Theodore S. Moran

The Fate of Antioxidant Enzymes in Bronchoalveolar Lavage Fluid Over 7 Days in Mice with Acute Lung Injury

Characterization of lung injury is important if timely therapeutic intervention is to be used properly and successfully. In this study, lung injury was defined as the progressive formation of pulmonary edema. Our model gas was phosgene, a pulmonary edemagenic compound. Phosgene, widely used in industry, can produce life-threatening pulmonary edema within hours of exposure. Four groups of 40 CD-1 male mice were exposed whole-body to either air or a concentration × time (c × t) amount of 32-42 mg/m 3 (8-11 ppm) phosgene for 20 min (640-840 mg·min/m 3) . Groups of air- or phosgene-exposed mice were euthanized 1, 4, 8, 12, 24, 48, or 72 h or 7 days postexposure. The trachea was excised, and 800 µl saline was instilled into the lungs and washed back and forth 5 times to collect bronchoalveolar lavage fluid (BALF). The antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), total glutathione (GSH), and protein were determined at each time point. Phosgene exposure significantly enhanced both GPx and GR in phosgene-exposed mice compared with air-exposed mice from 4 to 72 h, p ≤ .01 and p ≤ .005, respectively. BALF GSH was also significantly increased, p ≤ .01, from 4 to 24 h after exposure, in comparison with air-exposed. BALF protein, an indicator of air/blood barrier integrity, was significantly higher than in air-exposed mice 4 h to 7 days after exposure. In contrast, BALF SOD was reduced by phosgene exposure from 4 to 24 h, p ≤ .01, versus air-exposed mice. Except for protein, all parameters returned to control levels by 7 days postexposure. These data indicate that the lung has the capacity to repair itself within 24-48 h after exposure by reestablishing a functional GSH redox system despite increased protein leakage. SOD reduction during increased leakage may indicate that barrier integrity is affected by superoxide anion production.

Development of a Microinstillation Model of Inhalation Exposure to Assess Lung Injury Following Exposure to Toxic Chemicals and Nerve Agents in Guinea Pigs

Respiratory disturbances due to chemical warfare nerve agents (CWNAs) are the starting point of mass casualty and the primary cause of death by these weapons of terror and mass destruction. However, very few studies have been implemented to assess respiratory toxicity and exacerbation induced by CWNAs, especially methylphosphonothioic acid S-(2-(bis(1-methylethyl)amino)ethyl)O-ethyl ester (VX). In this study, we developed a microinstillation technique of inhalation exposure to assess lung injury following exposure to CWNAs and toxic chemicals. Guinea pigs were gently intubated by placing a microcatheter into the trachea 1.5 to 2.0 cm centrally above the bifurcation. This location is crucial to deliver aerosolized agents uniformly to the lungs lobes. The placement of the tube is calculated by measuring the distance from the upper front teeth to the tracheal bifurcation, which is typically 8.5 cm for guinea pigs of equivalent size and a weight range of 250 g to 300 g. The catheter is capable of withstanding 100 psi pressure; the terminus has five peripheral holes to pump air that aerosolizes the nerve agent that is delivered in the central hole. The microcatheter is regulated by a central control system to deliver the aerosolized agent in a volume lower than the tidal volume of the guinea pigs. The average particle size of the nerve agent delivered was 1.48 ± 0.07 micrometer. The microinstillation technology has been validated by exposing the animals to Coomassie brilliant blue, which showed a uniform distribution of the dye in different lung lobes. In addition, the concentration of the dye in the lungs correlated with the dose/time of exposure. Furthermore, histopathological analysis confirmed the absence of barotraumas following micoinstillation. This novel technique delivers the agent safely, requires less amount of agent, avoids exposure to skin, pelt, and eye, and circumvents the concern of deposition of the particles in the nasal and palette due to the switching of breathing from nasal to oronasal in whole-body dynamic chamber or nose only exposure. Currently, we are using this inhalation exposure technique to investigate lung injuries and respiratory disturbances following direct exposure to VX.

A simple method for accurate endotracheal placement of an intubation tube in Guinea pigs to assess lung injury following chemical exposure.

ABSTRACT Guinea pigs are considered as the animal model of choice for toxicology and medical countermeasure studies against chemical warfare agents (CWAs) and toxic organophosphate pesticides because of the low levels of carboxylesterase compared to rats and mice. However, it is difficult to intubate guinea pigs without damaging the larynx to perform CWA inhalation experiments. We describe an easy technique of intubation of guinea pigs for accurate endotracheal placement of the intubation tube. The technique involves a speculum made by cutting the medium-size ear speculum in the midline leaving behind the intact circular connector to the otoscope. Guinea pigs were anesthetized with Telazol/meditomidine, the tongue was pulled using blunt forceps, and an otoscope attached with the specially prepared speculum was inserted gently. Insertion of the speculum raises the epiglottis and restrains the movements of vocal cord, which allows smooth insertion of the metal stylet-reinforced intubation tube. Accurate endotracheal placement of the intubation tube was achieved by measuring the length from the tracheal bifurcation to vocal cord and vocal cord to the upper front teeth. The average length of the trachea in guinea pigs (275 ± 25 g) was 5.5 ± 0.2 cm and the distance from the vocal cord to the front teeth was typically 3 cm. Coinciding an intubation tube marked at 6 cm with the upper front teeth accurately places the intubation tube 2.5 cm above the tracheal bifurcation. This simple method of intubation does not disturb the natural flora of the mouth and causes minimum laryngeal damage. It is rapid and reliable, and will be very valuable in inhalation exposure to chemical/biological warfare agents or toxic chemicals to assess respiratory toxicity and develop medical countermeasures.

Acute toxic effects of nerve agent VX on respiratory dynamics and functions following microinsillation inhalation exposure in guinea pigs.

Exposure to a chemical warfare nerve agent (CWNA) leads to severe respiratory distress, respiratory failure, or death if not treated. We investigated the toxic effects of nerve agent VX on the respiratory dynamics of guinea pigs following exposure to 90.4 μg/m3 of VX or saline by microinstillation inhalation technology for 10 min. Respiratory parameters were monitored by whole-body barometric plethysmography at 4, 24, and 48 h, 7 d, 18 d, and 4 wk after VX exposure. VX-exposed animals showed a significant decrease in the respiratory frequency (RF) at 24 and 48 h of recovery (p value.0329 and.0142, respectively) compared to the saline control. The tidal volume (TV) slightly increased in VX exposed animals at 24 and significantly at 48 h (p = .02) postexposure. Minute ventilation (MV) increased slightly at 4 h but was reduced at 24 h and remained unchanged at 48 h. Animals exposed to VX also showed an increase in expiratory (Te) and relaxation time (RT) at 24 and 48 h and a small reduction in inspiratory time (Ti) at 24 h. A significant increase in end expiratory pause (EEP) was observed at 48 h after VX exposure (p = .049). The pseudo lung resistance (Penh) was significantly increased at 4 h after VX exposure and remained slightly high even at 48 h. Time-course studies reveal that most of the altered respiratory dynamics returned to normal at 7 d after VX exposure except for EEP, which was high at 7 d and returned to normal at 18 d postexposure. After 1 mo, all the monitored respiratory parameters were within normal ranges. Bronchoalveolar lavage (BAL) 1 mo after exposure showed virtually no difference in protein levels, cholinesterase levels, cell number, and cell death in the exposed and control animals. These results indicate that sublethal concentrations of VX induce changes in respiratory dynamics and functions that over time return to normal levels.

Abdominal bloating and irritable bowel syndrome like symptoms following microinstillation inhalation exposure to chemical warfare nerve agent VX in guinea pigs

While assessing the methylphosphonothioic acid S-(2-(bis(1-methylethyl)amino)ethyl)O-ethyl ester (VX) induced respiratory toxicity and evaluating therapeutics against lung injury, we observed that the animals were experiencing abnormal swelling in the abdominal area. Nerve agent has been known to increase salivary, nasal and gastrointestinal secretion and cause diarrhea. This study was initiated to investigate the effect of VX on the gastrointestinal tract (GI) since abdominal pathology may affect breathing and contribute to the on going respiratory toxicity. The mid-abdominal diameter and the size of the lower left abdomen was measured before and after 27.3 mg/m3 VX exposure by microinstillation and at 30min intervals up to 2h post-VX exposure. Both VX and saline exposed animals exhibited a decrease in circumference of the upper abdomen, although the decrease was slightly higher in VX-exposed animals up to 1 h. The waist diameter increased slightly in VX-exposed animals from 60 to 90min post-VX exposure but was similar to saline controls. The lower left abdomen near to the cecum, 6 cm below and 2 cm to the right of the end of the sternum, showed an increase in size at 30—60 min that was significantly increased at 90—120 min post-VX exposure. In addition, VX-exposed animals showed loose fecal matter compared to controls. Necropsy at 24 h showed an increased small intestine twisting motility in VX-exposed animals. Body tissue AChE assay showed high inhibition in the esophagus and intestine in VX-exposed animals indicating that a significant amount of the agent is localized to the GI following microinstillation exposure. These results suggest that microinstillation inhalation VX exposure induces gastrointestinal disturbances similar to that of irritable bowel syndrome and bloating.

Acute microinstillation inhalation exposure to soman induces changes in respiratory dynamics and functions in guinea pigs

We investigated the toxic effects of the chemical warfare nerve agent (CWNA) soman (GD) on the respiratory dynamics of guinea pigs following microinstillation inhalation exposure. Male Hartley guinea pigs were exposed to 841 mg/m3 of GD or saline for 4 min. At 24 and 48 h post GD exposure, respiratory dynamics and functions were monitored for 75 min after 1 h of stabilization in a barometric whole-body plethysmograph. GD-exposed animals showed a significant increase in respiratory frequency (RF) at 24 h postexposure compared to saline controls. The 24-h tidal volume (TV) increased in GD-exposed animals during the last 45 min of the 75-min monitoring period in the barometric whole-body plethysmograph. Minute ventilation also increased significantly at 24 h post GD exposure. The peak inspiratory flow (PIF) increased, whereas peak expiratory flow (PEF) decreased at 24 h and was erratic following GD exposure. Animals exposed to GD showed a significant decrease in expiratory (Te) and inspiratory time (Ti). Although end inspiratory pause (EIP) and end expiratory pause (EEP) were both decreased 24 h post GD exposure, EEP was more evident. Pause (P) decreased equally during the 75-min recording in GD-exposed animals, whereas the pseudo lung resistance (Penh) decreased initially during the monitoring period but was near control levels at the end of the 75-min period. The 48-h respiratory dynamics and function parameter were lower than 24 post GD exposures. These results indicate that inhalation exposure to soman in guinea pigs alters respiratory dynamics and function at 24 and 48 h postexposure.

BHA diet enhances the survival of mice exposed to phosgene: the effect of BHA on glutathione levels in the lung.

Phosgene-induced pulmonary edema formation has been under investigation for many years. One mechanism of protection may involve the use of antioxidants. Previously, it has been shown that butylated hydroxyanisole (BHA) treatment can enhance glutathione (GSH) levels. The present study focused on dietary supplementation in mice using BHA, a phenolic compound used in food preservation. Three groups of male CD-1 mice were studied: group 1, control animals fed with Purina rodent chow 5002; group 2, fed 0.75% BHA (w/w) in 5002; and group 3, fed 1.5% BHA (w/w) in 5002. Mice were fed for 22 days. On day 23 mice were exposed to 32 mg/m(3) phosgene for 20 min in a whole-body exposure chamber. Survival rate (SR) and odds ratio (OR) were determined at 12 and 24 h. In mice that died within 12 h, the lungs were removed immediately and lung wet weights (WW), dry weights (DW), lung wet weight/body weight ratio (LWW/BW), and lung tissue total glutathione (GSH) were assessed. For 12-h data, 6 mice from the 1.5% BHA group were sacrificed for lung tissue measurements. The SR for 0.75% BHA was 80% at 12 h and 55% at 24 h, compared with 36% and 23%, respectively, for controls. For 1.5% BHA, the 12- and 24-h SR were 100% and 92%, respectively. Odds ratios of 6.9 for 0.75% BHA and 46.6 for 1.5% BHA at 12 h and 4.0 and 42 for 0. 75% and 1.5% BHA, respectively, at 24 h were significantly (chi2) higher than control diet phosgene-exposed mice. Dietary pretreatment with 0.75% and 1.5% BHA significantly enhanced lung tissue GSH, 1.8-fold (p < or =.01) and 5.8-fold (p < or =.01), respectively, compared with phosgene-exposed control diet. Both BHA-supplemented diets significantly reduced WW. Only 1.5% BHA reduced DW, a measure of lung hyperaggregation. and LWW/BW compared with control diet. In air-exposed controls, BHA induced a dose-responsive decrease in WW, DW, LWW/BW ratio, and GSH. In conclusion, dietary pretreatment with BHA at the two dose levels reduced lung edema and lethality by enhancing lung tissue GSH in mice exposed to phosgene.