Pentoxifylline inhibits intercellular adhesion molecule-1 (ICAM-1) and lung injury in experimental phosgene-exposure rats.

Phosgene inhalation results in acute lung injury (ALI) mostly, pulmonary edema and even acute respiratory distress syndrome, but there is no specific antidote. Inflammatory cells play an important role in the ALI caused by phosgene. Intercellular adhesion molecule-1 (ICAM-1) is a critical factor for inflammatory organ injury. We hypothesized that pentoxifylline (PTX), an inhibitor of leukocyte activation, would have a protective effect on experimental phosgene-induced lung injury rats by inhibiting ICAM-1. To prove this hypothesis, we used rat models of phosgene (400 ppm x 1 min)-induced injury to investigate: (1) the time course of lung injury (control 1, 3, 6, 12, 24, and 48 h group), including pathological changes in hematoxylin and eosin staining and transmission electron microscope, myeloperoxidase (MPO) activity by colorimetric method and ICAM-1 protein level detected by western blot, (2) At 3 h after phosgene exposure, protective effects of different dosages of PTX (50 mg/kg and 100 mg/kg) administration were evaluated by MPO activity, ICAM-1 differential expression and WBC count in bronchoalveolar lavage fluid. The results showed that inflammatory cells emerged out of lung blood vessels at 3 h after phosgene exposure. The MPO activity of lung tissue increased significantly from 3 to 48 h after phosgene exposure (P < 0.05) and ICAM-1 expression presented a similar change, especially at 3 h and 24 h (P < 0.05). After pretreatment and treatment with PTX (100 mg/kg), significant protective effects were shown (P < 0.05). These data supported our hypothesis that PTX reduced phosgene-induced lung injury, possibly by inhibiting ICAM-1 differential expression.

Genomic analysis of murine pulmonary tissue following carbonyl chloride inhalation.

Carbonyl chloride (phosgene) is a toxic industrial compound widely used in industry for the production of synthetic products, such as polyfoam rubber, plastics, and dyes. Exposure to phosgene results in a latent (1-24 h), potentially life-threatening pulmonary edema and irreversible acute lung injury. A genomic approach was utilized to investigate the molecular mechanism of phosgene-induced lung injury. CD-1 male mice were exposed whole body to either air or a concentration x time amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg x min/m3). Lung tissue was collected from air- or phosgene-exposed mice at 0.5, 1, 4, 8, 12, 24, 48, and 72 h postexposure. RNA was extracted from the lung and used as starting material for the probing of oligonucleotide microarrays to determine changes in gene expression following phosgene exposure. The data were analyzed using principal component analysis to determine the greatest sources of data variability. A three-way analysis of variance based on exposure, time, and sample was performed to identify the genes most significantly changed as a result of phosgene exposure. These genes were rank ordered by p values and categorized based on molecular function and biological process. Some of the most significant changes in gene expression reflect changes in glutathione synthesis and redox regulation of the cell, including upregulation of glutathione S-transferase alpha-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as gamma-glutamyl cysteine synthetase). This is in agreement with previous observations describing changes in redox enzyme activity after phosgene exposure. We are also investigating other pathways that are responsive to phosgene exposure to identify mechanisms of toxicity and potential therapeutic targets.

Colchicine decreases airway hyperreactivity after phosgene exposure.

Phosgene (COCl(2)) exposure affects an influx of inflammatory cells into the lung, which can be reduced in an animal model by pretreatment with colchicine. Inflammation in the respiratory tract can be associated with an increase in airway hyperreactivity. We tested the hypotheses that (1) phosgene exposure increases airway reactivity and (2) colchicine can decrease this elevation. Sprague Dawley rats (70 d old; male) were exposed to 1 ppm COCl(2) for 1 h. Airway reactivity was tested at 0, 4, and 24 h postexposure by infusing anesthetized animals intravenously with acetylcholine and assessing expiratory resistance and dynamic compliance. Immediately and 4 h postexposure, a significant change in expiratory resistance and dynamic compliance was observed in those animals exposed to COCl(2), while at 24 h this response was greater. A second experiment was performed in rats pretreated with colchicine (1 mg/kg) or saline given intraperitoneally, exposed to 1 ppm COCl(2) for 1 h, with both expiratory resistance and dynamic compliance assessed at 24 h. After exposure, cell differentials and protein in lavage were also quantitated. The results indicate that colchicine decreased neutrophil influx, protein accumulation, and changes in both expiratory resistance and dynamic compliance after COCl(2) exposure. Colchicine may affect injury and changes in expiratory resistance and dynamic compliance by diminishing the incursion of inflammatory cells, but other properties of this medication may also be responsible for the observed results.

Chronological changes in electrolyte levels in arterial blood and bronchoalveolar lavage fluid in mice after exposure to an edemagenic gas.

Detection of acute lung injury is important if therapeutic medical countermeasures are to be used to reduce toxicity in a timely manner. Indicators of injury may aid in the eventual treatment course and enhance the odds of a positive outcome following a toxic exposure. This study was designed to investigate the effects of a toxic exposure to the industrial irritant gas phosgene on the electrolyte levels in arterial blood and bronchoalveolar lavage fluid (BALF). Phosgene is a well-known chemical intermediate capable of producing life-threatening pulmonary edema within hours after exposure. Four groups of 40 Crl:CD-1(ICR)BR male mice were exposed whole-body to either air or phosgene at a concentration x time (c x t) amount of 32-42 mg/m(3) (8-11 ppm) phosgene for 20 min (640-840 mg x min/m(3)). BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, or 72 h postexposure. After euthanasia, the trachea was excised, and 800 microl saline was instilled into the lungs. The lungs were washed 5x. Eighty microliters of BALF was placed in a cartridge and inserted into a clinical i-STAT analyzer. Na(+), Cl(-), K(+), and ionized Ca(2+) were analyzed. Arterial blood electrolyte levels were also analyzed in four additional groups of air- or phosgene-exposed mice. The left lung was removed to determine wet weight (WW), an indicator of pulmonary edema. Na(+) was significantly higher in air than in phosgene-exposed mice at 4, 8, and 12 h postexposure. Temporal changes in BALF Cl(-) in phosgene mice were not statistically different from those in the air mice. Both Ca(2+) and K(+) were significantly higher than in the air-exposed mice over 72 h, p < or = 0.03 and p < or = 0.001 (two-way analysis of variance, ANOVA), respectively. Significant changes in BALF K(+) and Ca(2+) occurred as early as 4 h postexposure in phosgene, p < or = 0.005, versus air-exposed mice. Over time, there were no significant changes in arterial blood levels of Na(+), Cl(-), or Ca(2+) for animals exposed to air versus phosgene. However, arterial K(+) concentrations were significantly higher, p < or = 0.05, than in air-exposed mice across all time points, with the highest K(+) levels of 7 mmol/L occurring at 8 h and 24 h after exposure. Phosgene caused a time-dependent significant increase in WW from 4 to 12 h, p < or = 0.025, compared with air-exposed mice. These data demonstrate that measuring blood K(+) levels 1 h after exposure along with BALF Na(+), K(+), and Ca(2+) may serve as an alternate indicators of lung injury since both K(+) and Ca(2+) follow temporal increases in air-blood barrier permeability as measured by wet weight.

Pathophysiological responses following phosgene exposure in the anaesthetized pig.

This study aimed to develop a reproducible model of phosgene-induced lung injury in the pig to facilitate the future development of therapeutic strategies. Ten female young adult large white pigs were used. Following induction of anaesthesia using a halothane/oxygen/nitrous oxide mixture, arterial and venous catheters were inserted together with a pulmonary artery thermodilution catheter, and a suprapubic urinary catheter by laparotomy. Anaesthesia was maintained throughout the experiment by intravenous infusion of ketamine, midazolam and alfentanil. On completion of surgery the animals were allowed to equilibrate for 1 h and then were divided into two groups. Group 1 (n = 5) was exposed to phosgene for 10 min (mean Ct = 2443 +/- 35 mg min m(-3)) while spontaneously breathing, whereas control animals (Group 2 n = 5) were exposed to air. At 30 min post-exposure, anaesthesia was deepened in order to allow the initiation of intermittent positive pressure ventilation and the animals were monitored for up to 24 h. Cardiovascular and respiratory parameters were monitored every 30 min and blood samples were taken for arterial and mixed venous blood gas analysis and clinical chemistry. A detailed post-mortem and histopathology was carried out on all animals following death or euthanasia at the end of the 24-h monitoring period. Control animals (Group 2) all survived until the end of the 24-h monitoring period with normal pathophysiological parameters. Histopathology showed only minimal passive congestion of the lung. Following exposure to phosgene (Group 1) there was one survivor to 24 h, with the remainder dying between 16.5 and 23 h (mean = 20 h). Histopathology from these animals showed areas of widespread pulmonary oedema, petechial haemorrhage and bronchial epithelial necrosis. There was also a significant increase in lung wet weight/body weight ratio (P < 0.001). During and immediately following exposure, a transient decrease in oxygen saturation and stroke volume index was observed. From 6 h there were significant decreases in arterial pH (P < 0.01), P(a)O(2) (P < 0.01) and lung compliance (P < 0.01), whereas oxygen delivery and consumption was reduced from 15 h onwards in phosgene-exposed animals. Mean pulmonary artery pressure of phosgene-exposed animals was increased from 15 h post-exposure, with periods of increased pulmonary vascular resistance index being recorded from 9 h onwards. We have developed a reproducible model of phosgene-induced lung injury in the anaesthetized pig. We have followed changes in cardiovascular and pulmonary dynamics for up to 24 h after exposure in order to demonstrate evidence of primary acute lung injury from 16 h post-exposure. Histopathology showed evidence of widespread damage to the lung and there was also a significant increase in lung wet weight/body weight ratio (P < 0.001).

Effect of dietary treatment with n-propyl gallate or vitamin E on the survival of mice exposed to phosgene.

Phosgene, widely used in industrial processes, can cause life-threatening pulmonary edema and acute lung injury. One mechanism of protection against phosgene-induced lung injury may involve the use of antioxidants. The present study focused on dietary supplementation in mice using n-propyl gallate (nPG)--a gallate acid ester compound used in food preservation--and vitamin E. Five groups of male mice were studied: group 1, control-fed with Purina rodent chow 5002; group 2, fed 0.75% nPG (w/w) in 5002; group 3, fed 1.5% nPG (w/w) in 5002; group 4 fed 1% (w/w) vitamin E in 5002; and group 5, fed 2% (w/w) vitamin E also in 5002. Mice were fed for 23 days. On day 23 mice were exposed to 32 mg m-3 (8 ppm) phosgene for 20 min (640 mg. min m-3) in a whole-body exposure chamber. Survival rates were determined at 12 and 24 h. In mice that died within 12 h, the lungs were removed and lung wet weights, dry weights, wet/dry weight ratios, lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and glutathione (GSH) were assessed. Vitamin E had no positive effect on any outcome measured. There was no significant difference between 1.5% nPG and any parameter measured or survival rate compared with 5002 + phosgene. However, dietary treatment with 0.75% nPG significantly increased survival rate (P

Posttreatment with ETYA protects against phosgene-induced lung injury by amplifying the glutathione to lipid peroxidation ratio.

Exposure to phosgene has been shown to cause severe and life-threatening pulmonary edema. There is evidence that successful treatment of phosgene-induced acute lung injury may be related to increased antioxidant activity. Acetylenic acids such as 5,8,11, 14-eicosatetraynoic acid (ETYA) have been shown to be effective in preventing pulmonary edema formation (PEF). In phosgene-exposed guinea pigs, we examined the effects of ETYA on PEF. Lipid peroxidation (thiobarbituric acid-reactive substance, TBARS) and total glutathione (GSH) were measured in lung tissue from isolated, buffer-perfused guinea pig lungs at 180 min after start of exposure. Guinea pigs were challenged with 175 mg/m(3) (44 ppm) phosgene for 10 min (1750 mg( small middle dot)min/m(3)). Five minutes after removal from the exposure chamber, guinea pigs were treated, ip, with 200 microl of 100 microM ETYA in ethanol (ETOH). Two hundred microliters of 50 microM ETYA in ETOH was added to the 200 ml perfusate every 40 min beginning at 60 min after start of exposure (t = 0). There were four groups in this study: air-exposed, phosgene-exposed, phosgene + ETYA-posttreated, and air + ETYA-posttreated. Posttreatment with ETYA prevented GSH depletion, 2. 7 +/- 0.5 micromol/mg protein versus 1 +/- 0.2 micromol/mg protein, for the untreated phosgene-exposed lungs (p < or =.05). ETYA posttreatment also significantly decreased PEF (p

Efficacy of ibuprofen and pentoxifylline in the treatment of phosgene-induced acute lung injury.

Phosgene, a highly reactive former warfare gas, is a deep lung irritant which produces adult respiratory distress syndrome (ARDS)-like symptoms following inhalation. Death caused by phosgene involves a latent, 6-24-h, fulminating non-cardiogenic pulmonary edema. The following dose-ranging study was designed to determine the efficacy of a non-steroidal anti-inflammatory drug, ibuprofen (IBU), and a methylxanthine, pentoxifylline (PTX). These drugs were tested singly and in combination to treat phosgene-induced acute lung injury in rats. Ibuprofen, in concentrations of 15-300 mg kg-1 (i.p.), was administered to rats 30 min before and 1 h after the start of whole-body exposure to phosgene (80 mg m-3 for 20 min). Pentoxifylline, 10-120 mg kg-1 (i.p.), was first administered 15 min prior to phosgene exposure and twice more at 45 and 105 min after the start of exposure. Five hours after phosgene inhalation, rats were euthanized, the lungs were removed and wet weight values were determined gravimetrically. Ibuprofen administered alone significantly decreased lung wet weight to body weight ratios compared with controls (P < or = 0.01) whereas PTX, at all doses tested alone, did not. In addition, the decrease in lung wet weight to body weight ratio observed with IBU+PTX could be attributed entirely to the dose of IBU employed. This is the first study to show that pre- and post-treatment with IBU can significantly reduce lung edema in rats exposed to phosgene.

Enhanced and prolonged pulmonary influenza virus infection following phosgene inhalation.

Animal infectivity models have been important in the demonstration of enhanced susceptibility to viral and bacterial infection as a result of low-level toxicant exposure. This study demonstrated an enhanced and prolonged viral infection using an influenza virus infectivity model in the rat following exposure to the toxicant gas phosgene. Fischer-344 rats exposed to either air or a sublethal concentration of phosgene demonstrated peak pulmonary influenza virus titers 1 d after infection. Virus titers in rats exposed to air declined rapidly falling below detectable levels by 4 d after infection. However, a significantly enhanced and prolonged pulmonary influenza virus infection was observed on d 3 and 4 after infection in rats exposed to phosgene. Virus was cleared below detectable limits on d 5 after infection in animals exposed to phosgene. Thus, inhalation of sublethal concentrations of phosgene resulted in an increased severity of pulmonary influenza virus infection. This study provides a demonstration of the effective use of a rat viral infectivity model to detect the immunotoxicity of inhaled pollutants. This model will allow future studies to focus on the immunological mechanism(s) responsible for the enhanced and prolonged pulmonary influenza virus infection.

Natural killer activity in Fischer-344 rat lungs as a method to assess pulmonary immunocompetence: immunosuppression by phosgene inhalation.

Phosgene, also known as carbonyl chloride, carbon oxychloride, and chloroformyl chloride, is a toxic air pollutant and a potential occupational health hazard. Studies were initiated (a) to evaluate the measurement of pulmonary natural killer (NK) activity as a method to assess pulmonary immunocompetence, and (b) to determine whether exposure to phosgene resulted in local pulmonary or systemic immune dysfunction. Fischer-344 male rats were exposed either to filtered air or to 1.0 ppm phosgene gas for four hours. The effect of phosgene on lung NK activity was quantified at different times after acute phosgene exposure. Pulmonary NK activity was measured by mincing lung tissue into small pieces prior to incubation with collagenase. Whole-lung homogenate was assayed for NK activity utilizing a 4 hour 51-Cr-release assay with YAC-1 cells as target cells. Acute phosgene exposure resulted in a suppressed pulmonary NK activity on days 1, 2, and 4 after exposure; however, normal levels of biological activity were observed 7 days after exposure. The suppressed NK activity was not restored after removal of adherent cells from the lung homogenate, thus indicating that the effect of phosgene on NK activity was not due to immunosuppression via mobilization of suppressor alveolar macrophages. Pulmonary immunotoxicity was also observed after exposure at 0.5 ppm, while no adverse effects were observed at 0.1 ppm phosgene. Systemic immunotoxic effects were observed for NK activity in the spleen, but not in the peripheral blood. It is thus important in pulmonary immunotoxicology to evaluate systemic immune functions, since secondary effects--distant to the original interaction--may occur with potentially serious consequences. Cells exhibiting natural killer activity comprise a part of the nonspecific innate immunity that is important in defense against both neoplastic and viral diseases. Any perturbation of this important nonspecific immunological mechanism may result in a compromised host more susceptible to infectious and neoplastic disease.