Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.     

Acute head-only exposure of dogs to phosgene. Part III. Comparison of indicators of lung injury in dogs and rats

To better understand the relevance of phosgene-induced changes in bronchoalveolar lavage (BAL) fluid protein observed in acutely exposed rats, groups of beagle dogs were similarly exposed for 30 min to phosgene using a head-only mode of exposure. The actual exposure concentrations were 9, 16.5, and 35 mg/m3, with resultant C x t products of 270, 495, and 1050 mg/m3 x min. In rats, a C x t product of 270 mg/m3 x min caused a significant elevation of protein in the bronchoalveolar lavage (BAL) fluid, while the nonlethal threshold concentration (LCt01) was estimated to be 1075 mg/m3 x min. The endpoints examined in dogs focused on changes in BAL, lung weights, arterial blood gases, and lung histopathology approximately 24 h postexposure. Mortality did not occur at any C x t product. Increased lung weights and elevations in protein, soluble collagen, and polymorphonuclear leukocyte (PMN) counts in BAL were observed at 1050 mg/m3 x min with borderline changes at 495 mg/m3 x min. Following exposure to 1050 mg/m3 x min, the analysis of arterial blood gases provided evidence of a significantly decreased arterial pO2. Histopathology revealed a mild, although distinctive, inflammatory response at the bronchoalveolar level at 495 mg/m3 x min, whereas serofibrinous exudates and edema were observed at 1050 mg/m3 x min. The magnitude of effects correlated with the individual dogs' respiratory minute volume and breathing patterns (panting). Collectively, phosgene-induced indicators of acute lung injury appeared to be characterized best by protein in BAL fluid. With regard to both the inhaled dose and the associated increase of protein in BAL, the responses obtained in dogs appear to be more similar to humans. In contrast, elevations in BAL protein occurred in rats at three-fold lower concentrations when compared to dogs. The results of this study demonstrate that the magnitude of elevations of plasma exudate in BAL fluid following acute exposure to the pulmonary irritant phosgene is markedly more pronounced in rats when compared to the dog which is considered more human-like than rats. This is believed to be associated with the higher ventilation of small rodents and with rodent-specific sensory bronchopulmonary defense reflexes.     

Acute Head-Only Exposure of Dogs to Phosgene. Part III. Comparison of Indicators of Lung Injury in Dogs and Rats

To better understand the relevance of phosgene-induced changes in bronchoalveolar lavage (BAL) fluid protein observed in acutely exposed rats, groups of beagle dogs were similarly exposed for 30 min to phosgene using a head-only mode of exposure. The actual exposure concentrations were 9, 16.5, and 35 mg/m³, with resultant C × t products of 270, 495, and 1050 mg/m³ × min. In rats, a C × t product of 270 mg/m³ × min caused a significant elevation of protein in the bronchoalveolar lavage (BAL) fluid, while the nonlethal threshold concentration (LCt₀₁) was estimated to be 1075 mg/m³ × min. The endpoints examined in dogs focused on changes in BAL, lung weights, arterial blood gases, and lung histopathology approximately 24 h postexposure. Mortality did not occur at any C × t product. Increased lung weights and elevations in protein, soluble collagen, and polymorphonuclear leukocyte (PMN) counts in BAL were observed at 1050 mg/m³ × min with borderline changes at 495 mg/m³ × min. Following exposure to 1050 mg/m³ × min, the analysis of arterial blood gases provided evidence of a significantly decreased arterial pO₂. Histopathology revealed a mild, although distinctive, inflammatory response at the bronchoalveolar level at 495 mg/m³ × min, whereas serofibrinous exudates and edema were observed at 1050 mg/m³ × min. The magnitude of effects correlated with the individual dogs' respiratory minute volume and breathing patterns (panting). Collectively, phosgene-induced indicators of acute lung injury appeared to be characterized best by protein in BAL fluid. With regard to both the inhaled dose and the associated increase of protein in BAL, the responses obtained in dogs appear to be more similar to humans. In contrast, elevations in BAL protein occurred in rats at three-fold lower concentrations when compared to dogs. The results of this study demonstrate that the magnitude of elevations of plasma exudate in BAL fluid following acute exposure to the pulmonary irritant phosgene is markedly more pronounced in rats when compared to the dog which is considered more human-like than rats. This is believed to be associated with the higher ventilation of small rodents and with rodent-specific sensory bronchopulmonary defense reflexes.     

Inhaled nitric oxide aggravates phosgene model of acute lung injury

The principal acute mode of action of inhaled phosgene gas is related to an increase alveolar fluid exudation under pathologic conditions. This paper considers some aspects in modeling phosgene-induced acute lung injury (ALI) in an acute rat bioassay and whether edema formation can be modulated by inhaled nitric oxide (iNO). Protein analysis in bronchoalveolar lavage (BAL) fluid is amongst the most sensitive method to quantify the phosgene-induced non-cardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum concentrations in BAL-protein occur within one day postexposure, typically within a latency period up to about 15?h as a consequence of an increasingly exhausted lymphatic drainage. An almost similar sensitivity was given by the functional endpoint ‘enhanced pause (Penh)’ when measured by non-invasive whole-body barometric plethysmography over a time period of 20?h. The magnitude of edema formation follows a concentration x time (C¹xt) relationship, although animal model-specific deviations may occur at very short exposure durations (1–20?min) due to a rodent-specific, reflexively induced transient decreased ventilation. This has to be accounted for when simulating accidental exposure scenarios to study the mechanisms involved in pharmacological modulation of fluid transport in this type of ALI. Therefore, a special focus has to be given to the dosimetry of inhaled phosgene, otherwise any change in effect magnitude, as a result of under-dosing of phosgene, may be misconceived as promising therapy. This study demonstrates that accidental exposures can be modeled best in rats by exposure durations of at least 20–30?min. Lung function measurements (Penh) show that pathophysiological effects appear to occur concomitant with the exposure to phosgene; however, its full clinical manifestation requires a gross imbalance of pulmonary fluid clearance. When applying this concept, post-phosgene exposure iNO at 1.5?ppm?×?6?h or 15 pm?×?20?h led to an aggravation of edema formation while L-NAME, a non-selective inhibitor of nitric oxide synthase, led to attenuation. Ethyl pyruvate, given either prophylactically or therapeutically, was ineffective.     

Inhaled nitric oxide aggravates phosgene model of acute lung injury

The principal acute mode of action of inhaled phosgene gas is related to an increase alveolar fluid exudation under pathologic conditions. This paper considers some aspects in modeling phosgene-induced acute lung injury (ALI) in an acute rat bioassay and whether edema formation can be modulated by inhaled nitric oxide (iNO). Protein analysis in bronchoalveolar lavage (BAL) fluid is amongst the most sensitive method to quantify the phosgene-induced non-cardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum concentrations in BAL-protein occur within one day postexposure, typically within a latency period up to about 15 h as a consequence of an increasingly exhausted lymphatic drainage. An almost similar sensitivity was given by the functional endpoint 'enhanced pause (Penh)' when measured by non-invasive whole-body barometric plethysmography over a time period of 20 h. The magnitude of edema formation follows a concentration x time (C1xt) relationship, although animal model-specific deviations may occur at very short exposure durations (1-20 min) due to a rodent-specific, reflexively induced transient decreased ventilation. This has to be accounted for when simulating accidental exposure scenarios to study the mechanisms involved in pharmacological modulation of fluid transport in this type of ALI. Therefore, a special focus has to be given to the dosimetry of inhaled phosgene, otherwise any change in effect magnitude, as a result of under-dosing of phosgene, may be misconceived as promising therapy. This study demonstrates that accidental exposures can be modeled best in rats by exposure durations of at least 20-30?min. Lung function measurements (Penh) show that pathophysiological effects appear to occur concomitant with the exposure to phosgene; however, its full clinical manifestation requires a gross imbalance of pulmonary fluid clearance. When applying this concept, post-phosgene exposure iNO at 1.5?ppm?×?6?h or 15 pm?×?20 h led to an aggravation of edema formation while L-NAME, a non-selective inhibitor of nitric oxide synthase, led to attenuation. Ethyl pyruvate, given either prophylactically or therapeutically, was ineffective.     

Effects Of Acute Exposure To Phosgene On Pulmonary Host Defenses And Resistance To Infection

Abstract

Phosgene is a toxic gas widely used in industrial processes. The most sensitive endpoint for phosgene toxicity in mice is decreased resistance to challenge with bacterial infection or tumor cells. These effects were attributed to impaired alveolar macrophage (AM) and pulmonary natural killer cell (NK) function. The purpose of this study was to investigate whether similar effects occurred in Fischer 344 rats. Intrapulmonary killing of bacteria was impaired and the inflammatory response enhanced in rats infected by aerosol with Streptococcus zooepidemicus immediately after a 6-/1 exposure to 0.1 or 0.2 ppm phosgene as compared to air controls. Also, ingestion of latex beads in vitro by AM obtained from bronchoalveolar lavage (BAD fluid of rats exposed to 0.2 ppm phosgene was significantly less than controls. If infection or BAL were delayed until 18 h after exposure, there was no difference between phosgene and air exposed rats. In uninfected rats polymorphonuclear leukocytes were increased in BAL fluid 18 h after exposure to 0.5 ppm but not lower concentrations of phosgene. Pulmonary NK activity was suppressed immediately and 18 h after exposure to 0.5 ppm but not 0.1 ppm. The data indicate that, as in the mouse, intrapulmonary killing of bacteria is the most sensitive endpoint for phosgene toxicity in the rat, although recovery from acute phosgene exposure is rapid. Consequences of repeated chronic exposure remain to be elucidated.     

Pulmonary Host Defenses and Resistance to Infection Following Subchronic Exposure to Phosgene

Abstract

Acute exposure to phosgene, a toxic gas widely used in industrial processes, decreases resistance to bacteria in mice and rats and enhances susceptibility to B16 tumor cell challenge in mice. These effects appear to be due to impaired alveolar macrophage and natural killer (NK) cell activity, respectively. In this study effects of repeated phosgene exposures on bacterial infection and NK activity were determined. Rats were exposed for 4 or 12 wk, 6 h/day, 5 days/wk, to 0.1 or 0.2 ppm phosgene or 2 days/wk to 0.5 ppm and infected by aerosol with Streptococcus zooepidemicus immediately after the last exposure. An additional group was also infected after 4 wk of recovery following the 12-wk exposure regimens. Bronchoalveolar lavage (BAL) fluid was assessed 0, 6, and 24 h postinfection for bacteria and inflammatory cells. Differential cell counts in BAL and pulmonary NK activity were also determined in uninfected rats 18 Is after the last exposure. All phosgene exposures impaired clearance of bacteria from the lungs and caused an increase in polymorphonuclear leukocytes (PMNs) in BAL of infected rats. Effects in the 0.5 ppm exposure group were greatest, and were significantly different from those in the 0.2 ppm exposure group, although the product of concentration × time was the same. BAL cell counts and bacterial clearance were normal in rats assessed 4 wk after the 12-wk phosgene exposures. Bacterial clearance and the PMN response to infection following repeated exposure were similar to those observed after a single exposure; that is, for these endpoints, effects due to repetitive exposure were neither additive nor attenuated. In contrast, NK activity was suppressed only at the 0.5 ppm level, and the magnitude of suppression was much less than that following acute exposure, suggesting that attenuation of this effect did occur with repeated exposure. The data indicate that susceptibility to streptococcal infection is a sensitive endpoint for phosgene toxicity following subchronic exposure.     

SHORT-TERM INHALATION TOXICITY OF POLYISOCYANATE AEROSOLS IN RATS: COMPARATIVE ASSESSMENT OF IRRITANT-THRESHOLD CONCENTRATIONS BY BRONCHOALVEOLAR LAVAGE

The object of this study was to compare the relative potency of respirable aerosols of the aliphatic hexamethylene 1,6-diisocyanate homopolymer of the isocyanurate type (HDI-IC) and the aromatic polymeric methylenediphenyl-4,4'-diisocyanate (pMDI) to elicit early changes in bronchoalveolar lavage fluid (BALF). The validity of the concentration × time (C × t) concept was addressed in rats exposed to concentrations from 3.4 to 58.1 mg pMDI/m 3 and exposure durations of 6 h to 23 min, respectively (C × t ≈ 1200 mg/m 3 -min). One additional group of rats was exposed to 2.7 mg MDA/m 3 for 1 × 6 h, a putative product of hydrolysis of pMDI. In rats repeatedly exposed to 12.9 mg pMDI/m 3 (6 h/day, 5 days/wk for 14 days), cumulative exposure-related changes were examined. Results show that total protein and angiotensin-converting enzyme (ACE) in BALF were among the most sensitive endpoints to probe early effects caused by exposure to irritant polyisocyanate aerosols. In the repeated-exposure study, BALF protein was maximal after the first exposure day. Based on these most sensitive endpoints in BALF, a benchmark no-effect threshold concentration of 0.5 and 3 mg/m 3 was estimated for the pMDI and HDI-IC aerosol, respectively. The slope of the concentration-effect curve was steeper following exposure to HDI-IC than to pMDI. These estimated acute no-observed-effect levels (NOELs) were almost identical to those observed in longer term inhalation studies using conventional endpoints. It is concluded that pulmonary irritation caused by polyisocyanate aerosols can readily be quantified in an acute rat bioassay by the analysis of total protein in BALF.     

Attempts to counteract phosgene-induced acute lung injury by instant high-dose aerosol exposure to hexamethylenetetramine,cysteine or glutathione

Phosgene is an important high-production-volume intermediate with widespread industrial use. Consistent with other lung irritants causing ALI (acute lung injury), mode-of-action-based countermeasures remain rudimentary. This study was conducted to analyze whether extremely short high-level exposure to phosgene gas could be mitigated using three different inhaled nucleophiles administered by inhalation instantly after exposure to phosgene. Groups of young adult male Wistar rats were acutely exposed to carbonyl chloride (phosgene) using a directed-flow nose-only mode of exposure of 600?mg/m³ for 1.5?min (225?ppm?×?min). Immediately after exposure to phosgene gas the rats were similarly exposed to three strong nucleophiles with and without antioxidant properties for 5 or 15?min. The following nucleophiles were used: hexamethylenetetramine (HMT), l-cysteine (Cys), and l-glutathione (GSH). The concentration of the aerosol (mass median aerodynamic diameter 1.7–2?µm) was targeted to be in the range of 1?mg/L. Cys and GSH have antioxidant properties in addition. The calculated alveolar molar dosage of phosgene was 9 µmol/kg. At 15-min exposure duration, the respective inhaled dose of HMT, Csy, and GSH were 111, 103, and 46 µmol/kg, respectively. The alveolar dose of drugs was ~10-times lower. The efficacy of treatment was judged by protein concentrations in bronchoalveolar lavage fluid (BALF) collected 1 day post-exposure. In spite of using optimized aerosolization techniques, none of the nucleophiles chosen had any mitigating effect on BALF-protein extravasation. This finding appear to suggest that inhaled phosgene gas acylates instantly nucleophilic moieties at the site of initial deposition and that the resultant reaction products can not be reactivated even following instant inhalation treatment with competing nucleophilic agents. In spite of using maximal technically attainable concentrations, it appears to be experimentally challenging to deliver such nucleophiles to the lower respiratory tract at high dosages.     

Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage

The object of this study was to compare the relative acute pulmonary irritant potency of respirable aerosols of a variety of non-volatile polyisocyanates. The types of polyisocyanates examined included aliphatic homopolymers and mixed aliphatic-aromatic polyisocyanates consisting of the following monomers: HDI (hexamethylene 1,6-diisocyanate), IPDI (isophorone diisocyanate), MDI (methylene-diphenyl-4,4'-diisocyanate) and TDI (toluene diisocyanate). For reference purposes, the pulmonary irritant polyisocyanate aerosols were compared with monomeric IPDI, a semi-volatile respiratory tract (airway) irritant. In the substances tested, the concentration of free isocyanate moieties ranged from 11% to 38%. The relative potency to elicit pulmonary irritation was assessed by measurements of lung weights and total protein and lactate dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) following a single 6-h exposure of male rats. The time course of changes was analysed 3 h and 1, 3 and 7 days after exposure. When exposed to irritant concentrations of aerosol, BALF protein was maximal on post-exposure day 1 and returned to the level of the controls on post-exposure day 7. In contrast, rats exposed to sub-lethal concentrations of monomeric IPDI experienced a time-related increase in BALF protein. Based on this most sensitive endpoint, extrapolated no-observed-effect concentrations (NOECs) were in the range of 2-3 mg m(-3) for most polyisocyanates examined. The NOECs from all the substances investigated were in the range 1-50 mg m(-3). Thus, this methodology is adequate to rank the pulmonary irritant potency of polyisocyanate aerosols and to differentiate pulmonary from airway irritants. For pulmonary irritants the estimated acute NOECs were essentially similar to the no-observed-adverse effect concentrations (NOAECs) from long-term, repeated-exposure inhalation studies available for some of the polyisocyanates. A clear dependence of the NOAECs on the content of free isocyanate moieties was not observed. In summary, it is concluded that pulmonary irritation caused by polyisocyanate aerosols can be quantified readily in an acute rat bioassay by analysis of total protein in BALF. Moreover, this experimental evidence suggests that the NOECs of pulmonary irritants based on this endpoint are predictive of the NOAECs observed after subacute/subchronic inhalation exposure, suggesting that acute pulmonary irritation governs the outcome of repeated inhalation studies with such aerosols. However, for isocyanates where airway irritation predominates the pulmonary irritation, long(er)-term inhalation studies appear to be indispensable. The content of free NCO per se appears to be a poor predictor of the pulmonary irritant potency of polyisocyanate aerosols.     

Inhalation toxicity of 1,6-hexamethylene diisocyanate homopolymer (HDI-IC) aerosol: results of single inhalation exposure studies.

The early acute pulmonary response of female Wistar rats exposed nose-only to a mixture of 1,6-hexamethylene diisocyanate homopolymer (HDI-IC) aerosol was examined. This study was designed to investigate the time course of the relationship between acute pulmonary irritation and ensuing disturbances of the air/blood barrier in rats exposed to concentrations of 3.9, 15.9, 54.3, or 118. 1 mg HDI-IC/m(3). The duration of exposure was 6 h, followed by serial sacrifices 0 h, 3 h, 1 day, 3 days, and 7 days postexposure. Concentrations were selected based on the results of a 4-h acute inhalation study in rats (LC(50) = 462 mg/m(3)). Bronchoalveolar lavage (BAL) fluid was analyzed for markers indicative of injury of the bronchoalveolar region, including phospholipids as proxy of altered surfactant homeostasis. Glutathione (GSH) was determined in BAL fluid and lung tissue. BAL cells with increased intracellular phospholipids were observed on day 1 and especially day 3, with some residual increase on day 7. Increased intracellular phospholipids and activity of acid phosphatases appear to suggest that phagocytized phospholipids may transiently affect lysosomal function. Following exposure to 15.9 mg/m(3), changes returned almost entirely to the level of the air-exposed control on day 7. Especially at higher exposure concentrations, lung weights and total number of cells in BAL were still statistically significantly elevated at this time point. Experimental evidence suggests that markers indicative of a dysfunction of the air/blood barrier, such as angiotensin-converting enzyme, total protein, and phospholipids engulfed by alveolar macrophages, were most sensitive to probe this type of changes. Although GSH in BALF was increased following exposure, there was an apparent depletion of tissue GSH immediately after cessation of exposure. In summary, this study suggests that respirable HDI-IC aerosol appears to cause a transient dysfunction of the air/blood barrier indicated by an increased extravasation of plasma constituents. Despite the remarkable extent of effects observed, most changes were reversible within a postexposure period as short as 7 days. First evidence of increased leakage of pulmonary epithelial barrier was observed at 3.9 mg/m(3). With respect to changes of early markers of pulmonary epithelial barrier dysfunction, approximately 3 mg HDI-IC/m(3) was considered to be the threshold concentration for acute pulmonary irritation.     

The Temporal Profile of Cytokines in the Bronchoalveolar Lavage Fluid in Mice Exposed to the Industrial Gas Phosgene

Diagnosis of an exposure to airborne toxicants can be problematic. Phosgene is used widely in industry for the production of many synthetic products, such as polyfoam rubber, plastics, and dyes. Although nearly 100% of the gas is consumed during processing, there is the potential problem of accidental or even intentional exposure to this irritant/choking agent. Exposure to phosgene has been known to cause latent life-threatening pulmonary edema. A major problem is that there is a clinical latency phase from 3 to 24 h in people before irreversible acute lung injury occurs. Assessment of markers of acute lung injury after a suspected exposure would be useful in developing rational treatment strategies. These experiments were designed to assess bronchoalveolar lavage fluid (BALF) for the presence of the early markers of exposure to phosgene in mice from 1 to 72 h after exposure. Separate groups of 40 CD-1 male mice (Crl:CD-1(ICR)BR) weighing 29 ± 1 g were exposed whole-body to either air or a concentration × time (c × t) amount of 32 mg/m 3 (8 ppm) phosgene for 20 min (640 mg·min/m 3) . BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, and 72 h postexposure. After euthanasia, the trachea was excised, and 800 µl saline was instilled into the lungs and washed 5 ×. BALF was assessed for interleukin (IL)-4, IL-6, tumor necrosis factor (TNF)α, IL-1α, macrophage inflammatory protein (MIP)-2, and IL-10. At 4 h postexposure, IL-6 was 15-fold higher for phosgene-exposed mice than for the time-matched air-exposed control group. At 8 and 12 h, IL-6, IL-1β, MIP-2, and IL-10 were significantly higher in phosgene-exposed mice than in time-matched air-exposed controls, p ≤ .05 to p ≤ .001, whereas TNFα reached peak significance from 24 to 72 h. IL-4 was significantly lower in the phosgene-exposed mice than in the air-exposed mice from 4 to 8 h after exposure. These data show that BALF is an important tool in assessing pro- and anti-inflammatory markers of phosgene-induced acute lung injury and that knowledge of these temporal changes may allow for timely treatment strategies to be applied.     

Characterization of a nose-only inhaled phosgene acute lung injury mouse model

Abstract

Context: Phosgene’s primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6‐24 h post-exposure.

Objective: To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures.

Methods: A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8?ppm) or air (sham). An inhalation lethality study was conducted to determine the 8?ppm median lethal exposure (LCt₅₀) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt₅₀. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life.

Results and discussion: The 24-hour LCt₅₀ was 226?ppm*min (8?ppm for 28.2?min) and the 48-hour LCt₅₀ was 215?ppm*min (8?ppm for 26.9?min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt₅₀ phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies.

Conclusion: An 8?ppm phosgene exposure for 34?min (1.2?×?LCt₅₀) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant.     

Bone marrow-derived mesenchymal stem cells attenuate phosgene-induced acute lung injury in rats

Abstract

Accidental phosgene exposure could result in acute lung injury (ALI), effective therapy is needed for the patients with phosgene-induced ALI. As a type of cells with therapeutic potential, mesenchymal stem cells (MSCs) have been showed its efficacy in multiple diseases. Here, we assessed the therapeutic potential of MSCs in phosgene-induced ALI and explored the related mechanisms. After isolation and characterization of rat bone marrow MSCs (BMMSCs), we transplanted BMMSCs into the rats exposed to phosgene and observed significant improvement on the lung wet-to-dry ratio and partial oxygen pressure (PaO₂) at 6, 24, 48?h after phosgene exposure. Histological analyses revealed reduced sign of pathological changes in the lungs. Reduced level of pro-inflammatory tumor necrosis factor α and increased level of anti-inflammatory factor interleukin-10 were found in both bronchoalveolar lavage and plasma. Significant increased expression of epithelial cell marker AQP5 and SP-C was also found in the lung tissue. In conclusion, treatment with MSC markedly decreases the severity of phosgene-induced ALI in rats, and these protection effects were closely related to the pulmonary air blood barrier repairment and inflammatory reaction regulation.     

TWO-WEEK INHALATION TOXICITY OF POLYMERIC DIPHENYLMETHANE-4,4'-DIISOCYANATE (PMDI) IN RATS: Analysis of Biochemical and Morphological Markers of Early Pulmonary Response

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m³ (6 h/day, 15 exposures). The level of 13.7 mg/m³ was actually a combination of an initial target concentration of 10 mg/m³ in wk 1, which was raised to 16 mg/m³ in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m³ experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m³, and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m³ and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m³ and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13.7 mg/m³ resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.     

INFLAMMATORY RESPONSES AND MUCUS SECRETION IN RATS WITH ACUTE BRONCHIOLITIS INDUCED BY NICKEL CHLORIDE

The effects of inhaled particulate matter in the workplace and outdoor environment on sensitive subpopulations are not sufficiently investigated in human and animal models. Thus, animal models for pulmonary diseases are necessary for appropriate risk assessment of toxic materials. We studied biochemical characteristics of an acute inflammatory process induced by inhalation of nickel chloride aerosols in rats. Acute bronchiolitis was induced by inhalation of nickel chloride aerosols for 5 days in Wistar rats according to the method described by Kyono et al. (1999). Deterioration and recovery from inflammatory responses were evaluated by analyzing markers of inflammation in bronchoalveolar lavage (BAL) fluid. Experimental animals were sacrificed during and after the nickel aerosol exposure period. The number of neutrophils markedly increased to approximately 0.5 × 10 3 cells/µl BAL fluid during nickel aerosol exposure, accompanied by increase of total protein, soluble L-selectin, cytokine-induced neutrophil chemoattractant/growth-regulated gene products (CINC/GRO), elastolytic activity, trypsin inhibitory capacity,β -glucuronidase activity, fucose, and sialic acid in BAL fluid compared with those of the control group. There was correlation between number of leukocytes and soluble L-selectin concentration. The number of pulmonary macrophages in BAL fluid decreased to approximately 15% of those of the control group on the days of nickel aerosol exposure. The level of CINC/GRO recovered to that of the control group on day 3 after cessation of the nickel aerosol exposure. However, other inflammatory markers remained at the elevated levels. Changes in the markers of inflammation during and after the nickel aerosol exposure were consistent with previously reported morphological findings. The results indicated that this animal model is potentially useful as an acute bronchiolitis model.     

Attempts to counteract phosgene-induced acute lung injury by instant high-dose aerosol exposure to hexamethylenetetramine, cysteine or glutathione

Phosgene is an important high-production-volume intermediate with widespread industrial use. Consistent with other lung irritants causing ALI (acute lung injury), mode-of-action-based countermeasures remain rudimentary. This study was conducted to analyze whether extremely short high-level exposure to phosgene gas could be mitigated using three different inhaled nucleophiles administered by inhalation instantly after exposure to phosgene. Groups of young adult male Wistar rats were acutely exposed to carbonyl chloride (phosgene) using a directed-flow nose-only mode of exposure of 600?mg/m3 for 1.5?min (225?ppm?×?min). Immediately after exposure to phosgene gas the rats were similarly exposed to three strong nucleophiles with and without antioxidant properties for 5 or 15?min. The following nucleophiles were used: hexamethylenetetramine (HMT), l-cysteine (Cys), and l-glutathione (GSH). The concentration of the aerosol (mass median aerodynamic diameter 1.7-2?μm) was targeted to be in the range of 1?mg/L. Cys and GSH have antioxidant properties in addition. The calculated alveolar molar dosage of phosgene was 9 μmol/kg. At 15-min exposure duration, the respective inhaled dose of HMT, Csy, and GSH were 111, 103, and 46 μmol/kg, respectively. The alveolar dose of drugs was ~10-times lower. The efficacy of treatment was judged by protein concentrations in bronchoalveolar lavage fluid (BALF) collected 1 day post-exposure. In spite of using optimized aerosolization techniques, none of the nucleophiles chosen had any mitigating effect on BALF-protein extravasation. This finding appear to suggest that inhaled phosgene gas acylates instantly nucleophilic moieties at the site of initial deposition and that the resultant reaction products can not be reactivated even following instant inhalation treatment with competing nucleophilic agents. In spite of using maximal technically attainable concentrations, it appears to be experimentally challenging to deliver such nucleophiles to the lower respiratory tract at high dosages.     

Correlation Between sPLA2-llA and Phosgene-Induced Rat Acute Lung Injury

Secreted phospholipase A₂ of group IIA (sPLA₂-IIA) has been involved in a variety of inflammatory diseases, including acute lung injury. However, the specific role of sPLA₂-IIA in phosgene-induced acute lung injury remains unidentified. The aim of the present study was to investigate the correlation between sPLA₂-IIA activity and the severity of phosgene-induced acute lung injury. Adult male rats were randomly exposed to either normal room air (control group) or a concentration of 400 ppm phosgene (phosgene-exposed group) for there are 5 phosgene-exposed groups altogether. For the time points of 1, 3, 6, 12 and 24 h post-exposure, one phosgene-exposed group was sacrificed at each time point. The severity of acute lung injury was assessed by Pa_O2/F_IO2 ratio, wet-to-dry lung-weight ratio, and bronchoalveolar lavage (BAL) fluid protein concentration. sPLA₂-IIA activity in BAL fluid markedly increased between 1 h and 12 h after phosgene exposure, and reached its highest level at 6 h. Moreover, the trend of this elevation correlated well with the severity of lung injury. These results indicate that sPLA₂-IIA probably participates in phosgene-induced acute lung injury.     

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.     

Pulmonary Alterations in Rats Due to Acute Phosgene Inhalation

Pulmonary Alterations in Rats Due to Acute Phosgene Inhalation.CURRIE, W. D., HATCH, G. E., AND FROSOLONO, M. F. (1987). Fundam.Appl. Toxicol. 8, 107–114. This study evaluated the relationshipbetween low-level phosgene (COCl₂) exposure and pulmonary changeor damage. Male Sprague–Dawley rats were exposed to phosgenefor 4 hr at concentrations of 0.125 to 1.0 ppm (30,60, 120,and 240 ppm.min). We examined the dose-related changes in bodyweight, lung wet and dry weights, lavage fluid protein concentrations(LFP), total cell count, and cell differential in rats exposedto phosgene under carefully controlled conditions. These parameterswere measured at the conclusion of single acute exposures andfor 3 days postexposure. Significant changes in lung weights(wet and dry) were observed following exposure to 120 and 240ppm.min phosgene and the LFP was significantly altered at 60ppm.min. The changes in lung wet and dry weights pooled overall times and phosgene concentrations each correlated significantlywith the change in LFP induced by phosgene. The total numberof cells in the lavage fluid of phosgene-exposed rats was increased,and the most sensitive cellular indicator of phosgene inhalationwas the increase in the percentage of polymorphonuclear leukocytes(PMNs). These results confirm that LFP concentration and cellulardifferentials can be used as an index of lung damage due tophosgene. A dose-response relationship for the measured parameterswas observed. Over the dosage range studied, the return of allmeasured parameters to near control levels within 3 days followingexposure showed that the pulmonary damage was reversible orrapidly reparable. Although the acute effects were shown tobe reversible, studies on chronic, low-level phosgene exposuresare necessary to determine safe levels for industrial employees.