infused into control in situ preparations to observe intact bronchial circulations. Lungs were prepared for routine evaluation by light microscopy. In situ MB was observed in all pulmonary and bronchial vessels. In IPL, MB was observed only in far terminal airway-associated vessels. No differences were observed in MB distribution between bolus (end-experiment) and recirculated (time zero) applications. NO2 exposure produced no effect on MB distribution. We conclude that in rat IPL: (1) negligible anastomotic flow occurs from the pulmonary into the bronchial circulation, (2) nonedemagenic NO2 exposures do not alter existing perfusate distribution, and (3) the perfusate appearance of inhalation-derived species results from gas phase deposition only in distal sites which have ready accessibility to the pulmonary circulation.
Assuntos
Brônquios/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Óxidos de Nitrogênio/toxicidade , Circulação Pulmonar/efeitos dos fármacos , Animais , Brônquios/irrigação sanguínea , Técnicas Histológicas , Técnicas In Vitro , Pulmão/irrigação sanguínea , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Absorption of inhaled NO2 across the pulmonary gas/tissue interface is principally governed by chemical reaction(s) rather than by physical solubility. While the kinetics of NO2 transfer into reactant-containing aqueous solutions appear to be bulk phase independent, it is unclear whether unreacted NO2 diffuses appreciably through the epithelial lining fluid (ELF) to cellular compartments. We avoided the difficulties associated with directly quantifying NO2 dissolved in biological fluids by indirectly determining the potential for NO2 penetration to underlying tissues. An in vitro system was developed which horizontally suspended a wettable, gas permeable, fibrous material between two gas chambers. Aqueous substrates were applied to the sieve material and NO2 (10.9 ppm) was introduced into one chamber and sampled for in the other. O2 served as a tracer gas. We determined the influence of ELF, a model biochemical (reduced glutathione; GSH), and PO4 buffer (control) on NO2 transfer as evaluated by "breakthrough time." (A) Both O2 and NO2 rapidly diffused through the sieve material when dry. Under PO4 wetted conditions, O2 continued to penetrate rapidly but NO2 transfer was slightly inhibited relative to O2. (B) Addition of GSH (1 mM) significantly prolonged NO2 breakthrough time. Increasing initial [GSH] resulted in concomitant prolongation of NO2 breakthrough time. (C) We observed a direct correlation between oxidation of sieve GSH and NO2 breakthrough. (D) Freshly harvested rat ELF inhibited NO2 transfer in a concentration-dependent manner similar to GSH. These data suggest that in the presence of reactant solutes, unreacted NO2 does not penetrate through the ELF layer. Reactive absorption must, therefore, occur primarily within the ELF compartment so that reaction products which induce subsequent toxicity are generated as a result of the initial uptake interactions.
Assuntos
Pulmão/metabolismo , Dióxido de Nitrogênio/farmacocinética , Animais , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Epitélio/metabolismo , Filtração , Masculino , Dióxido de Nitrogênio/análise , Permeabilidade , Ratos , Ratos EndogâmicosRESUMO
Using an isolated rat lung model, we investigated the characteristics of pulmonary O3 absorption, including the contributory role of chemical reaction vs physical solubility. Due to the physicochemical similarities between O3 and NO2, we utilized investigational strategies analogous to those previously employed to characterize NO2 absorption kinetics. The effects of vascular perfusion, temperature, inspired concentration ([O3]i), surface area, and minute ventilation (tidal volume (Vt) times ventilation frequency (f)) on air space O3 clearance during quasi-steady-state exposures were investigated using fractional uptakes (%U) and reactive uptake coefficients (k') as endpoints. We found the following: (1) At 1 ppm [O3]i (37 degrees C), %U (95 +/- 5%) was perfusion independent (60 min). (2) %U displayed temperature dependence (r = 0.99). Activation energies (Ea) and Q10 were computed from Arrhenius plots (ln k' vs 1/T; r = -0.99). For 1 ppm (11-37 degrees C), Ea = 4140 kcal/g.mol and Q10 = 1.23. (3) Absorption demonstrated [O3]i dependence. At 25 degrees C, < or = 1 ppm displayed %U = 86 +/- 4% with k' = 234 ml/min. Exposures > 1 ppm resulted in decreasing %U and k' (5 ppm %U = 60 +/- 3% and k' = 121 ml/min). (4) To evaluate epithelial damage, lactate dehydrogenase (LDH) activity was quantified in cell-free bronchoalveolar lavage fluid. For exposures < or = 1 ppm LDH equaled control, while for exposures > 1 ppm LDH steadily increased to a four-fold maximum at 5 ppm. (5) O3 uptake was independent of functional residual capacity-induced changes in air space surface area. (6) Absorption was proportional to Vt (r = 0.99) and displayed notable ventilation frequency-dependent decline above 70 breaths per minute. Based on the perfusion independence, temperature dependence, and the Ea and Q10, we conclude that O3 absorption in isolated lungs involves a reactive component. While k' remained stable from 0 to 1 ppm O3, at concentrations above 1 ppm other contributory factors such as O3/substrate reaction kinetics, epithelial damage, and solute O3 backpressure may affect the overall net absorption rate. In addition, the data suggest that O3 uptake may be principally localized to the conducting airways.
Assuntos
Pulmão/metabolismo , Ozônio/farmacocinética , Absorção , Administração por Inalação , Animais , Epitélio/efeitos dos fármacos , Técnicas In Vitro , Cinética , Pulmão/efeitos dos fármacos , Masculino , Ozônio/administração & dosagem , Ozônio/toxicidade , Ratos , Ratos Sprague-Dawley , Respiração , TemperaturaRESUMO
Previous studies have suggested that the rate of inhaled O3 absorption from the intrapulmonary gas phase is principally mediated by reaction-dependent mechanisms rather than by physical solubility, tissue diffusion, or blood flow (Postlethwait et al., 1994, Toxicol. Appl. Pharmacol. 125, 77-89). The initial site of interaction between O3 and the lung surface occurs at the gas/liquid interface of the epithelial lining fluid (ELF). Therefore, we investigated (a) whether reactive uptake by ELF constituents could account for pulmonary uptake and (b) whether selected constituents acted as O3-specific absorption targets. Rat ELF was harvested by bronchoalveolar lavage. By injecting the same lavage fluid a second [(BALF)2] and/or third [(BALF)3] time into fresh lungs, a more concentrated form of ELF was obtained. Controlled quasi-steady-state exposures (O3 in air; 30-min duration) of cell-free BALF and model substrates (reduced glutathione, GSH) were utilized. Results were based on temperature-specific fractional and normalized uptake rates (r). We observed the following: (1) Buffer pH substantially influenced O3 absorption by GSH but by BALF only modestly. (2) Uptake displayed significant [BALF] and [GSH] dependence. (3) Fractional uptake decreased (BALF and GSH) with increasing [O3] although absolute uptake increased. (4) Absorption demonstrated temperature dependence. Arrhenius plots [ln(r) vs 1/T] were used to compute activation energies (Ea) and Q10. (BALF)1 Ea = 3387 cal/g mol with Q10 = 1.20. GSH (1 mM) Ea = 2240 with Q10 = 1.13. (5) Increasing flow reduced fractional uptake in a nonlinear fashion. (6) Dialysis (1000-molecular-weight cutoff) reduced uptake by (BALF)1 moderately (-30%). Sulfhydryl depletion produced minimal effect (-10%), while ascorbate depletion (-37%) and combined sulfhydryl and ascorbate depletion (-39%) were the most effective. Treatments produced lesser effects on (BALF)3. We conclude that the pH, aqueous substrate, and temperature-dependence and the Ea and Q10 are consistent with reaction-dependent O3 uptake by ELF. The analogous absorption characteristic between the ELF and intact lung (temperature, [O3], contact time) suggests that the ELF represents the primary site for O3-reactive absorption. Reduced sulfhydryls do not appear to substantially interact with inhaled O3. Principal absorption targets may include ascorbate, phospholipids, and other moderate to large molecular weight constituents.
Assuntos
Pulmão/metabolismo , Ozônio/farmacocinética , Absorção/efeitos dos fármacos , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/química , Epitélio/metabolismo , Pulmão/efeitos dos fármacos , Masculino , Ozônio/administração & dosagem , Ozônio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The influence of age on O3 effects in the lung was studied in 8 groups of Sprague-Dawley rats: 7, 12, and 18 d of age (neonatal); 24, 30, and 45 d of age (infant); and 60 and 90 d of age (adult). Lung weight, total lung protein and DNA contents, and a series of marker enzyme activities in lung tissue were determined. After exposure of rats from each group to 0.8 ppm (1568 microgram/m3) O3 continuously for 3 d, a biphasic effect was noted. The biochemical parameters, expressed per lung, in O3-exposed rats relative to their corresponding controls decreased in the 7- and 12-d-old groups, increased or remained unchanged in the 18-d-old group, and increased in the 24- to 90-d-old groups. However, the increases were much greater for 60- to 90-d-old rats than for 24- to 30-d-old rats. The increase in lung biochemical parameters is thought to occur in response to lung injury and subsequent repair processes, and greater increases in the lungs of older rats suggest that they are more responsive to O3 exposure than younger rats. The decrease in lung biochemical parameters and increased mortality in 7- and 24-d-old neonatal rats suggest that they are more susceptible to O3 stress than infant and adult rats.
Assuntos
Envelhecimento , Pulmão/efeitos dos fármacos , Ozônio/farmacologia , Animais , Animais Recém-Nascidos , Masculino , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Changes in O2 tension such as those associated with hypoxic ischemia or hyperoxia may potentially modulate purine nucleotide turnover and production of associated catabolites. We used an isolated perfused rat lung preparation to evaluate the effect of O2 tension on pulmonary uric acid production. Three O2 concentrations (21%, normoxia; 95%, hyperoxia; 0%, hypoxia) were utilized for both pulmonary ventilation and equilibration of recirculating perfusate. All gas mixtures contained 5% CO2 and were balanced with N2. We used Certified Virus Free Sprague-Dawley male rats weighting 250-300 g, four to five rats in each exposure regimen. After a 10-min equilibration period, we measured uric acid levels at 0 and 60 min in lung perfusate and at 60 min in lung tissue. After 60 min of ventilation/perfusion, we observed significant uric acid accumulation in both lung tissue (25-60%) and perfusate (8- to 10-fold) for all three O2 regimens. However, hypoxia produced substantially greater net uric acid concentrations (net = the difference between zero and 60 min) than either normoxia or hyperoxia (1.5-fold in lung tissue, and 2-fold in perfusate, respectively). The data suggest that pulmonary hypoxia results in greater purine catabolism leading to increased uric acid production. Vascular space uric acid, as measured in the recirculating perfusate, was proportional to lung weight changes (r = 0.99) with hypoxia exhibiting the greatest values, possibly reflecting a linkage between tissue perturbation and uric acid release. Thus, measurement of uric acid may serve as a useful marker of adenine nucleotide turnover and lung injury.