Robyn B. Lee

TEMPORAL CHANGES IN RESPIRATORY DYNAMICS IN MICE EXPOSED TO PHOSGENE

One hallmark of phosgene inhalation toxicity is the latent formation of life-threatening, noncardiogenic pulmonary edema. The purpose of this study was to investigate the effect of phosgene inhalation on respiratory dynamics over 12 h. CD-1 male mice, 25-30 g, were exposed to 32 mg/m 3 (8 ppm) phosgene for 20 min (640 mg min/m 3) followed by a 5-min air washout. A similar group of mice was exposed to room air for 25 min. After exposure, conscious mice were placed unrestrained in a whole-body plethysmograph to determine breathing frequency (f), inspiration (Ti) and expiration (Te) times, tidal volume (TV), minute ventilation (MV), end inspiratory pause (EIP), end expiratory (EEP) pause, peak inspiratory flows (PIF), peak expiratory flows (PEF), and a measure of bronchoconstriction (Penh). All parameters were evaluated every 15 min for 12 h. Bronchoalveolar lavage fluid (BALF) protein concentration and lung wet/dry weight ratios (W/D) were also determined at 1, 4, 8, and 12 h. A treatment × time repeated-measures two-way analysis of variance (ANOVA) revealed significant differences between air and phosgene for EEP, EIP, PEF, PIF, TV, and MV, p ≤ .05, across 12 h. Phosgene-exposed mice had a significantly longer mean Ti, p ≤ .05, compared with air-exposed mice over time. Mice exposed to phosgene showed marked increases (approximately double) in Penh across all time points, beginning at 5 h, when compared with air-exposed mice, p ≤ .05. BALF protein, an indicator of air/blood barrier integrity, and W/D were significantly higher, 10- to 12-fold, in phosgene-exposed than in air-exposed mice 4-12 h after exposure, p ≤ .001 and p ≤ .05, respectively. These results indicate that exposure to phosgene causes early bronchoconstriction, a temporal obstructivelike injury pattern, and disruption of mechanical rhythm largely regulated by the progressive production of pulmonary edema on airway flow. Potential therapeutic intervention may include compounds that produce bronchodilation and mechanical ventilation support if warranted.

Cutaneous Uptake of 14C-Hd Vapor By The Hairless Guinea Pig

The hairless guinea pig (HGP) is used by our laboratory to model the human cutaneous response to sulfur mustard (HD), bis(2-chloroethylsulfide), exposure. We determined the HD content in the skin of HGP after a 7-min exposure to vapors saturated with a mixture of HD and 14C-HD. Concentration/time (CT) values in the range of 2 micrograms/cm2/min were determined by counting skin 14C disintegrations per min (dpm) in animals euthanized immediately after exposure. These values are similar to human penetration rates obtained by other investigators. A rate curve monitoring the reduction in skin 14C dpm was developed for animals euthanized between 0 and 24 hr post- exposure. This curve showed the greatest change after 1 hr. The epidermal (62%) to dermal (38%) ratio of 14C at 24 hr was measured for two animals. We saw no site preference for HD penetration among the 8 sites used. The 14C content of template adhesive tape was determined to follow HD distribution. These results contribute to a better understanding of the cutaneous response to HD in the HGP model.

Determination of LCt50s in Anesthetized Rats Exposed to Aerosolized Nerve Agents

Nerve agents pose a threat to the respiratory tract with exposure that could result in acute compromised lung performance and death. The determination of toxicity by inhalation is important for the rational development of timely therapeutic countermeasures. This study was designed to deliver aerosolized dilute nerve agents in a dose-response manner to investigate the extent of lethality of nerve agents: soman, sarin, VX and VR. Male rats (240–270 g) were anesthetized intramuscularly with 10 mg/kg xylazine and 90 mg/kg ketamine. Following anesthesia, rats were intubated with a glass endotracheal tube (ET) and placed in a glove box. The ET was connected to a closed circuit nebulizer system (Aeroneb, Aerogen, Inc.) that delivered a particle size of < 2.0 µm and was in series between the ventilator and the ET. Nerve agents were delivered by a small animal ventilator set for a volume of 2.5 mL × 60–80 breaths/min. VX or VR were nebulized and delivered in concentrations ranging from 6.25–800 µg/kg over a 10-min exposure time period. Sarin (GB) or soman (GD), 6.5–1250 µg/kg, were delivered in a similar manner. Lethality by inhalation occurred either during the 10-min exposure period or less than 15 min after the cessation of exposure. Survivors were euthanized at 24 h postexposure. LCt50 estimates (± 95% confidence intervals [CIs]) were obtained from the sequential stage-wise experiments using the probit analysis. Probit analysis revealed that the LD50 for VX was 110.7 µg/kg (CI: 73.5–166.7), VR 64.2 µg/kg (CI: 42.1–97.8); soman (GD), 167 µg/kg (CI: 90–310), and sarin (GB), 154 µg/kg (CI: 98–242), respectively. Although VR is a structural isomer of VX, the compounds appear to be markedly different in terms of toxicity when delivered by aerosol. These relationships were converted to actual 10 min LCt50 equivalents: VX = 632.2, VR = 367, GD = 954.3 and GB = 880 mg·min/m3. Validation of exposure was verified by the determination of blood levels of acetylcholinesterase (AChE) across doses for the agent VR.

A Combination Drug Treatment Against Ocular Sulfur Mustard Injury

The eye is considered to be one of the most sensitive organs to sulfur mustard [bis(2‐chloroethyl) sulfide (SM)], with injuries ranging from mild conjunctivitis to advanced corneal disease. Even mild ocular involvement from sulfur mustard exposure can result in both physical and psychological incapacitation. In this study we explored the use of Food and Drug Administration (FDA) approved medications (prednisolone acetate ophthalmic suspension, triamcinolone, and cefazolin) as ocular treatments for sulfur mustard injury. Female New Zealand White rabbits were divided into a SM positive control group (n = 8) and a single treatment group (n = 7). At 10, 20, 30, 60, 90, and 120 min after SM exposure, two drops of prednisolone acetate ophthalmic suspension was administered to each treatment group rabbit while the control group received saline drops. At 120 min after SM exposure, each treatment group animal received a single 1.0 mL sub‐Tenons injection containing 20 mg triamcinolone and 50 mg cefazolin. Control group rabbits did not receive an injection. Rabbits were observed for a total of 16 weeks after SM exposure. Corneal thickness, corneal stromal injury, neovascularization (NV), eyelid notching, and chemosis were recorded weekly for 6 consecutive weeks and on week 16 after exposure. The SM treatment group at weeks 2, 3, and 4 had a significantly lower index value for corneal thickness than the SM positive control group. For corneal stromal injury, NV, eyelid notching, and chemosis, significant evidence of a protective effect due to treatment was seen at weeks 4, 5, and 6. In addition, corneal stromal injury was reduced at weeks 2 and 3 and notching at week 2. By week 3, all SM positive control animals developed NV in contrast to 1 of 7 treatment animals. By week 6 all positive control animals still exhibited NV compared to 2 of 7 treatment animals. These data suggest that prednisolone acetate suspension dosed for the first 2 h after SM exposure followed by a single sub‐Tenons injection of a triamcinolone/cefazolin combination is effective in treating the early stages of corneal injury from SM exposure.

Comparison of hematologic consequences and efficacy of p-aminophenones in mice

Controlled methemoglobin (MHb) formation is one strategy employed to counter cyanide (CN) toxicity. Currently available MHb formers present certain drawbacks and limitations. The purpose of this study was to characterize, in mice, the hematologic effects of the MHb-forming compound p-aminopropiophenone (PAPP), and two structurally-related p-aminophenones, p-aminoheptanoylphenone (PAHP) and p-aminooctanoylphenone (PAOP). Although these three p-aminophenones have been shown previously to be efficacious as pretreatments against CN, a more complete understanding of their hematologic effects is lacking. In addition, because the active form of PAPP has been shown to be its N-hydroxy metabolite, the N-hydroxy metabolites of PAPP, PAHP and PAOP were also tested. Using a hemoximeter, blood samples obtained -2 to +180 min relative to intramuscular (i.m.) or intraperitoneal (i.p.) drug injections were evaluated. Sodium nitrite (NaNO(2)) and the appropriate solvents served as the positive and negative controls, respectively. Dose-, time-, route-, and compound-related effects were observed. MHb and sulfhemoglobin levels increased, whereas levels of those parameters related to oxygen-carrying capacity of the blood, such as, oxygen saturation and oxyhemoglobin decreased. In general, the effects of PAHP and PAOP were longer lasting than those of PAPP and NaNO(2). Furthermore, PAPP and NaNO(2) were equally effective with either route of administration. Conversely, PAHP and PAOP showed larger effects when administered i.p. versus i.m. The animals treated with N-hydroxy metabolites of the p-aminophenones also showed similar changes in the hematological parameters measured. N-hydroxy PAPP was shown to be the most rapidly acting MHb-forming compound examined in this series. It could achieve therapeutic concentrations of MHb within 2 min and thus may be considered as a treatment for CN intoxication. Although additional work is needed, these data provide information that will be useful for the successful development of improved anti-CN MHb formers.

Younger rats are more susceptible to the lethal effects of sarin than adult rats: 24 h LC50 for whole-body (10 and 60 min) exposures

Abstract Chemical warfare nerve agents (CWNA) inhibit acetylcholinesterase and are among the most lethal chemicals known to man. Children are predicted to be vulnerable to CWNA exposure because of their smaller body masses, higher ventilation rates and immature central nervous systems. While a handful of studies on the effects of CWNA in younger animals have been published, exposure routes relevant to battlefield or terrorist situations (i.e. inhalation for sarin) were not used. Thus, we estimated the 24 h LC50 for whole-body (10 and 60 min) exposure to sarin using a stagewise, adaptive dose design. Specifically, male and female Sprague-Dawley rats were exposed to a range of sarin concentrations (6.2–44.0 or 1.6–12.5 mg/m³) for either 10 or 60 min, respectively, at six different times during their development (postnatal day [PND] 7, 14, 21, 28, 42 and 70). For male and female rats, the lowest LC50 values were observed for PND 14 and the highest LC50 values for PND 28. Sex differences were observed only for PND 42 for the 10 min exposures and PND 21 and 70 for the 60 min exposures. Thus, younger rats (PND 14) were more susceptible than older rats (PND 70) to the lethal effects of whole-body exposure to sarin, while adolescent (PND 28) rats were the least susceptible and sex differences were minimal. These results underscore the importance of controlling for the age of the animal in research on the toxic effects associated with CWNA exposure.

Age-Related Susceptibility to Epileptogenesis and Neuronal Loss in Male Fischer Rats Exposed to Soman and Treated With Medical Countermeasures

Elderly individuals compose a large percentage of the world population; however, few studies have addressed the efficacy of current medical countermeasures (MCMs) against the effects of chemical warfare nerve agent exposure in aged populations. We evaluated the efficacy of the anticonvulsant diazepam in an old adult rat model of soman (GD) poisoning and compared the toxic effects to those observed in young adult rats when anticonvulsant treatment is delayed. After determining their respective median lethal dose (LD50) of GD, we exposed young adult and old adult rats to an equitoxic 1.2 LD50 dose of GD followed by treatment with atropine sulfate and the oxime HI-6 at 1 min after exposure, and diazepam at 30 min after seizure onset. Old adult rats that presented with status epilepticus were more susceptible to developing spontaneous recurrent seizures (SRSs). Neuropathological analysis revealed that in rats of both age groups that developed SRS, there was a significant reduction in the density of mature neurons in the piriform cortex, thalamus, and amygdala, with more pronounced neuronal loss in the thalamus of old adult rats compared with young adult rats. Furthermore, old adult rats displayed a reduced density of cells expressing glutamic acid decarboxylase 67, a marker of GABAergic interneurons, in the basolateral amygdala and piriform cortex, and a reduction of astrocyte activation in the piriform cortex. Our observations demonstrate the reduced effectiveness of current MCM in an old adult animal model of GD exposure and strongly suggest the need for countermeasures that are more tailored to the vulnerabilities of an aging population.