Alveolar Epithelial Cell Damage Research Articles

NNOS and Nox4 go nuclear: nNOS-derived and NADPH oxidase-derived reactive oxygen/nitrogen species promote oxidative nuclear damage in alveolar epithelial cells

nNOS and Nox4 go nuclear: nNOS-derived and NADPH oxidase-derived reactive oxygen/nitrogen species promote oxidative nuclear damage in alveolar epithelial cells

Hypoxia upregulates hypoxia inducible factor (HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

The role of the hypoxia-inducible factor (HIF) subunits 1alpha and 2alpha in response to hypoxia is well established in lung epithelial cells, whereas little is known about HIF-3alpha with respect to transcriptional and translational regulation by hypoxia. HIF-3alpha and HIF-1alpha are two similar but distinct basic helix-loop-helix-PAS proteins, which have been postulated to activate hypoxia responsive genes in response to hypoxia. Here, we used quantitative real time RT-PCR and immunoblotting to determine the activation of HIF-3alpha vs. HIF-1alpha by hypoxia. HIF-3alpha was strongly induced by hypoxia (1% O2) both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation, whereas HIF-1alpha protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stability in A549 cells, as measured on mRNA and protein levels. Interestingly, HIF-3alpha and HIF-1alpha exhibited strikingly similar responses to a variety of activating or inhibitory pharmacological agents. These results demonstrate that HIF-3alpha is expressed abundantly in lung epithelial cells, and that the transcriptional induction of HIF-3alpha plays an important role in the response to hypoxia in vitro. Our findings suggest that HIF-3alpha, as a member of the HIF system, is complementary rather than redundant to HIF-1alpha induction in protection against hypoxic damage in alveolar epithelial cells.

Nitric oxide production by pulmonary leukocytes from induced sputum in patients with asthma and its effect on epithelial cell viability

Nitric oxide (NO) is one of many factors potentially involved in lung remodeling in asthma. The aim of the study was to assess the effect of pulmonary leukocytes from patients with bronchial asthma on alveolar epithelial cell damage in relation to NO production. Induced sputum samples were obtained from 25 patients with bronchial asthma and 10 healthy volunteers. Twelve asthmatics were on inhaled corticosteroid treatment and 13 were corticosteroid free. Type II-like alveolar epithelial (A549) cells were cultured for 48 h in the presence of cell-free media from a 24-h culture of leukocytes obtained from the induced sputa (IS-Su). The level of NO was measured in supernatants from the cell cultures and the viability of the A549 cells was established. The levels of NO in IS-Su from corticosteroid-free asthmatics were significantly higher (p = 0.001) than those in IS-Su from healthy controls. Furthermore, NO production by A549 cells exposed to IS-Su from steroid-free asthmatics (group A) was significantly higher than that from asthmatics on corticosteroid therapy (group cA) as well as from healthy controls (p = 0.01 and p = 0.001, respectively). Lower viability of the epithelial cells exposed to IS-Su was observed in group A compared with controls (median: 72% vs. 97.5%; p < 0.001). In addition, a negative correlation (R(S) = - 0.706, p < 0.001) was found between the levels of NO produced by pulmonary leukocytes and the viability of epithelial cells. The results suggest that in the course of asthma, pulmonary leukocytes may interact with alveolar epithelial cells by inducing an excessive production of NO which, in turn, may contribute to epithelium impairment.

Elastolytic activity and alveolar epithelial type-1 cell damage after chronic LPS inhalation: Effects of dexamethasone and rolipram

This study investigated whether a correlation between leukocyte-derived elastolytic activity, alveolar epithelial type-1 cell damage, and leukocyte infiltration of the airways existed in guinea-pigs chronically exposed to inhaled lipopolysaccharide (LPS). The airway pathology of this model, notably the neutrophilia, resembles chronic obstructive pulmonary disease (COPD). The effect of the corticosteroid, dexamethasone, or the phosphodiesterase-4 (PDE4)-inhibitor, rolipram, on these features was studied. Conscious guinea-pigs were exposed for 1 h to single or repeated (nine) doses of LPS (30 μg ml −1). Dexamethasone (20 mg kg −1, ip) or rolipram (1 mg kg −1, ip) was administered 24 and 0.5 h before the first exposure and daily thereafter. Bronchoalveolar lavage fluid (BALF) was removed and elastolytic activity determined as the elastase-like release of Congo Red from impregnated elastin. The presence of the specific epithelial cell type-1 protein (40–42 kDa) RT1 40 in BALF was identified by Western blotting using a rat monoclonal antibody and semi-quantified by dot-blot analysis. The antibody was found to identify guinea-pig RT1 40. BALF inflammatory cells, particularly neutrophils and macrophages, and elastolytic activity were increased in chronic LPS-exposed guinea-pigs, the latter by 90%. Chronic LPS exposure also increased (10.5-fold) RT1 40 levels, indicating significant alveolar epithelial type-1 cell damage. Dexamethasone or rolipram treatment reduced the influx of inflammatory cells, the elastolytic activity (by 40% and 38%, respectively), and RT1 40 levels (by 50% and 57%, respectively). In conclusion, chronic LPS-exposed guinea-pigs, like COPD, exhibit elastolytic lung damage. This was prevented by a PDE4 inhibitor and supports their development for suppressing this leukocyte-mediated pathology.

Endothelial Cell Injury of Carmustine (BCNU) in Hepatic Sinusoids: Contribution of Oxidation and Immune Reaction.

Background: Carmustine (BCNU) is used for the treatment of brain tumors and is included in conditioning regimens of stem cell transplantation. It has been long believed that BCNU causes hepatotoxicity by inducing pericholangitis and intrahepatic cholestasis leading to biliary cirrhosis. Either hepatocytes or biliary ductal cells have been thought to be the target in BCNU-induced liver toxicity. However, the mechanisms operational at cell level have not been well elucidated. Possible explanations include glutathione depletion in cells, DNA damage accompanied by cellular oxidative stress and/or acceleration of apoptosis. It has been shown that BCNU treatment inhibits hepatic glutathione reductase activity in rats. Recently, a two-step process involving free radical formation and immune reaction has been proposed as a mechanism of BCNU-induced alveolar epithelial cell damage by our group (Savasan S, Ozdemir O, Buck S, et al., 95th annual meeting of the AACR, Orlando, March 27–31, 2004. Abstract#: 1133, 2004). In this study, we have investigated BCNU effect on human liver sinusoid endothelial cells (HLS-EC) in vitro.Material and Methods: Cytotoxicity in HLS-EC was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6(3) /PI staining, further quantification of fluorosphere-adjusted events and trypan blue exclusion test in repeated experiments. The generation of reactive oxygen species (ROS) was measured by flow cytometric DCF staining. Lymphokine activated killer (LAK) cell-mediated cytotoxicity against BCNU-treated and untreated HLS-EC was measured by flow cytometric cell mediated cytotoxicity assay. The influence ROS-scavenging compound butylated hydroxyanisole (BHA) and anti-inflammatory dexamethasone (DXM) on BCNU treatment was also studied.Results: Severe HLS-EC damage occurred following BCNU treatment shown by four different cytotoxicity methods. This effect was associated with oxidative stress demonstrated by significant increase in DCF staining. Although pre-treatment of cells with DXM did not affect BCNU cytotoxicity, BHA suppressed BCNU cytotoxicity by 25% (p < 0.05). BCNU treatment also resulted in increased LAK cell-mediated elimination of HLS-EC (65%) compared to untreated cells (16%). Similar to its effect on direct BCNU cytotoxicity, BHA prevented BCNU treated HLS-EC killing by LAK cells by 25%. However, DXM did not influence LAK cell-mediated killing of BCNU-treated HLS-EC cells. These results suggest that both ROS generation and immune reaction against perturbed endothelial cells play a central role in BCNU-associated HLS-EC injury. In contrary to our findings on human alveolar epithelial cells, BHA ameliorated BCNU-induced direct and immune-mediated cytotoxicity on HLS-EC, but not DXM.Conclusion: Reactive oxygen species scavenging may be useful in prevention of liver toxicity of BCNU.

Neutrophil Elastase Induces IL-8 Synthesis by Lung Epithelial Cells via the Mitogen-Activated Protein Kinase Pathway

The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01–0.5 µg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 µg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-ĸB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-ĸB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-ĸB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-ĸB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.

Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway.

The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01-0.5 microg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 microg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kappaB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kappaB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.

KL-6 levels are elevated in plasma from patients with acute respiratory distress syndrome

The acute respiratory distress syndrome (ARDS) is an extreme form of lung injury characterised by disruption to the alveolar epithelium. KL-6 is a mucin-like glycoprotein expressed on type II pneumocytes. Circulating levels of KL-6 have diagnostic and prognostic significance in a number of interstitial lung diseases, and when elevated are thought to indicate disruption of the alveolar epithelial lining. In this study, the authors sought to determine whether plasma KL-6 levels were elevated in patients with ARDS and whether these were associated with aetiology, disease severity, outcome or ventilatory strategy. Plasma samples were collected from 28 patients with ARDS, nine ventilated controls of matched illness severity and 10 healthy individuals. KL-6 concentrations were measured by enzyme-linked immunosorbent assay. Patients with ARDS had higher plasma levels of KL-6 (median 537 U x mL(-1), interquartile range (IQR) 383-1,119), as compared to ventilated controls (median 255 U x mL(-1), IQR 83-338) and normal individuals (median 215 U x mL(-1), IQR 149-307). In patients with ARDS, plasma KL-6 levels were higher in nonsurvivors than survivors, and correlated positively with oxygenation index and negatively with arterial oxygen tension:inspiratory oxygen fraction ratio. There were also significant positive correlations with mean and peak airway pressures. Elevated levels of plasma KL-6 may provide a useful marker for acute respiratory distress syndrome in ventilated patients and have possible prognostic significance. Alveolar epithelial cell damage may be influenced by the nature of mechanical ventilatory support.

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.

Clara-Cell Hyperplasia After Quartz and Coal-Dust Instillation in Rat Lung

Bronchiolo-alveolar hyperplasia of type II cells in rat lungs after particle exposure is a well-known preneoplastic lesion. The Clara cell, stem cell of the bronchiolar epithelium and the main carrier of cytochrome P-450 isoenzyme system in the lung, has barely been evaluated with regard to this effect. The aim of this study was to examine Clara-cell hyperplasia after particle exposure and to characterize cell proliferation and its normal function. Female Wistar rats were intratracheally instilled with coal dust samples of variable quartz content, quartz (DQ12), titanium dioxide, or saline solution containing 0.5% Tween 80. After 126-129 wk, all coal mine dust- and quartz-exposed animals developed Clara-cell hyperplasia: up to 0.48% of the total lung area, which was significantly increased compared to titanium dioxide (p < .05) and control (p < .03) animals. Proliferation and hyperplasia of bronchiolar Clara cells by coal dusts was independent of their quartz content. The lack of proliferating cell nuclear antigen staining in most of the hyperplastic Clara cells suggests that following damage of alveolar epithelial cells, Clara cells migrate in and remodulate the alveolar epithelium. After the migration they keep their function in the xenobiotic metabolism, as shown by expansion of CYP2E1 active Clara cells. The minor development of Clara-cell hyperplasia in titanium dioxide-treated rats indicates that this is not a general particle effect, and is possibly due to its lower toxicity to epithelial cells.

Neutrophils cause oxidative DNA damage in alveolar epithelial cells

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2′deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H 2O 2, cause oxidative DNA damage in epithelial cells.

The roles of diesel exhaust particle extracts and the promotive effects of NO2 and/or SO2 exposure on rat lung tumorigenesis

This experiment was carried out to clarify the roles of diesel exhaust particle (DEP) extracts and the promotive effects of nitrogen dioxide (NO2) and/or sulfur dioxide (SO2) exposure on rat lung tumorigenesis. F344 male rats were intratracheally administered DEP extract-coated carbon black particles (DEcCBP) and exposed to 6 ppm NO2 and/or 4 ppm SO2 for 10 months. At 18 months after starting the experiment, lung lesions were histopathologically investigated and DNA in rat lungs was analyzed for the presence of adducts using the 32P-postlabeling assay. Infiltration of alveolar macrophages, which was significant in the lungs of rats administered carbon black particles, was not prominent in those administered DEcCBP. DEcCBP occasionally formed small hyaline masses in the alveolar ducts and alveolar bronchiolization developed in the epithelium of alveolar ducts near the masses. Lung tumorigenesis and DNA aduct formation were observed in the animals administered DEcCBP with exposure to NO2 and/or SO2, but not in those administered DEcCBP alone. The results of the present study suggested that DEP extracts eluting from the small masses cause DNA damage in alveolar epithelial cells and alveolar epithelial cell proliferation, and that NO2 and/or SO2 exposure promote lung tumor induction by DEP extracts.

The involvement of Fas-Fas ligand pathway in fibrosing lung diseases.

Pulmonary fibrosis begins with alveolitis, which progresses to destruction of lung tissue and excess collagen deposition. This process could be the result of DNA damage and a form of apoptosis. Therefore, we hypothesized that Fas ligand (FasL), which induces apoptosis in cells expressing Fas antigen (Fas), is associated with pulmonary fibrosis. We examined frozen lung tissues from seven patients with idiopathic pulmonary fibrosis (IPF), and bronchoalveolar lavage fluid (BALF) cells from 19 patients with IPF and from 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP). We used five frozen lungs with normal lung parenchyma and BALF cells from 10 patients with solitary pulmonary nodule as controls. Reverse transcription-polymerase chain reaction (RT-PCR) showed that FasL messenger RNA (mRNA) was expressed in BALF cells from all patients with IPF and from 15 of 16 patients with CVD-IP. FasL mRNA was not detected in BALF cells except in one of 10 controls. RT in situ PCR detected FasL mRNA in inflammatory cells in BALF from patients with IPF. Immunohistochemistry detected FasL protein in infiltrating lymphocytes and granulocytes in all of seven frozen lung tissues of IPF, but in none of five control lung tissues. Additionally, the expression of Fas appeared to be upregulated in bronchiolar and alveolar epithelial cells in IPF compared with normal lung parenchyma by immunohistochemistry. We conclude that Fas and FasL were upregulated in fibrosing lung diseases and may associate with DNA damage or apoptosis of bronchiolar and alveolar epithelial cells in this disorder.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells.

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Molecular Mechanisms in Acute Lung Injury

This chapter explores the experience in the use of lung inflammatory models, which have provided information on the role of various inflammatory mediators and their sequence of engagement. This information has provided insights into potential interventional approaches for the suppression of inflammatory processes in humans. The use of animal models of lung inflammation has allowed for the probing of pathways of inflammation responsible for injury. Although the models do not necessarily extrapolate to lung inflammatory diseases in humans, they have permitted a detailed dissection of events that ultimately lead to damage of interstitial capillary endothelial cells and alveolar epithelial cells. In this chapter, models of inflammatory injury in rat lung are discussed: injury occurring after systemic activation of complement and injury developing after alveolar deposition of either IgG or IgA immune complexes. In each model, there are similarities but also distinctive differences that have provided insights into diverse pathways by which the inflammatory response causes tissue injury. This chapter also demonstrates how the in vitro use of endothelial cell monolayers has provided an approach to understanding the mechanisms by which activated neutrophils injure endothelial cells

Effect of ICRF-187 on the pulmonary damage induced by hyperoxia in the rat

Histological and ultrastructural studies were made of the lungs of rats that were exposed to 100% oxygen of 60 h and were treated with either normal saline or with ICRF-187, a bis-diketopiperazine derivative of EDTA that has the capacity to chelate iron. This metal is thought to be needed to catalyze the formation of toxic oxygen free radicals. ICRF-187 (20 mg/kg) was given intraperitoneally at approximately 12 h intervals (5 doses) during the 60 h exposure. Seven of the ten saline-treated rats exposed to oxygen died prior to the end of the study whereas only one of the 10 rats in the ICRF-187-treated group died. This difference in mortality is found to be statistically significant ( P < 0.05). All saline-treated rats showed light and electron microscopic evidence of pulmonary damage. ICRF-187 attenuated the morphologic alterations observed by light microscopy (intra-alveolar edema, inflammatory exudates and bronchiolar epithelial cell swelling and hyperplasia; P < 0.05). In addition, electron microscopic evaluation revealed that capillary thrombi, endothelial cell alterations and alveolar epithelial cell damage also were less severe in ICRF-187-treated rats. It is concluded that ICRF-187 may provide a new and useful approach for the prevention of hyperoxia-induced pulmonary damage.

Bioactivation of 3-methylindole by isolated rabbit lung cells

3-Methylindole (3MI) is a pneumotoxin that causes selective lung lesions indicative of Clara cell and alveolar epithelial cell damage in ruminants and rodents. The present study examined the cytotoxicity of 3MI to isolated rabbit Clara cells, type II alveolar epithelial cells, and alveolar macrophages. 3MI produced a dose-dependent cytotoxicity to Clara cells detectable within 1 hr of incubation at 37°C which reached a maximum at 3 hr. Concentrations of 0.25 and 0.5 m m 3MI were cytotoxic to Clara cells, while type II and alveolar macrophages required 1 m m 3 MI before cytotoxicity was observed. The cytochrome P450 suicide substrate inhibitor, 1-aminobenzotriazole, inhibited 3MI-induced cytotoxicity in Clara cells, type II cells, and alveolar macrophages. These observations were consistant with a cytochrome P450-mediated bioactivation of 3MI to a toxic intermediate. Studies with a trideuteromethyl analog of 3MI demonstrated a much reduced cytotoxicity to Clara cells as well as to type II cells, and macrophages. The deuterium isotope effect suggested that CH bond breakage at the 3-methyl group is a requisite oxidative transformation in the bioactivation of 3MI to a selective lung cell cytotoxin. The selectivity of cellular cytotoxicity is probably associated with higher rates of bioactivation by Clara cell cytochrome P450 monooxygenases compared to those of type II cells and macrophages. These studies demonstrate that 3MI is bioactivated in isolated pulmonary cells without the intervention of other organs and that bioactivation requires functional cytochrome P450 enzymes.

Potentiation of the cell specific toxicity of paraquat by 1,3-BIS(2-chloroethyl)-1-nitrosourea (BCNU): Implications for the heterogeneous distribution of glutathione (GSH) in rat lung

In order to study oxidative stress in the lung, we have developed a rat lung slice model with compromised oxidative defences. Lung slices with markedly inhibited glutathione reductase activity (∼80% inhibition) were prepared by incubating slices, with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 μM) in an amino acid-rich medium for 45 min at 37°. These lung slices had similar levels of GSH and ATP and polyamine uptake (a marker of alveolar epithelial type I and II cell function) to control rat lung slices. We have utilized these BCNU pretreated slices to study the effects of the herbicide, paraquat, in comparison to those of 2,3-dimethoxy-1,4-naphthoquinone, a potent redox cycler. Paraquat (10–100 μM) caused only minimal changes in the levels of GSH or ATP in control or compromised slices. In contrast, 2,3-dimethoxy-1,4-naphthoquinone caused a decrease in GSH in control slices but a markedly enhanced decrease in both GSH and ATP in compromised slices. Both compounds had only limited effects on putrescine and spermidine uptake in control slices. However, they caused a marked inhibition in compromised slices. Paraquat had little effect on 5-hydroxytryptamine uptake (a marker of endothelial cell function) in either control or compromised slices whereas the quinone inhibited uptake in the compromised slices. Thus, the lack of effect of paraquat on GSH and ATP does not support the involvement of oxidative stress in its toxicity. In contrast, using polyamine uptake, as a functional marker of alveolar epithelial cell damage, suggests a role for redox cycling. As paraquat is known to be accumulated primarily in alveolar type I and II cells (a small fraction of the lung cell population), our data suggest that only a small proportion of pulmonary GSH and ATP is present in alveolar epithelial type I and II cells but that much larger amounts may be present in endothelial cells. These studies highlight the problem of gross tissue measurements in heterogeneous tissues such as the lung.

Experimental pulmonary fibrosis induced by paraquat plus oxygen in rats: A morphologic and biochemical sequential study

Changes in lung structure and collagen metabolism were studied at 1, 2, 3, 4, 6, and 8 weeks in a model of pulmonary fibrosis induced in rats with paraquat plus hyperoxia. Morphologic examination of the lungs revealed that the earliest lesions consisted of severe and irreversible endothelial and alveolar epithelial cell damage. Afterward, an inflammatory process took place, initially dominated by polymorphonuclear leukocytes and then by mononuclear cells, but with the constant presence of granulocytes. From the fourth week on there were fibroblast proliferation and a moderate increase of mast cells. In the early stages alveolitis was focal, but from the second week the lungs were diffusely affected with severe distortion of the architecture. Collagen content was moderately increased in the first 2 weeks and then showed a progressive increment until the end of the experiment. Collagen synthesis was significantly elevated from the fourth week, coinciding with interstitial fibroblast proliferation, although there were some animals that showed increased collagen production from the first week. Collagenolytic activity occurred in 3 stages: at 2 weeks there was increased collagen degradation, at 3, 4, and 6 weeks the values showed a trimodal behavior, and at 8 weeks almost all experimental rats presented an important decrease of collagenolysis. Thus, the development of lung fibrosis was associated first with increased rates of collagen synthesis and later with a decrease of collagen degradation.

Neutrophil-derived epoxide, 9,10-epoxy-12-octadecenoate, induces pulmonary edema.

We have observed that neutrophils biosynthesize linoleate epoxide, 9,10-epoxy-12-octadecenoate, and have named it leukotoxin because of its cytotoxic effect. In this experiment, the effect of leukotoxin on the lung was investigated. Acute effect of leukotoxin: Using Wistar rats, leukotoxin (100 mumol/kg) was injected intravenously for the leukotoxin group, and linoleate (100 mumol/kg) for the linoleate group. Physiological saline was injected as the control. Ten min after injection, rats were divided into 3 groups: (1) lungs were isolated, and lung wet weight, and dry weight were measured; (2) lung lavages were performed, and albumin concentration and activity of angiotensin converting enzyme (ACE) were measured; (3) morphological changes were studied by light and electron microscope. After administration of leukotoxin, lung wet weight/body weight ratios and dry weight/wet weight ratios were increased. Albumin concentration and ACE activity in lung lavages were also increased. Pulmonary edema was also confirmed by light microscopic findings. Alveolar epithelial cell damage and endothelium damage were also observed. Linoleate had no significant effect on these biochemical parameters and morphological findings. Subacute effect of leukotoxin: Twelve hr after administration of leukotoxin (50 mumol/kg) or linoleate (50 mumol/kg), the same studies were performed as in the acute experiments. Immediately after administration of leukotoxin, no significant effect was observed. However, 12 hr later similar changes were observed as in the acute experiments. Linoleate did not show any significant effect 12 hr after injection. These results indicate that leukotoxin biosynthesized by neutrophils might be closely related to the genesis of inflammatory edema.