Shira Chapman

Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits

Purpose. To establish an experimental model for sulfur mustard-induced acute and delayed ocular lesions in rabbits. Methods. Rabbit eyes were exposed to sulfur mustard (HD) vapor (370, 420 µg/l) for a period of two minutes. A three months follow-up study was carried out, based on the evaluation of clinical, biochemical and histological parameters. Results. HD exposure initiated typical clinical symptoms within 2–6 hrs, characterized by eye closure, eyelid swelling, conjunctival hyperemia, corneal erosions and inflammation. The clinical signs were significantly dose-dependent and reached a peak at 24–72 hrs post exposure. Biochemical evaluation of the aqueous humor exhibited an inflammatory reaction and oxidative stress at 4 hrs after exposure, subsiding at 28 hrs after exposure. Histological examination of corneas at 48 hrs revealed epithelial denudation and marked stromal edema, accompanied by cellular infiltration. Epithelial regeneration started after 72 hrs, and recovery was almost completed within 1Ð2 weeks, depending on the HD dose. A second phase of pathological processes started as early as two weeks post exposure and was characterized by corneal edema, opacity, recurrent erosions and neovascularization. The delayed injuries were found in 25 and 40% of the eyes respectively, and when appearing, were more severe than the initial ones. Conclusions. The development of HD-induced ocular lesions in rabbits is similar to the lesions described in human casualties. Quantitative analysis of the various clinical parameters emphasizes the contribution of each tissue to the overall toxic picture. Our experimental model is useful for studying the pathological mechanisms of HD-ocular lesions, and may serve for testing potential therapies.

BENEFICIAL EFFECTS OF TOPICAL ANTI-INFLAMMATORY DRUGS AGAINST SULFUR MUSTARD-INDUCED OCULAR LESIONS IN RABBITS

Ocular injuries following sulfur mustard (HD) exposure are characterized by an inflammatory response, observed as eyelid swelling, conjunctivitis, corneal oedema and cellular infiltration starting 1–4 h after exposure, depending on dose. These effects heal partially during the first 1–2 weeks after exposure, with the later appearance of neovascularization, recurrent erosions and recurrent oedema of the cornea (delayed response). We have shown previously that topically applied steroid treatment, administered after HD exposure, attenuated the extent of neovascularization, one of the characteristics of delayed ocular pathology in rabbits.

Single whole-body exposure to sarin vapor in rats: Long-term neuronal and behavioral deficits

Freely moving rats were exposed to sarin vapor (34.2+/-0.8 microg/l) for 10 min. Mortality at 24 h was 35% and toxic sings in the surviving rats ranged from sever (prolonged convulsions) through moderate to almost no overt signs. Some of the surviving rats developed delayed, intermittent convulsions. All rats were evaluated for long-term functional deficits in comparison to air-exposed control rats. Histological analysis revealed typical cell loss at 1 week post inhalation exposure. Neuronal inflammation was demonstrated by a 20-fold increase in prostaglandin (PGE(2)) levels 24 h following exposure that markedly decreased 6 days later. An additional, delayed increase in PGE(2) was detected at 1 month and continued to increase for up to 6 months post exposure. Glial activation following neural damage was demonstrated by an elevated level of peripheral benzodiazepine receptors (PBR) seen in the brain 4 and 6 months after exposure. At the same time muscarinic receptors were unaffected. Six weeks, four and six months post exposure behavioral evaluations were performed. In the open field, sarin-exposed rats showed a significant increase in overall activity with no habituation over days. In a working memory paradigm in the water maze, these same rats showed impaired working and reference memory processes with no recovery. Our data suggest long lasting impairment of brain functions in surviving rats following a single sarin exposure. Animals that seem to fully recover from the exposure, and even animals that initially show no toxicity signs, developed some adverse neural changes with time.

Sulfur mustard toxicity in macrophages: effect of dexamethasone

Cells from the murine macrophage‐like cell line J774A.1 (J774) and cultures of primary alveolar macrophages (PAM) obtained from guinea pigs were exposed to sulfur mustard (HD, 50‐200 μM) and treated with dexamethasone (2.5 μM) 10 min after HD exposure. Cell cultures were studied at 3 and 24 h after exposure by the cleavage of Thiazolyl blue reaction (MTT) reaction and crystal violet staining (viability assays), by morphological observation and by [3H]thymidine incorporation. Exposure of J774 cells to HD caused a dose‐dependent decrease in viability that was evident at 24 h. Although no significant change in viability was observed at 3–4 h after HD exposure, a dose‐dependent decrease in [3H]thymidine incorporation was observed. Treatment with dexamethasone caused a dose‐dependent decrease in viability. However, the combined exposure to HD and dexamethasone had a synergistic effect on the decrease of cell viability. This synergistic effect is not due to a change in DNA synthesis rate because [3H]thymidine incorporation was not affected by dexamethasone. In PAM cultures, HD caused some ‘activating’ effect on [3H]thymidine incorporation and an increase in cell number at the lower dose (100 μM) but this was less at 200 μM. Both effects were reduced by dexamethasone treatment. We conclude that macrophages derived from different sources exhibit a different responsiveness to immunomodulators (HD and dexamethasone) and that dexamethasone can reduce the ‘inflammatory’ effect of HD in PAM. Published in 2000 by John Wiley & Sons, Ltd.

Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro

1 The present study characterizes the role of extracellularly added glutathione in protection against sulfur mustard (HD) toxicity in a macrophage monocyte cell line J774. 2 Toxic effects of HD depend on dose and duration of exposure with an ED50 of 50 and 75 mM for dividing and confluent cells respectively. 3 Exposure to HD, 100-200 mM caused *15% decrease in the cellular glutathione (GSH) content 2 h after exposure, pretreatment with GSH, 0.2-10 mM, elevated cellular GSH *61.5. 4 GSH pretreatment increased cell viability after HD 2-3-fold. Similar protective effects of GSH treatment were found in a human epidermoid carcinoma cell line (KB). 5 Protection by post treatment with GSH was apparent even 60 min post HD exposure. 6 No protection was afforded when the intracellular GSH concentration was elevated prior to exposure and the extracellular GSH had been washed out. However, GSH depleted cells were more sensitive to HD than normal cells, and were also protected by addition of GSH to the growth medium, although the intracellular GSH content remained low. 7 We conclude that it is essential for the GSH to be present extracellularly in order to protect cells from HD toxicity. 8 Our findings have therapeutic implications in particular for the protection of lungs after inhalation exposure to HD vapor.

Treatment of skin injuries induced by sulfur mustard with calmodulin antagonists, using the pig model.

Sulfur mustard (HD) is a potent cutaneous vesicant that penetrates rapidly through the skin, causing prolonged injuries and leading to severe incapacitation. Although there has been long and intensive efforts to find a treatment for HD skin lesions, no effective treatment is available for HD‐induced skin injuries. Recently, ointments containing calmodulin antagonists were found to be effective in preventing skin injuries induced by HD in hairless mice.

Sarin-induced brain damage in rats is attenuated by delayed administration of midazolam.

Sarin poisoned rats display a hyper-cholinergic activity including hypersalivation, tremors, seizures and death. Here we studied the time and dose effects of midazolam treatment following nerve agent exposure. Rats were exposed to sarin (1.2 LD50, 108 μg/kg, im), and treated 1 min later with TMB4 and atropine (TA 7.5 and 5 mg/kg, im, respectively). Midazolam was injected either at 1 min (1 mg/kg, im), or 1 h later (1 or 5 mg/kg i.m.). Cortical seizures were monitored by electrocorticogram (ECoG). At 5 weeks, rats were assessed in a water maze task, and then their brains were extracted for biochemical analysis and histological evaluation. Results revealed a time and dose dependent effects of midazolam treatment. Rats treated with TA only displayed acute signs of sarin intoxication, 29% died within 24h and the ECoG showed seizures for several hours. Animals that received midazolam within 1 min survived with only minor clinical signs but with no biochemical, behavioral, or histological sequel. Animals that lived to receive midazolam at 1h (87%) survived and the effects of the delayed administration were dose dependent. Midazolam 5 mg/kg significantly counteracted the acute signs of intoxication and the impaired behavioral performance, attenuated some of the inflammatory response with no effect on morphological damage. Midazolam 1mg/kg showed only a slight tendency to modulate the cognitive function. In addition, the delayed administration of both midazolam doses significantly attenuated ECoG compared to TA treatment only. These results suggest that following prolonged seizure, high dose midazolam is beneficial in counteracting adverse effects of sarin poisoning.

Effects of CBDP and MEPQ on the toxicity and distribution of [3H]-soman in mice

Soman poisoning presents a problem in terms of its detailed pathophysiology and its detoxification mechanism(s). The present study was designed to evaluate the role of carboxylesterases (CaE) and cholinesterase (ChE) in the distribution and detoxification of soman in vivo. Mice were injected (i.v.) with 0.06–1.0 LD50 of [3H]-soman, 60 min following pretreatment with either 2-O-cresyl-4H-1∶2∶3 benzodioxa-phosphorine-2-oxide (CBDP), which blocks CaE or 7-(methylethoxyphosphinyloxy)-1-methyl quinolinium iodide (MEPQ), which selectively inhibits intravascular ChE. One hour after [3H]-soman administration animals were sacrificed and whole body autoradiography was performed. High concentrations of [3H]-soman were found in lung and kidney in control mice, and low concentrations were found in central nervous system. Pretreatment with CBDP caused a 93% decrease in radioactive labelling in the lung, and a minor decrease in overall labelling, whereas pretreatment with MEPQ did not change the distribution pattern of [3H]-soman. It is concluded that lung is a major target organ for soman detoxification and that it exerts this effect by means of enzymatic reaction with soman through the abundant amounts of CaE which are present in the lung. Intravascular ChE has little (if any) effect on the distribution and detoxification of soman in vivo.

Early changes in M2 muscarinic acetylcholine receptors (mAChRs) induced by sarin intoxication may be linked to long lasting neurological effects

HighlightsThe affinity of the M2 mAChR was significantly decreased (KD value increased) in the cortex 1 & 7 days after exposing rats to 1XLD50 sarin.The significant change in the KD of the M2 mAChR was not accompanied by a significant change in Bmax 1 & 7 days post exposure to sarin (1XLD50).No gender difference was seen in the reduction of the affinity of M2 mAChR following sarin, as the KDs were elevated similarly in male and female rats. ABSTRACT The effect of sarin on the binding parameters (KD & Bmax) of M2 muscarinic acetylcholine receptor (mAChR) was studied 24 h and 1 week post exposure. Male & female Sprague‐Daweley rats were poisoned with 1XLD50 sarin (80 &mgr;g/kg, im) followed by treatment of trimedoxime bromide and atropine (7.5:5 mg/kg, im) 1 min later. Brains were removed and analyzed for M2 mAChR binding, using [3H]AFDX384, an M2 selective antagonist. A significant increase in KD of M2 mAChR was found in the cortex 24 h post poisoning, displaying elevation from 4.65 ± 1.16 to 8.45 ± 1.06 nM and 5.24 ± 0.93 to 9.29 ± 1.56 nM in male and female rats, respectively. A rise in KD was also noted 1 week following exposure from 5.04 ± 1.20 to 11.75 ± 2.78 and from 5.37 ± 1.02 to 11.66 ± 1.73 nM, presenting an added increase of 51 and 40% (compared to 24 h) in males and females, respectively. Analysis of M2 receptor density (Bmax) revealed a significant reduction of 68% in males and insignificant reduction of 22% in females, 24 h after sarin exposure which was followed by 37% recovery in males and 100% recovery in females, 1 week later. These results indicate that sarin induces a long‐term decreased affinity in M2 mAChR (elevated KDs) and a transient effect on the number of this receptor subtype (Bmax). We hypothesize that the reduced affinity of the M2 receptors (negative auto‐regulatory receptors) may cause long‐term brain deficits by impairing the normal regulation release of ACh into the synaptic cleft.

Dry Blood Spot sample collection for post-exposure monitoring of chemical warfare agents – In vivo determination of phosphonic acids using LC-MS/MS

Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots (DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1-100 ng/ml for GB and GD metabolites, and 3-100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment, after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and compatible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this technique is highly suitable for mass-casualty incidents.