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Inhalation exposure to sulfur mustard in the guinea pig model: Clinical, biochemical and histopathological characterization of respiratory injuries
Toxicology and Applied Pharmacology 241 (2009) 154–162
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Inhalation exposure to sulfur mustard in the guinea pig model: Clinical, biochemical and histopathological characterization of respiratory injuries Nahum Allon ⁎, Adina Amir, Eliau Manisterski, Ishay Rabinovitz, Shlomit Dachir, Tamar Kadar Department of Pharmacology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 74100, Israel
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Article history: Received 11 June 2009 Revised 2 August 2009 Accepted 4 August 2009 Available online 12 August 2009 Keywords: Sulfur mustard Guinea pig LCt50 FTIR Respiratory parameters Clinical symptoms Histopathological evaluation BAL Abnormal epithelial regeneration Recurrent lung injury
a b s t r a c t Guinea pigs (GP) were exposed (head only) in individual plethysmographs to various concentrations of sulfur mustard vapor, determined online, using FTIR attached to flow chamber. The LCt50 and the inhaled LD50 were calculated at different time points post exposure. Surviving animals were monitored for clinical symptoms, respiratory parameters and body weight changes for up to 30 days. Clinical symptoms were noted at 3 h post exposure, characterized by erythematic and swelling nose with extensive mucous secretion (with or without bleeding). At 6 h post exposure most of the guinea pigs had breathing difficulties, rhonchi and dyspnea and few deaths were noted. These symptoms peaked at 48 h and were noted up to 8 days, associated with few additional deaths. Thereafter, a spontaneous healing was noted, characterized by recovery of respiratory parameters and normal weight gain with almost complete apparent healing within 2 weeks. Histopathological evaluation of lungs and trachea in the surviving GPs at 4 weeks post exposure revealed a dose-dependent residual injury in both lung and trachea expressed by abnormal recovery of the tracheal epithelium concomitant with a dose-dependent increase in cellular volume in the lungs. These abnormal epithelial regeneration and lung remodeling were accompanied with significant changes in protein, LDH, differential cell count and glutathione levels in the bronchoalveolar lavage (BAL). It is suggested that the abnormal epithelial growth and cellular infiltration into the lung as well as the continuous lung inflammation could cause recurrent lung injury similar to that reported for HD exposed human casualties. © 2009 Elsevier Inc. All rights reserved.
Introduction Sulfur mustard (HD) is a potent nucleophile that alkylates cellular and extracellular components of the living tissue. This causes complex cellular events including cell cycle arrest, DNA (Langenberg et al., 1998; Niu et al., 1996; Rao et al., 1999) and protein alkylation (Fidder et al., 1996; Noort et al., 1997) and release of inflammatory mediators (Casillas et al., 1997; Amir et al., 2000; Dachir et al., 2002) that result in skin, lung and ocular injuries. Inhalation exposure of sulfur mustard results in severe acute and recurrent respiratory injury lasting for months or even years after the initial exposure (Beheshti et al., 2006). The acute phase of the injury starts 2–4 h post exposure, characterized by chest tightness, lacrimation, rhinorrhea and hacking cough, and peaks at 24–72 h with severe dyspnea, pulmonary edema and bronchopneumonia. The delayed effects are chronic bronchitis, asthma, laryngitis and recurrent pneumonia that last for years and occur in about 80% of the casualties (Somani and Babu, 1989; Balali Mood and Hefazi, 2006). This, together with increased incidents of laryngeal and bronchial carcinoma (Balali Mood and Hefazi, 2006)
and absence of efficient therapy make the inhalation exposure to mustard highly problematic. Data collected from human casualties and animal models suggest high involvement of the inflammatory response in the development of pulmonary injury (Freitag et al., 1991). Most of our knowledge about lung injury induced by HD vapor was collected from casualties of the Iran–Iraq war and from accidental exposures (Somani and Babu, 1989; Beheshti et al., 2006). There are only few studies describing the effects of inhalation exposure to HD in animal models (Allon et al., 1993; Calvet et al., 1994; Vijayaraghavan, 1997). The aim of this study was to establish an animal model for acute inhalation exposure to HD vapor in guinea pigs (GP), to determine its LCt50 and to characterize the time course of lung injury. This database will be further used to evaluate the efficacy of various treatments and for the development of optimal therapeutic regimen. We used a battery of clinical, physiological, biochemical and histological techniques to characterize the damage and recovery of the pulmonary system following inhalation of HD vapor.
Methods ⁎ Corresponding author. Fax: +972 8 9381559. E-mail address: [email protected] (N. Allon). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.08.006
Materials. Sulfur mustard is a hazardous compound and all work with neat agent was carried out in a well-ventilated hood under
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suitable safety precautions. The purity of HD (N95%) was determined by NMR spectroscopy. Animals. Male albino Dunkin–Hartley guinea pigs weighing 270– 300 g (age 4–5 weeks) were purchased from IFFA-CREDO (France). Care and maintenance were in accordance with the principles described in the “guide for Care and Use of Laboratory Animals” (NIH publication 85-23, 1985). Animals were housed in the animal facilities of the IIBR and housing environment was controlled with constant temperature of 21 ± 1 °C, constant humidity, and a 12h light/dark cycle. Food and water were available ad-libitum. All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996, and were approved by the Institutional animal Care and Use Committee. Inhalation exposure. Forty-eight GPs were subjected to inhalation exposure of 6 different concentrations of HD vapor and their clinical symptoms were monitored up to 30 days. Death occurred at different time intervals after exposure. Guinea pigs were exposed head only to HD vapor in an inhalation apparatus designed and built as described elsewhere (Bitron and Aharonson, 1978; Allon et al., 1998). The exposure system was built in a hood and operated under subatmospheric pressure (- 3 cm water), contained two separate air passes of clean and HD-contaminated air. HD vapor was introduced into the system by pneumatic valves following 10 min of animals' adaptation to a flow of clean air and switched back to clean air at the end of the exposure. The major modification was introducing plethysmographs to the system. Pre-calibration of the plethysmographs was carried out with a hydraulic pump that simulated normal tidal volume (TV) and respiratory rate (RR) values of guinea pigs. The plethysmographs enabled the online measurements of TV, RR and the calculation of minute volume (MV) during the 10 min exposure. Moreover, the actual inhaled dose (ID) of HD for individual GP was calculated, assuming that all the inhaled HD was retained in the system. The respiratory parameters: TV, RR and MV were calculated online by a computer-aided program (Mezada, Israel). Four animals were exposed simultaneously through individual glass mask in separate chambers connected with parallel flow lines. Each chamber served as a whole body plethysmograph that enabled the monitoring of the respiratory parameters, before, during and after the exposure. The head mask compartment was secluded from the plethysmograph by inflatable latex sealing collar. Animals were exposed to HD for 10 min and the inhaled air flow was switched back to clean air for an additional 10 min period. All the experiments were conducted at 22± 1 °C and 50± 15% relative humidity. At the end of each exposure, animals were transferred to their home cage for monitoring of gross behavior and clinical signs. Animals exposed to non-contaminated fresh air, served as controls. Analysis of HD concentrations. During each exposure, HD vapor was collected at a rate of 5 l/min into four sinter glass containers (sampling close to the animal's breathing zone) containing 15 ml of diethylphtaleate (DEP) (Aldrich AR). The retention of the HD vapor in DEP was N96% and HD remained stable in the solution for more than 4 weeks. Chemical analysis was performed according to Geckel (1951) by alkylating the HD with 4-(4-nitro-benzyl)-pyridin (DB3). The pH of the alkylated HD was then adjusted to pH = 7.4 and read at 570 nm. HD calibration had linear sensitivity between 1 and 35 μg/ml and about 10% variations. The variability between the HD concentrations sampled from various breathing zones never exceeds 10%. Online HD analysis using FTIR. The concentration of HD vapor in the inhaled air was monitored online using FTIR (Bruker, Germany) equipped with a gas chamber (InfraRed, England). The peak values (area under the curve) determined from the FTIR measurements were
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calibrated against chemical analysis of HD concentration, (correlation coefficient of N0.99). The use of FTIR as a detector for HD vapor enabled online measurement of HD before the exposure and adjustment of the concentration whenever needed and during the exposure for validation. Calculation of LCt50. Forty-eight GPs were exposed for 10 min in groups of 8 to one of the following HD concentrations: 0, 700, 1100, 2200, 2350, 3100 μg/l. The LCt50 was calculated at 24 h, 8 days and 30 days post exposure. Calculation of the inhaled median lethal dose (ILD50). The inhaled volume of six out of eight GPs in each concentration group was measured, during exposure, using individual plethysmographs. The inhaled dose was normalized to body weight for each GP and then grouped into four dose groups. The surviving ratio for 24 h, 8 days and 30 days was calculated. Clinical evaluation. Clinical evaluation included monitoring of animal body weight, physiological and clinical observations performed at various time points post exposure (0, 2, 5, 14, 21, and 28 days). Biochemical, physiological and histopathological evaluations were conducted at the end of the experiment. Bronchalveolar lavage (BAL). Bronchoalveolar lavage was performed on anesthetized animals using routine procedures (Henderson et al., 1988). Saline was introduced into the lungs (2× 3 ml) and recovered fluid was centrifuged. Pelleted cells were differentially counted on Giemza stained slides. The supernatant fluid was used to determine the levels of protein and albumin (Bradford, 1976), lactic dehydrogenase activity (LDH) (sigma diagnostics no. 500), β-glucoronidase activity (β-GLU) (rate of dissociation of para-nitrophenolglucoronide under pH = 5 in the presence of 0.01% Triton X-100), alkaline phosphatase (ALK) (sigma diagnostics no. 245) (idell 1989) and glutathione (GSH) (Anderson, 1985). Histopathology. Histological evaluation of lungs and trachea was performed on 6 μm thick paraffin sections stained by either hematoxylin and eosin (H&E) for general morphology or by a combination of PAS, hematoxylin and light green staining (PHG) and included microscopic evaluation and quantitative morphometric measurements. The quantitative analysis was performed on pictures taken from trachea and lung sections using computerized image analysis system (Galai, Migdal Haemek, Israel). The percentage of epithelial coverage expressed the extent of regeneration in the trachea and was conducted on three consecutive sections in the middle of the tracheal tract. In the lungs, the relative area that was covered by cells (cellular volume) was measured. In each animal, a total of 40 fields (2× 106 μm2 each) in two main lobes were measured. Statistical analysis. Two-way ANOVA analyses were performed using prizma software. Specific post hoc comparisons were carried out using the Bonferroni posttests for dependent variables. Data are presented as mean ± S.E.M. and are considered statistically different when p b 0.05. Results Online analysis of HD vapor concentrations Control of HD concentration in the inhaled air is the essential factor for reproducible and reliable experiments. Proper concentration control can be achieved only by online monitoring of the HD levels. In order to achieve this goal we developed an online analysis system for HD vapor employing FTIR attached in parallel to the vapor chamber. Air was sampled continuously, into FTIR gas chamber, at a constant rate of 10 l/min and absorption at 730 nm was monitored
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Fig. 1. Online analysis of HD vapor using FTIR: Various concentrations (10 l/min) of HD vapor were bypassed through FTIR instrument attached to long path (6 m) gas cell. The area under the highest absorbance peak (AUC), 730 cm-1 was correlated with HD concentration (A). Calibration curve between the area under the peak and HD concentration determined by chemical analysis (correlation coefficient of r = 0.999) is presented in B. (C) Depiction of HD concentration measured with FTIR during 10 min exposure. HD concentration was calculated from average peak area during exposure based on calibration curve presented in (B).
every 2 s. The area under the curve of the peak absorbance for HD vapor (Fig. 1A) was correlated to the values of HD determined by chemical analysis in the collected sinter-glass samplers. The calibration curve (Fig. 1B) (r = 0.999) was loaded into the computer and online HD concentration was determined for each exposure (Fig. 1C) calculating the average concentration for the entire exposure. Nevertheless, in each exposure HD vapor was collected at a rate of 5 l/min into sinter glass containers containing 15 ml of DEP for chemical analysis. The analysis served as a backup for the FTIR and the
Table 1 Determination of LCt50 values following exposure of GPs to HD vapor for 10 min. Ct (μg × min/l)
0 700 1100 2200 2530 3100
General characterization LCt50 of HD vapor Forty-eight GPs were subjected to inhalation exposure of six different concentrations of HD vapor and their clinical symptoms were monitored up to 30 days. Death occurred at different time intervals after exposure. LCt50 was defined for 1, 8 and 30 days post exposure as demonstrated in Table 1. Table 2 Determination of ILD50 values following exposure of GPs to HD vapor for 10 min.
Time post exposure (surviving ratio) 24 h
8 days
30 days
8/8 8/8 7/8 6/8 6/8 3/8
8/8 8/8 6/8 3/8 2/8 0/8
8/8 8/8 6/8 2/8 2/8 0/8
LCt50 (24 h) = 2440 (1890–3150) (μg × min/l), 95% confidence limit. LCt50 (8 days) = 1660 (1250–2200) (μg × min/l), 95% confidence limit. LCt50 (30 days) = 1580 (1220–2040) (μg × min/l), 95% confidence limit.
differences between those two methods never exceed the 10% variation of chemical analysis.
Dose (μg/kg)
250–540 540–775 775–1110 1110–1500
Time post exposure (surviving ratio) 24 h
8 days
30 days
11/12 4/6 4/7 3/6
11/12 3/6 3/7 0/6
11/12 3/6 1/7 0/6
ILD50 (24 h) = 995 (740–1340) (μg/kg), 95% confidence limit. ILD50 (8 days) = 660 (506–850) (μg/kg), 95% confidence limit. ILD50 (30 days) = 630 (485–810) (μg/kg), 95% confidence limit.
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Determination of Inhaled LD50 (ILD50) Measuring MV during exposure in 75% of the GPs enabled the calculation of individual inhaled HD doses and the calculation of the ILD50. The inhaled doses were grouped into four groups as shown in Table 2 and the ILD50 was calculated. Effect of exposure to HD vapor on respiratory parameters The TV, RR and MV of naïve GPs measured before the exposure were found to be within the range reported previously for untreated resting GPs i.e. 300–400 ml × min/GP (Guyton and Hall, 2005). Exposure of GPs to HD vapor caused an immediate decline in respiratory parameters that continued to decrease throughout the next 2 days following exposure and reached 17–20% of its initial values. Respiratory parameters were monitored for up to 30 days post exposure as demonstrated in Fig. 2. The most profound changes in respiratory parameters were noted during the first week followed by recovery to control levels starting from the second week. Changes comprised of a dose-dependent decrease in RR with a correlation coefficient of 0.959 and 0.968 for the second and fifth day post exposure respectively. Two-way ANOVA revealed significant time and dose interaction (p b 0.0001) with significant differences (Bonferroni posttest) at days 2 and 5 (p b 0.05). In spite of the variation in TV, a dose-dependent decrease in MV was observed following HD inhalation with a correlation coefficient of 0.88 and 0.94 for the second and fifth day post exposure respectively. Two-way ANOVA revealed significant time and dose interaction (p b 0.002) with significant differences (Bonferroni posttest) mainly at day 5 (p b 0.05). The most extreme changes observed were about 75% decrease in MV and RR on the fifth day following exposure to the highest dose. Effect of HD exposure on body weight Inhalation exposure to HD vapor caused a dose-dependent decrease in body weight during the first week following exposure as demonstrated in Fig. 3. A correlation of r = .973 was found between the HD concentration and the decrease in body weight 48 h post exposure (Fig. 3 insert). GPs exposed to Ct of 700μg × min/l (for 10 min) showed no change in body weight while those exposed to Ct of 1100 μg × min/l (for 10 min) showed 7% decrease in body-weight 48 h post exposure with recovery to control levels within 1 week. GPs exposed to Ct of either 2200 or 2530 μg × min/l (for 10 min) showed further decrease of 12–15% within 48 h post exposure. Recovery to normal weight started only about 10 days following exposure.
Fig. 2. The effect of inhalation exposure of guinea pigs to various concentrations of HD vapor on respiratory parameters. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l) while their respiratory parameters were measured with the aid of individual plethysmographs. Respiratory parameters; tidal volume (TV) and respiration rate (RR) were monitored pre, during and up to 28 days post inhalation exposure to HD vapor. Minute volume (MV) was calculated as MV = TV × RR. Note the significant (p b 0.05) decrease in RR and MV (two-way ANOVA) during the first week following exposure as well as the concentration-dependent changes mainly at day 5 (r = 0.97 and 0.94 respectively). Notice the complete recovery of respiratory parameters at 2 weeks post exposure.
Fig. 3. The effect of inhalation exposure of guinea pigs to various concentrations of HD vapor on weight gain. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). GPs' body weight was weighted at various times up to 28 days post exposure. Data presented as the percent changes from the individual weight before exposure. Note the high correlation between inhaled HD concentration and weight loss at 48 h post exposure (insert).
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Fig. 4. The effect of inhalation exposure of guinea pigs to various concentrations of inhaled HD on the recovery of trachea injury 1 month post exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on H&E stained trachea sections, 30 days post exposure. A. Control trachea; B. Trachea following inhalation of 700 μg × min/l HD; C. Trachea following inhalation of 700 μg × min/l HD; D. Trachea following inhalation of 1100 μg × min/l HD. Notice the hyperplasia of goblet cells following the lower dose (arrow in C) and the increase in micro-vesicles and the abnormal growth of the epithelium in B. At the higher doses 4D the increase in number of blood cells and fragments of detached epithelia accumulated in the trachea's lumen are clearly seen. Bar indicates 100 μm.
Fig. 5. The effect of inhalation exposure of guinea pigs to various concentrations of HD on the recovery of lung injury 1 month post exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on lung sections stained with H&E, 30 days post exposure. A. Control; B. Exposed lung following inhalation of 700 μg × min/l HD; C. Exposed lung following inhalation of 1100 μg × min/l HD; D. Exposed lung following inhalation of 2200 μg × min/l HD. Notice the dose-dependent increase in the cellular area and the decrease in the alveolar air space. Bar in a, b and d indicates 100 μm, in c indicates 500 μm.
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Clinical symptoms Clinical evaluation of the exposed guinea pigs was performed at 3 and 6 h following exposure and at different time intervals following exposure for up to 1 month. Exposing GPs to Ct of 700 μg × min/l HD induced only mild symptoms that subsided within 1 week after exposure. However, exposure to higher HD concentrations induced moderate to severe clinical symptoms that started as early as 1 h post exposure. The percentage of GPs with severe symptoms increased as the doses increased. GPs exposed to Ct of 700μg × min/l recovered within 5 days without any further visible symptoms. As a whole, except for the lower Ct group (700 μg × min/l) that showed only mild toxicity signs, animals exhibited similar toxicity signs with a dose-dependent variation in severity and death rate. Erythematic and swelling nose associated with extensive mucous secretion (with or without bleeding) were noted 3 h post exposure. At 6 h post exposure most of the GPs had breathing difficulties, rhonchi and dyspnea and even death (n = 1) was noted. One day post exposure five out of eight GPs died at the higher Ct (3100 μg × min/l) and one to two deaths occurred at each of the lower Cts groups. The GPs developed inflammatory response in ocular tissues, concomitant with extensive mucous secretion (mostly bloody) and nose swelling. Additional incidences of death occurred during the first week. Recovery was relatively fast. Detectable signs of recovery were evident seven or more days following exposure. At 2 weeks post exposure most of the survived GPs seemed to fully recover from the HD exposure. However, the physical condition of two of the GPs started to deteriorate again 3 weeks following exposure and one of them died on the 28th day. Thirty days following exposure the survived GPs were divided into two groups: one designated for histopathology and the other for biochemical evaluation of bronchoalveolar lavage and lung homogenate.
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Lung A dose-dependent increase of cellular infiltration was observed in the lung at 1 month following exposure (Fig. 5). As a result, the surface area covered by cells, extra cellular matrix and congested blood vessels increased in the lungs of GPs exposed to the higher HD concentrations (p b 0.025). Thus, lung volume available for air exchange reduced to 50% compared to 68% in the control animals (Fig. 6B). Biochemical evaluation of the BAL BAL was collected from half of the survived GPs 30 days following exposure. Evaluation of BAL content revealed no significant differences between the control group and the GPs exposed to the low Cts (700 or 1100 μg × min/l) in any of the parameters tested (Fig. 7). In two GPs that survived the exposure to HD concentration above the LCt50 a significant increase in BAL protein, LDH, total cell count and number of neutrophils was found 30 days following exposure (Fig. 7) indicating that despite the apparent clinical
Histopathology A clinical recovery of the animals started around 1 week following exposure to HD vapor. However the histological evaluation of tissues taken from lung and trachea 1 month following HD inhalation revealed an incomplete healing in both tissues. The extent of residual damage and pathological regeneration was dose-dependent. For practical purposes (mainly due to low survival rate at high doses) the GPs were divided into three groups according to the concentration of HD during exposure (control, 700–1100 μg × min/l (below LCt50), 2200–2530 μg × min/l (above LCt50)). Trachea The regenerative epithelium of the trachea varied from single layer (Fig. 4B) to stratified epithelium (Fig. 4C) displaying incomplete regeneration and the differentiation of the epithelium differed from control (Fig. 4A). A stratified epithelium was seen following exposure to the lower concentration (700 μg × min/l), however, even in this group most of the animals had only about 75% of the surface area covered by a multilayered epithelium. The new epithelium contained cilia and goblet cells but hyperplasia of the latter was often seen (Fig. 4C). In GPs exposed to higher concentrations, more severe pathological findings were observed, the regenerative epithelium consisted of one to three layers which were often sloughed off and microvesications were noted at the basal layer. The epithelium displayed large vacuoles and necrotic cells (Fig. 4B and D). Cilia were seen only in part of the superficial cells and the number of goblet cells was lower than in controls. Edema and blood congestion were found in the lamina propria (Fig. 4). Morphometric analysis of the extent of stratified epithelial coverage is depicted in Fig. 6A. As mentioned above, the regeneration of the epithelium was in reverse correlation with the inhaled concentration.
Fig. 6. Quantitative histopathological evaluation of the recovery of respiratory system from HD vapor exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on trachea and lung sections stained with H&E 30 days post exposure. A—The effect of inhaled HD concentration on the percentage of lung cellular volume 1 month post exposure. In every animal a total of 40 randomly picked fields in two main lobes were measured. Note the concentration-dependent increase in cellular volume. B—The effect of inhaled HD concentration on the degree of epithelial regeneration in the trachea 1 month following exposure. Measurements were conducted on three consecutive sections in the middle of each trachea. The areas with no epithelium were either from recurrent molting of fragments of lately regenerated epithelia due to the appearance of micro-blisters or to no recovery at all. We could not differentiate one from the other.
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Fig. 7. The effect of inhalation exposure of guinea pigs to various concentrations of inhaled HD on BAL parameters 1 month post exposure. BAL was collected from surviving GP, as described in the Methods section, under deep anesthesia, 30 days post exposure and analyzed according to the described methods. Notice the trend of increase in several parameters even for GPs exposed to the lower HD concentrations. Significant residual damage was detected only in the BAL in few parameters and only following exposure to the highest HD concentration. ⁎ indicates significant differences (p b 0.05) from control group.
healing, the lungs were inflamed and not fully recovered. No significant differences were noted in the lung homogenates, in any of the measured parameters in the exposed GP vs control GP, 30 days post exposure. Discussion The primary target organs for airborne chemicals are the respiratory tract and the lungs, due to their close proximity to the environment. Lung injuries are considered to be the main cause of long-term disability among patients exposed to sulfur mustard gas. Most of the accumulated data about HD injury and the various suggested treatments came from human casualties during World War I and the Iran–Iraq war. Although the data are based on large number of casualties and prolonged follow-up, there is no consensus on medical management of HD casualties other than the need for thorough decontamination and the supply of supportive care. In the case of war casualties, it is well understood that there is no way to estimate the inhaled doses and to correlate them with the extent of injury. We have shown in the current study that gross clinical evaluation during remission period was not a good predicator for future lung injury and did not accurately represent mild or even moderate changes occurring in the lungs. Since treatment is based
mainly upon supportive care, no treatment is expected to be provided during remission periods where no clinical adverse symptoms are noted. Thus, any quest for treatment of pulmonary injury, following HD inhalation, will require reliable information about changes occurring in the lungs and trachea during the remission periods. Several animal models have been reported in the literature. In most of them animals were subjected to whole-body HD exposure (Papirmeister et al., 1991 and the references cited therein; Rao et al., 1999). In the current study we have presented a reliable animal model for inhalation exposure to HD vapor using head only, individual plethysmographic exposure chambers, and utilizing online measurements of HD concentration and respiratory parameters. This enabled us to calculate the individual HD doses inhaled by each of the GPs at any time of the exposure. GPs have been selected as an animal model for inhalation toxicity since: 1. GPs are the most commonly used small animal species in preclinical studies related to lung diseases such as asthma and COPD (Canning and Chou, 2008). 2. GPs have been extensively used to study pulmonary alteration associated with inhalation of irritants (Castranova et al., 2002). 3. GPs are also a useful model for investigating the effects of drugs (West and Fernandez, 2004) their heart demonstrate similarity to human heart failure with respect to calcium cycling, myosin isoforms and myocardial function (Hasenfuss, 1998). 4. This together with the
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complexity of handling needed for the measuring respiratory parameters in awake restrained animals that required calm and easy manageable animals. These characteristics make the GP the most suitable animal for this research. Furthermore, the clinical symptoms that were observed in the GPs following exposure to HD vapor such as; cough, dyspnea, hemorrhage exudates from the nose and necrosis of upper and lower airways as well as their time course resembled those described for human patients (Somani and Babu, 1989; Freitag et al., 1991; Balali Mood and Hefazi, 2006). Additionally, the distribution of incidence of death occurring in the GPs model following inhalation exposure to HD is in consistent with other animal models see Papirmeister et al. (1991) and Rao et al. (1999). In humans, exposure to high doses of HD vapor was reported to cause damage to the terminal airways with productive cough, dyspnea, and possibly hemorrhage in the alveoli (Sohrabpour, 1984, 2004). Severe coughing with necrosis and inflammation of the mucosa were also noted (Balali, 1984). This was followed by obstruction of upper and lower airways (Freitag et al., 1991) leading to adult respiratory distress syndrome (ARDS) (Balali Mood et al., 2005; Balali Mood and Hefazi, 2006). Infection of the respiratory tract was a common complication that developed 36–48 h post exposure and was accompanied by a release of inflammatory mediators (Freitag et al., 1991; Balali Mood and Hefazi, 2006). Although infection is not considered a problem in the animal model, elevation of inflammatory mediators release following HD exposure were noted (Casillas et al., 1997; Amir et al., 2000; Dachir et al., 2002; Gao et al., 2007). This could develop into bronchopneumonia with death occurring between the second day and the fourth week after exposure (Papirmeister et al., 1991). In our GP model most of the severe symptoms occurred as early as 6 h post exposure. Most of the incidents of death occurred within the first 24 h followed by sporadic death for up to 4 weeks. GPs in our model seemed to be more sensitive than mice that started to die only 5 days after exposure to one or two LCt50 of HD (Kumar and Vijayaraghavan, 1998). In our model, during the first hours and up to a week post exposure, there was a dose-dependent progressive decrease in MV and RR. Guinea pigs exposed to the highest doses reduced RR down to 25% of its original values. This reduction together with severe lung edema that reduced oxygen diffusion into the blood (Allon, et al. unpublished results) might be the main cause of death following inhalation of HD. Recovery from lung injury in our GP model seemed to be fast, respiratory parameters returned to normal within 2 weeks as well as feeding behavior and weight gain. However, two GPs exhibited deterioration in their clinical conditions and one of them died 28 days post exposure. Fast recovery was also reported by Papirmeister et al. (1991) but with some residual irritations that were persistent for as long as 6 months. Although we could hardly find any residual irritation using clinical observations, histopathological and biochemical evaluation of the lungs and tracheas conducted 4 weeks post exposure, revealed dose-dependent residual injuries. The regeneration of the epithelial cells in the trachea was fast but incomplete with abnormal differentiation characterized by hyperplasia of goblet cells, with large vacuoles, and sloughing of the regenerated epithelium from the basal membrane. Indeed, epithelial debris are often observed in the lumen of the respiratory tract and may be the cause of congestion and blockage of patients' airways that required specific drastic therapeutic measures to relieve the recurrent congested trachea (Freitag et al., 1991; Rees et al., 1991). Late recurrent injuries were reported also in an experimental model of GPs following HD aerosol exposure (Van Helden et al., 2004; Beheshti et al., 2006). The abnormal histopathological changes reported in the current research are in corroboration with the changes in BAL biochemistry that were noted for most of the parameters tested. Although, significant changes were found only following exposure to the high HD concentrations, nonsignificant trends of changes were noted even following the lower concentrations of HD. This abnormal
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recovery may be partly due to cross-linkage of DNA strands (Rao et al., 1999), abnormal growth, recurrent inflammation, blistering and dissociation of pieces of epithelial tissue into the trachea. Recurrent inflammation has been considered to be the main cause for airway remodeling in asthmatic patients (Broide, 2008 and the references cited therein). Some of the airway remodeling changes closely resembled the features seen following HD recovery. Thus, it is suggested that some of the remodeling occurred during HD recovery were due to the continued inflammatory response of HD injury and that drugs affecting airway remodeling and/or inflammation may ameliorate HD recurrent injuries. Changes at the lower lung epithelium are probably the cause for the recurrent respiratory problems and of delayed destructive pulmonary sequels such as chronic bronchitis, asthma, bronchiectasis large airway narrowing, and pulmonary fibrosis described in human patients (Balali Mood and Hefazi, 2006). In summary, a reproducible inhalation exposure system for HD was presented using GPs as animal model. The system will be used to characterize the initial clinical changes following exposure and to test various potential treatments.
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Papirmeister, B., Feister, A.J., Robinson, S.I., Ford, R.D., 1991. Defense against mustard gas. Toxic Mechanisms and Pharmacological Implications. CRC Press, Boca Raton, FL, pp. 133–139. Rao, P.V.L., Vijayaraghavan, R., Bhaskar, A.S.B., 1999. Sulfur mustard induced DNA damage in mice after dermal and inhalation exposure. Toxicology 139, 39–51. Rees, J., Harper, P., Ellis, F., Mitchell, D., 1991. Mustard gas casualties. Lancet 337, 430. Sohrabpour, H., 1984. Clinical manifestations of chemical agents on Iranian combatants during Iran–Iraq conflict. Arch. Belg. Supp. 291–297. Sohrabpour, H., 2004. Observation and clinical manifestations of patients injured with mustard gas. Med. J. Islam. Repub. Iran 1408, 32–37. Somani, S.M., Babu, S.R., 1989. Toxicodynamics of sulfur mustard. Int. J. Clin. Pharmacol. Therapy Toxicol. 27, 419–435. Van Helden, H.P., Kuijpers, W.C., Diemel, R.V., 2004. Asthma-like symptoms following intra-tracheal exposure of guinea pigs to sulfur mustard aerosol: therapeutic efficacy of exogenous lung surfactant curosurf and salbutamol. Inhal. Toxicol. 16, 537–548. Vijayaraghavan, R., 1997. Modifications of breathing pattern induced by inhaled sulfur mustard in mice. Arch. Toxicol. 69, 35–42. West, K.L., Fernandez, M.L., 2004. Guinea pigs as models to study the hypocholesterolemic effect of drugs. Cardiovasc. Drug Rev. 22, 55–70.
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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Inhalation exposure to sulfur mustard in the guinea pig model: Clinical, biochemical and histopathological characterization of respiratory injuries Nahum Allon ⁎, Adina Amir, Eliau Manisterski, Ishay Rabinovitz, Shlomit Dachir, Tamar Kadar Department of Pharmacology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 74100, Israel
a r t i c l e
i n f o
Article history: Received 11 June 2009 Revised 2 August 2009 Accepted 4 August 2009 Available online 12 August 2009 Keywords: Sulfur mustard Guinea pig LCt50 FTIR Respiratory parameters Clinical symptoms Histopathological evaluation BAL Abnormal epithelial regeneration Recurrent lung injury
a b s t r a c t Guinea pigs (GP) were exposed (head only) in individual plethysmographs to various concentrations of sulfur mustard vapor, determined online, using FTIR attached to flow chamber. The LCt50 and the inhaled LD50 were calculated at different time points post exposure. Surviving animals were monitored for clinical symptoms, respiratory parameters and body weight changes for up to 30 days. Clinical symptoms were noted at 3 h post exposure, characterized by erythematic and swelling nose with extensive mucous secretion (with or without bleeding). At 6 h post exposure most of the guinea pigs had breathing difficulties, rhonchi and dyspnea and few deaths were noted. These symptoms peaked at 48 h and were noted up to 8 days, associated with few additional deaths. Thereafter, a spontaneous healing was noted, characterized by recovery of respiratory parameters and normal weight gain with almost complete apparent healing within 2 weeks. Histopathological evaluation of lungs and trachea in the surviving GPs at 4 weeks post exposure revealed a dose-dependent residual injury in both lung and trachea expressed by abnormal recovery of the tracheal epithelium concomitant with a dose-dependent increase in cellular volume in the lungs. These abnormal epithelial regeneration and lung remodeling were accompanied with significant changes in protein, LDH, differential cell count and glutathione levels in the bronchoalveolar lavage (BAL). It is suggested that the abnormal epithelial growth and cellular infiltration into the lung as well as the continuous lung inflammation could cause recurrent lung injury similar to that reported for HD exposed human casualties. © 2009 Elsevier Inc. All rights reserved.
Introduction Sulfur mustard (HD) is a potent nucleophile that alkylates cellular and extracellular components of the living tissue. This causes complex cellular events including cell cycle arrest, DNA (Langenberg et al., 1998; Niu et al., 1996; Rao et al., 1999) and protein alkylation (Fidder et al., 1996; Noort et al., 1997) and release of inflammatory mediators (Casillas et al., 1997; Amir et al., 2000; Dachir et al., 2002) that result in skin, lung and ocular injuries. Inhalation exposure of sulfur mustard results in severe acute and recurrent respiratory injury lasting for months or even years after the initial exposure (Beheshti et al., 2006). The acute phase of the injury starts 2–4 h post exposure, characterized by chest tightness, lacrimation, rhinorrhea and hacking cough, and peaks at 24–72 h with severe dyspnea, pulmonary edema and bronchopneumonia. The delayed effects are chronic bronchitis, asthma, laryngitis and recurrent pneumonia that last for years and occur in about 80% of the casualties (Somani and Babu, 1989; Balali Mood and Hefazi, 2006). This, together with increased incidents of laryngeal and bronchial carcinoma (Balali Mood and Hefazi, 2006)
and absence of efficient therapy make the inhalation exposure to mustard highly problematic. Data collected from human casualties and animal models suggest high involvement of the inflammatory response in the development of pulmonary injury (Freitag et al., 1991). Most of our knowledge about lung injury induced by HD vapor was collected from casualties of the Iran–Iraq war and from accidental exposures (Somani and Babu, 1989; Beheshti et al., 2006). There are only few studies describing the effects of inhalation exposure to HD in animal models (Allon et al., 1993; Calvet et al., 1994; Vijayaraghavan, 1997). The aim of this study was to establish an animal model for acute inhalation exposure to HD vapor in guinea pigs (GP), to determine its LCt50 and to characterize the time course of lung injury. This database will be further used to evaluate the efficacy of various treatments and for the development of optimal therapeutic regimen. We used a battery of clinical, physiological, biochemical and histological techniques to characterize the damage and recovery of the pulmonary system following inhalation of HD vapor.
Methods ⁎ Corresponding author. Fax: +972 8 9381559. E-mail address: [email protected] (N. Allon). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.08.006
Materials. Sulfur mustard is a hazardous compound and all work with neat agent was carried out in a well-ventilated hood under
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suitable safety precautions. The purity of HD (N95%) was determined by NMR spectroscopy. Animals. Male albino Dunkin–Hartley guinea pigs weighing 270– 300 g (age 4–5 weeks) were purchased from IFFA-CREDO (France). Care and maintenance were in accordance with the principles described in the “guide for Care and Use of Laboratory Animals” (NIH publication 85-23, 1985). Animals were housed in the animal facilities of the IIBR and housing environment was controlled with constant temperature of 21 ± 1 °C, constant humidity, and a 12h light/dark cycle. Food and water were available ad-libitum. All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996, and were approved by the Institutional animal Care and Use Committee. Inhalation exposure. Forty-eight GPs were subjected to inhalation exposure of 6 different concentrations of HD vapor and their clinical symptoms were monitored up to 30 days. Death occurred at different time intervals after exposure. Guinea pigs were exposed head only to HD vapor in an inhalation apparatus designed and built as described elsewhere (Bitron and Aharonson, 1978; Allon et al., 1998). The exposure system was built in a hood and operated under subatmospheric pressure (- 3 cm water), contained two separate air passes of clean and HD-contaminated air. HD vapor was introduced into the system by pneumatic valves following 10 min of animals' adaptation to a flow of clean air and switched back to clean air at the end of the exposure. The major modification was introducing plethysmographs to the system. Pre-calibration of the plethysmographs was carried out with a hydraulic pump that simulated normal tidal volume (TV) and respiratory rate (RR) values of guinea pigs. The plethysmographs enabled the online measurements of TV, RR and the calculation of minute volume (MV) during the 10 min exposure. Moreover, the actual inhaled dose (ID) of HD for individual GP was calculated, assuming that all the inhaled HD was retained in the system. The respiratory parameters: TV, RR and MV were calculated online by a computer-aided program (Mezada, Israel). Four animals were exposed simultaneously through individual glass mask in separate chambers connected with parallel flow lines. Each chamber served as a whole body plethysmograph that enabled the monitoring of the respiratory parameters, before, during and after the exposure. The head mask compartment was secluded from the plethysmograph by inflatable latex sealing collar. Animals were exposed to HD for 10 min and the inhaled air flow was switched back to clean air for an additional 10 min period. All the experiments were conducted at 22± 1 °C and 50± 15% relative humidity. At the end of each exposure, animals were transferred to their home cage for monitoring of gross behavior and clinical signs. Animals exposed to non-contaminated fresh air, served as controls. Analysis of HD concentrations. During each exposure, HD vapor was collected at a rate of 5 l/min into four sinter glass containers (sampling close to the animal's breathing zone) containing 15 ml of diethylphtaleate (DEP) (Aldrich AR). The retention of the HD vapor in DEP was N96% and HD remained stable in the solution for more than 4 weeks. Chemical analysis was performed according to Geckel (1951) by alkylating the HD with 4-(4-nitro-benzyl)-pyridin (DB3). The pH of the alkylated HD was then adjusted to pH = 7.4 and read at 570 nm. HD calibration had linear sensitivity between 1 and 35 μg/ml and about 10% variations. The variability between the HD concentrations sampled from various breathing zones never exceeds 10%. Online HD analysis using FTIR. The concentration of HD vapor in the inhaled air was monitored online using FTIR (Bruker, Germany) equipped with a gas chamber (InfraRed, England). The peak values (area under the curve) determined from the FTIR measurements were
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calibrated against chemical analysis of HD concentration, (correlation coefficient of N0.99). The use of FTIR as a detector for HD vapor enabled online measurement of HD before the exposure and adjustment of the concentration whenever needed and during the exposure for validation. Calculation of LCt50. Forty-eight GPs were exposed for 10 min in groups of 8 to one of the following HD concentrations: 0, 700, 1100, 2200, 2350, 3100 μg/l. The LCt50 was calculated at 24 h, 8 days and 30 days post exposure. Calculation of the inhaled median lethal dose (ILD50). The inhaled volume of six out of eight GPs in each concentration group was measured, during exposure, using individual plethysmographs. The inhaled dose was normalized to body weight for each GP and then grouped into four dose groups. The surviving ratio for 24 h, 8 days and 30 days was calculated. Clinical evaluation. Clinical evaluation included monitoring of animal body weight, physiological and clinical observations performed at various time points post exposure (0, 2, 5, 14, 21, and 28 days). Biochemical, physiological and histopathological evaluations were conducted at the end of the experiment. Bronchalveolar lavage (BAL). Bronchoalveolar lavage was performed on anesthetized animals using routine procedures (Henderson et al., 1988). Saline was introduced into the lungs (2× 3 ml) and recovered fluid was centrifuged. Pelleted cells were differentially counted on Giemza stained slides. The supernatant fluid was used to determine the levels of protein and albumin (Bradford, 1976), lactic dehydrogenase activity (LDH) (sigma diagnostics no. 500), β-glucoronidase activity (β-GLU) (rate of dissociation of para-nitrophenolglucoronide under pH = 5 in the presence of 0.01% Triton X-100), alkaline phosphatase (ALK) (sigma diagnostics no. 245) (idell 1989) and glutathione (GSH) (Anderson, 1985). Histopathology. Histological evaluation of lungs and trachea was performed on 6 μm thick paraffin sections stained by either hematoxylin and eosin (H&E) for general morphology or by a combination of PAS, hematoxylin and light green staining (PHG) and included microscopic evaluation and quantitative morphometric measurements. The quantitative analysis was performed on pictures taken from trachea and lung sections using computerized image analysis system (Galai, Migdal Haemek, Israel). The percentage of epithelial coverage expressed the extent of regeneration in the trachea and was conducted on three consecutive sections in the middle of the tracheal tract. In the lungs, the relative area that was covered by cells (cellular volume) was measured. In each animal, a total of 40 fields (2× 106 μm2 each) in two main lobes were measured. Statistical analysis. Two-way ANOVA analyses were performed using prizma software. Specific post hoc comparisons were carried out using the Bonferroni posttests for dependent variables. Data are presented as mean ± S.E.M. and are considered statistically different when p b 0.05. Results Online analysis of HD vapor concentrations Control of HD concentration in the inhaled air is the essential factor for reproducible and reliable experiments. Proper concentration control can be achieved only by online monitoring of the HD levels. In order to achieve this goal we developed an online analysis system for HD vapor employing FTIR attached in parallel to the vapor chamber. Air was sampled continuously, into FTIR gas chamber, at a constant rate of 10 l/min and absorption at 730 nm was monitored
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Fig. 1. Online analysis of HD vapor using FTIR: Various concentrations (10 l/min) of HD vapor were bypassed through FTIR instrument attached to long path (6 m) gas cell. The area under the highest absorbance peak (AUC), 730 cm-1 was correlated with HD concentration (A). Calibration curve between the area under the peak and HD concentration determined by chemical analysis (correlation coefficient of r = 0.999) is presented in B. (C) Depiction of HD concentration measured with FTIR during 10 min exposure. HD concentration was calculated from average peak area during exposure based on calibration curve presented in (B).
every 2 s. The area under the curve of the peak absorbance for HD vapor (Fig. 1A) was correlated to the values of HD determined by chemical analysis in the collected sinter-glass samplers. The calibration curve (Fig. 1B) (r = 0.999) was loaded into the computer and online HD concentration was determined for each exposure (Fig. 1C) calculating the average concentration for the entire exposure. Nevertheless, in each exposure HD vapor was collected at a rate of 5 l/min into sinter glass containers containing 15 ml of DEP for chemical analysis. The analysis served as a backup for the FTIR and the
Table 1 Determination of LCt50 values following exposure of GPs to HD vapor for 10 min. Ct (μg × min/l)
0 700 1100 2200 2530 3100
General characterization LCt50 of HD vapor Forty-eight GPs were subjected to inhalation exposure of six different concentrations of HD vapor and their clinical symptoms were monitored up to 30 days. Death occurred at different time intervals after exposure. LCt50 was defined for 1, 8 and 30 days post exposure as demonstrated in Table 1. Table 2 Determination of ILD50 values following exposure of GPs to HD vapor for 10 min.
Time post exposure (surviving ratio) 24 h
8 days
30 days
8/8 8/8 7/8 6/8 6/8 3/8
8/8 8/8 6/8 3/8 2/8 0/8
8/8 8/8 6/8 2/8 2/8 0/8
LCt50 (24 h) = 2440 (1890–3150) (μg × min/l), 95% confidence limit. LCt50 (8 days) = 1660 (1250–2200) (μg × min/l), 95% confidence limit. LCt50 (30 days) = 1580 (1220–2040) (μg × min/l), 95% confidence limit.
differences between those two methods never exceed the 10% variation of chemical analysis.
Dose (μg/kg)
250–540 540–775 775–1110 1110–1500
Time post exposure (surviving ratio) 24 h
8 days
30 days
11/12 4/6 4/7 3/6
11/12 3/6 3/7 0/6
11/12 3/6 1/7 0/6
ILD50 (24 h) = 995 (740–1340) (μg/kg), 95% confidence limit. ILD50 (8 days) = 660 (506–850) (μg/kg), 95% confidence limit. ILD50 (30 days) = 630 (485–810) (μg/kg), 95% confidence limit.
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Determination of Inhaled LD50 (ILD50) Measuring MV during exposure in 75% of the GPs enabled the calculation of individual inhaled HD doses and the calculation of the ILD50. The inhaled doses were grouped into four groups as shown in Table 2 and the ILD50 was calculated. Effect of exposure to HD vapor on respiratory parameters The TV, RR and MV of naïve GPs measured before the exposure were found to be within the range reported previously for untreated resting GPs i.e. 300–400 ml × min/GP (Guyton and Hall, 2005). Exposure of GPs to HD vapor caused an immediate decline in respiratory parameters that continued to decrease throughout the next 2 days following exposure and reached 17–20% of its initial values. Respiratory parameters were monitored for up to 30 days post exposure as demonstrated in Fig. 2. The most profound changes in respiratory parameters were noted during the first week followed by recovery to control levels starting from the second week. Changes comprised of a dose-dependent decrease in RR with a correlation coefficient of 0.959 and 0.968 for the second and fifth day post exposure respectively. Two-way ANOVA revealed significant time and dose interaction (p b 0.0001) with significant differences (Bonferroni posttest) at days 2 and 5 (p b 0.05). In spite of the variation in TV, a dose-dependent decrease in MV was observed following HD inhalation with a correlation coefficient of 0.88 and 0.94 for the second and fifth day post exposure respectively. Two-way ANOVA revealed significant time and dose interaction (p b 0.002) with significant differences (Bonferroni posttest) mainly at day 5 (p b 0.05). The most extreme changes observed were about 75% decrease in MV and RR on the fifth day following exposure to the highest dose. Effect of HD exposure on body weight Inhalation exposure to HD vapor caused a dose-dependent decrease in body weight during the first week following exposure as demonstrated in Fig. 3. A correlation of r = .973 was found between the HD concentration and the decrease in body weight 48 h post exposure (Fig. 3 insert). GPs exposed to Ct of 700μg × min/l (for 10 min) showed no change in body weight while those exposed to Ct of 1100 μg × min/l (for 10 min) showed 7% decrease in body-weight 48 h post exposure with recovery to control levels within 1 week. GPs exposed to Ct of either 2200 or 2530 μg × min/l (for 10 min) showed further decrease of 12–15% within 48 h post exposure. Recovery to normal weight started only about 10 days following exposure.
Fig. 2. The effect of inhalation exposure of guinea pigs to various concentrations of HD vapor on respiratory parameters. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l) while their respiratory parameters were measured with the aid of individual plethysmographs. Respiratory parameters; tidal volume (TV) and respiration rate (RR) were monitored pre, during and up to 28 days post inhalation exposure to HD vapor. Minute volume (MV) was calculated as MV = TV × RR. Note the significant (p b 0.05) decrease in RR and MV (two-way ANOVA) during the first week following exposure as well as the concentration-dependent changes mainly at day 5 (r = 0.97 and 0.94 respectively). Notice the complete recovery of respiratory parameters at 2 weeks post exposure.
Fig. 3. The effect of inhalation exposure of guinea pigs to various concentrations of HD vapor on weight gain. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). GPs' body weight was weighted at various times up to 28 days post exposure. Data presented as the percent changes from the individual weight before exposure. Note the high correlation between inhaled HD concentration and weight loss at 48 h post exposure (insert).
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Fig. 4. The effect of inhalation exposure of guinea pigs to various concentrations of inhaled HD on the recovery of trachea injury 1 month post exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on H&E stained trachea sections, 30 days post exposure. A. Control trachea; B. Trachea following inhalation of 700 μg × min/l HD; C. Trachea following inhalation of 700 μg × min/l HD; D. Trachea following inhalation of 1100 μg × min/l HD. Notice the hyperplasia of goblet cells following the lower dose (arrow in C) and the increase in micro-vesicles and the abnormal growth of the epithelium in B. At the higher doses 4D the increase in number of blood cells and fragments of detached epithelia accumulated in the trachea's lumen are clearly seen. Bar indicates 100 μm.
Fig. 5. The effect of inhalation exposure of guinea pigs to various concentrations of HD on the recovery of lung injury 1 month post exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on lung sections stained with H&E, 30 days post exposure. A. Control; B. Exposed lung following inhalation of 700 μg × min/l HD; C. Exposed lung following inhalation of 1100 μg × min/l HD; D. Exposed lung following inhalation of 2200 μg × min/l HD. Notice the dose-dependent increase in the cellular area and the decrease in the alveolar air space. Bar in a, b and d indicates 100 μm, in c indicates 500 μm.
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Clinical symptoms Clinical evaluation of the exposed guinea pigs was performed at 3 and 6 h following exposure and at different time intervals following exposure for up to 1 month. Exposing GPs to Ct of 700 μg × min/l HD induced only mild symptoms that subsided within 1 week after exposure. However, exposure to higher HD concentrations induced moderate to severe clinical symptoms that started as early as 1 h post exposure. The percentage of GPs with severe symptoms increased as the doses increased. GPs exposed to Ct of 700μg × min/l recovered within 5 days without any further visible symptoms. As a whole, except for the lower Ct group (700 μg × min/l) that showed only mild toxicity signs, animals exhibited similar toxicity signs with a dose-dependent variation in severity and death rate. Erythematic and swelling nose associated with extensive mucous secretion (with or without bleeding) were noted 3 h post exposure. At 6 h post exposure most of the GPs had breathing difficulties, rhonchi and dyspnea and even death (n = 1) was noted. One day post exposure five out of eight GPs died at the higher Ct (3100 μg × min/l) and one to two deaths occurred at each of the lower Cts groups. The GPs developed inflammatory response in ocular tissues, concomitant with extensive mucous secretion (mostly bloody) and nose swelling. Additional incidences of death occurred during the first week. Recovery was relatively fast. Detectable signs of recovery were evident seven or more days following exposure. At 2 weeks post exposure most of the survived GPs seemed to fully recover from the HD exposure. However, the physical condition of two of the GPs started to deteriorate again 3 weeks following exposure and one of them died on the 28th day. Thirty days following exposure the survived GPs were divided into two groups: one designated for histopathology and the other for biochemical evaluation of bronchoalveolar lavage and lung homogenate.
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Lung A dose-dependent increase of cellular infiltration was observed in the lung at 1 month following exposure (Fig. 5). As a result, the surface area covered by cells, extra cellular matrix and congested blood vessels increased in the lungs of GPs exposed to the higher HD concentrations (p b 0.025). Thus, lung volume available for air exchange reduced to 50% compared to 68% in the control animals (Fig. 6B). Biochemical evaluation of the BAL BAL was collected from half of the survived GPs 30 days following exposure. Evaluation of BAL content revealed no significant differences between the control group and the GPs exposed to the low Cts (700 or 1100 μg × min/l) in any of the parameters tested (Fig. 7). In two GPs that survived the exposure to HD concentration above the LCt50 a significant increase in BAL protein, LDH, total cell count and number of neutrophils was found 30 days following exposure (Fig. 7) indicating that despite the apparent clinical
Histopathology A clinical recovery of the animals started around 1 week following exposure to HD vapor. However the histological evaluation of tissues taken from lung and trachea 1 month following HD inhalation revealed an incomplete healing in both tissues. The extent of residual damage and pathological regeneration was dose-dependent. For practical purposes (mainly due to low survival rate at high doses) the GPs were divided into three groups according to the concentration of HD during exposure (control, 700–1100 μg × min/l (below LCt50), 2200–2530 μg × min/l (above LCt50)). Trachea The regenerative epithelium of the trachea varied from single layer (Fig. 4B) to stratified epithelium (Fig. 4C) displaying incomplete regeneration and the differentiation of the epithelium differed from control (Fig. 4A). A stratified epithelium was seen following exposure to the lower concentration (700 μg × min/l), however, even in this group most of the animals had only about 75% of the surface area covered by a multilayered epithelium. The new epithelium contained cilia and goblet cells but hyperplasia of the latter was often seen (Fig. 4C). In GPs exposed to higher concentrations, more severe pathological findings were observed, the regenerative epithelium consisted of one to three layers which were often sloughed off and microvesications were noted at the basal layer. The epithelium displayed large vacuoles and necrotic cells (Fig. 4B and D). Cilia were seen only in part of the superficial cells and the number of goblet cells was lower than in controls. Edema and blood congestion were found in the lamina propria (Fig. 4). Morphometric analysis of the extent of stratified epithelial coverage is depicted in Fig. 6A. As mentioned above, the regeneration of the epithelium was in reverse correlation with the inhaled concentration.
Fig. 6. Quantitative histopathological evaluation of the recovery of respiratory system from HD vapor exposure. Guinea pigs were exposed head only, for 10 min to various concentrations of inhaled HD (μg × min/l). Histological evaluation was performed on trachea and lung sections stained with H&E 30 days post exposure. A—The effect of inhaled HD concentration on the percentage of lung cellular volume 1 month post exposure. In every animal a total of 40 randomly picked fields in two main lobes were measured. Note the concentration-dependent increase in cellular volume. B—The effect of inhaled HD concentration on the degree of epithelial regeneration in the trachea 1 month following exposure. Measurements were conducted on three consecutive sections in the middle of each trachea. The areas with no epithelium were either from recurrent molting of fragments of lately regenerated epithelia due to the appearance of micro-blisters or to no recovery at all. We could not differentiate one from the other.
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Fig. 7. The effect of inhalation exposure of guinea pigs to various concentrations of inhaled HD on BAL parameters 1 month post exposure. BAL was collected from surviving GP, as described in the Methods section, under deep anesthesia, 30 days post exposure and analyzed according to the described methods. Notice the trend of increase in several parameters even for GPs exposed to the lower HD concentrations. Significant residual damage was detected only in the BAL in few parameters and only following exposure to the highest HD concentration. ⁎ indicates significant differences (p b 0.05) from control group.
healing, the lungs were inflamed and not fully recovered. No significant differences were noted in the lung homogenates, in any of the measured parameters in the exposed GP vs control GP, 30 days post exposure. Discussion The primary target organs for airborne chemicals are the respiratory tract and the lungs, due to their close proximity to the environment. Lung injuries are considered to be the main cause of long-term disability among patients exposed to sulfur mustard gas. Most of the accumulated data about HD injury and the various suggested treatments came from human casualties during World War I and the Iran–Iraq war. Although the data are based on large number of casualties and prolonged follow-up, there is no consensus on medical management of HD casualties other than the need for thorough decontamination and the supply of supportive care. In the case of war casualties, it is well understood that there is no way to estimate the inhaled doses and to correlate them with the extent of injury. We have shown in the current study that gross clinical evaluation during remission period was not a good predicator for future lung injury and did not accurately represent mild or even moderate changes occurring in the lungs. Since treatment is based
mainly upon supportive care, no treatment is expected to be provided during remission periods where no clinical adverse symptoms are noted. Thus, any quest for treatment of pulmonary injury, following HD inhalation, will require reliable information about changes occurring in the lungs and trachea during the remission periods. Several animal models have been reported in the literature. In most of them animals were subjected to whole-body HD exposure (Papirmeister et al., 1991 and the references cited therein; Rao et al., 1999). In the current study we have presented a reliable animal model for inhalation exposure to HD vapor using head only, individual plethysmographic exposure chambers, and utilizing online measurements of HD concentration and respiratory parameters. This enabled us to calculate the individual HD doses inhaled by each of the GPs at any time of the exposure. GPs have been selected as an animal model for inhalation toxicity since: 1. GPs are the most commonly used small animal species in preclinical studies related to lung diseases such as asthma and COPD (Canning and Chou, 2008). 2. GPs have been extensively used to study pulmonary alteration associated with inhalation of irritants (Castranova et al., 2002). 3. GPs are also a useful model for investigating the effects of drugs (West and Fernandez, 2004) their heart demonstrate similarity to human heart failure with respect to calcium cycling, myosin isoforms and myocardial function (Hasenfuss, 1998). 4. This together with the
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complexity of handling needed for the measuring respiratory parameters in awake restrained animals that required calm and easy manageable animals. These characteristics make the GP the most suitable animal for this research. Furthermore, the clinical symptoms that were observed in the GPs following exposure to HD vapor such as; cough, dyspnea, hemorrhage exudates from the nose and necrosis of upper and lower airways as well as their time course resembled those described for human patients (Somani and Babu, 1989; Freitag et al., 1991; Balali Mood and Hefazi, 2006). Additionally, the distribution of incidence of death occurring in the GPs model following inhalation exposure to HD is in consistent with other animal models see Papirmeister et al. (1991) and Rao et al. (1999). In humans, exposure to high doses of HD vapor was reported to cause damage to the terminal airways with productive cough, dyspnea, and possibly hemorrhage in the alveoli (Sohrabpour, 1984, 2004). Severe coughing with necrosis and inflammation of the mucosa were also noted (Balali, 1984). This was followed by obstruction of upper and lower airways (Freitag et al., 1991) leading to adult respiratory distress syndrome (ARDS) (Balali Mood et al., 2005; Balali Mood and Hefazi, 2006). Infection of the respiratory tract was a common complication that developed 36–48 h post exposure and was accompanied by a release of inflammatory mediators (Freitag et al., 1991; Balali Mood and Hefazi, 2006). Although infection is not considered a problem in the animal model, elevation of inflammatory mediators release following HD exposure were noted (Casillas et al., 1997; Amir et al., 2000; Dachir et al., 2002; Gao et al., 2007). This could develop into bronchopneumonia with death occurring between the second day and the fourth week after exposure (Papirmeister et al., 1991). In our GP model most of the severe symptoms occurred as early as 6 h post exposure. Most of the incidents of death occurred within the first 24 h followed by sporadic death for up to 4 weeks. GPs in our model seemed to be more sensitive than mice that started to die only 5 days after exposure to one or two LCt50 of HD (Kumar and Vijayaraghavan, 1998). In our model, during the first hours and up to a week post exposure, there was a dose-dependent progressive decrease in MV and RR. Guinea pigs exposed to the highest doses reduced RR down to 25% of its original values. This reduction together with severe lung edema that reduced oxygen diffusion into the blood (Allon, et al. unpublished results) might be the main cause of death following inhalation of HD. Recovery from lung injury in our GP model seemed to be fast, respiratory parameters returned to normal within 2 weeks as well as feeding behavior and weight gain. However, two GPs exhibited deterioration in their clinical conditions and one of them died 28 days post exposure. Fast recovery was also reported by Papirmeister et al. (1991) but with some residual irritations that were persistent for as long as 6 months. Although we could hardly find any residual irritation using clinical observations, histopathological and biochemical evaluation of the lungs and tracheas conducted 4 weeks post exposure, revealed dose-dependent residual injuries. The regeneration of the epithelial cells in the trachea was fast but incomplete with abnormal differentiation characterized by hyperplasia of goblet cells, with large vacuoles, and sloughing of the regenerated epithelium from the basal membrane. Indeed, epithelial debris are often observed in the lumen of the respiratory tract and may be the cause of congestion and blockage of patients' airways that required specific drastic therapeutic measures to relieve the recurrent congested trachea (Freitag et al., 1991; Rees et al., 1991). Late recurrent injuries were reported also in an experimental model of GPs following HD aerosol exposure (Van Helden et al., 2004; Beheshti et al., 2006). The abnormal histopathological changes reported in the current research are in corroboration with the changes in BAL biochemistry that were noted for most of the parameters tested. Although, significant changes were found only following exposure to the high HD concentrations, nonsignificant trends of changes were noted even following the lower concentrations of HD. This abnormal
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recovery may be partly due to cross-linkage of DNA strands (Rao et al., 1999), abnormal growth, recurrent inflammation, blistering and dissociation of pieces of epithelial tissue into the trachea. Recurrent inflammation has been considered to be the main cause for airway remodeling in asthmatic patients (Broide, 2008 and the references cited therein). Some of the airway remodeling changes closely resembled the features seen following HD recovery. Thus, it is suggested that some of the remodeling occurred during HD recovery were due to the continued inflammatory response of HD injury and that drugs affecting airway remodeling and/or inflammation may ameliorate HD recurrent injuries. Changes at the lower lung epithelium are probably the cause for the recurrent respiratory problems and of delayed destructive pulmonary sequels such as chronic bronchitis, asthma, bronchiectasis large airway narrowing, and pulmonary fibrosis described in human patients (Balali Mood and Hefazi, 2006). In summary, a reproducible inhalation exposure system for HD was presented using GPs as animal model. The system will be used to characterize the initial clinical changes following exposure and to test various potential treatments.
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