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Title: Use of epr spin-trapping techniques to detect radicals from rat lung lavage fluid following sulfur mustard vapor exposure

Abstract

Although well known for skin vesicating properties, pulmonary damage and associated infections account for most of the mortality associated with sulfur mustard (HD). We have employed an in vivo HD vapor exposure model, bronchoalveolar lavage and histopathology in conjunction with electron paramagnetic resonance (EPR) techniques to provide evidence for HD-induced (free radical/lipid peroxidation associated) lung injury. Anesthetized rats were intratracheally intubated and exposed to 0.35 mg HD vapor over 50 min. Immediately, 1 hr or 24 hr after exposure, lungs were lavaged with the spin trap, alpha-phenyl-t-butyl nitrone (PBN; 0.35 mg/ml). Recovered lavage fluid was assayed by EPR spectroscopy for radical spin adducts. Airway lipid extracts were assayed for thiobarbituric acid reactive products (TBARs); while separate groups of rats were used to evaluate histopathology. EPR results show the presence of an ascorbyl radical at 1 and 24 hr, and a carbon centered PBN spin adduct at 24 hr, both indicative of lipid peroxidation. TBAR (A532nm) formation was also detected at 24 hr. Histopathology revealed multifocal separation of the bronchial epithelium from the submucosa with little or no alveolar involvement at 24 hrs. These studies provide evidence that HD may affect lungs by a free radical mechanism which produces membrane andmore » other tissue damage.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Army Medical Research Inst. of Chemical Defense, Aberdeen Proving Ground, MD (United States)
OSTI Identifier:
7239943
Alternate Identifier(s):
OSTI ID: 7239943
Report Number(s):
AD-P-008764/3/XAB
Resource Type:
Technical Report
Resource Relation:
Other Information: This article is from 'Proceedings of the Medical Defense Bioscience Review (1993) Held in Baltimore, Maryland on 10-13 May 1993. Volume 1', AD-A275 667, p113-121
Country of Publication:
United States
Language:
English
Subject:
45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEFENSE; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; CHEMICAL WARFARE AGENTS; TOXICITY; WEAPONS 450600* -- Military Technology, Weaponry, & National Defense-- Chemical & Biological-- (1990); 560300 -- Chemicals Metabolism & Toxicology

Citation Formats

Anderson, D.R., Yourick, J.J., Arroyo, C.M., Young, G.D., and Harris, L.W. Use of epr spin-trapping techniques to detect radicals from rat lung lavage fluid following sulfur mustard vapor exposure. United States: N. p., 1993. Web.
Anderson, D.R., Yourick, J.J., Arroyo, C.M., Young, G.D., & Harris, L.W. Use of epr spin-trapping techniques to detect radicals from rat lung lavage fluid following sulfur mustard vapor exposure. United States.
Anderson, D.R., Yourick, J.J., Arroyo, C.M., Young, G.D., and Harris, L.W. Thu . "Use of epr spin-trapping techniques to detect radicals from rat lung lavage fluid following sulfur mustard vapor exposure". United States. doi:.
@article{osti_7239943,
title = {Use of epr spin-trapping techniques to detect radicals from rat lung lavage fluid following sulfur mustard vapor exposure},
author = {Anderson, D.R. and Yourick, J.J. and Arroyo, C.M. and Young, G.D. and Harris, L.W.},
abstractNote = {Although well known for skin vesicating properties, pulmonary damage and associated infections account for most of the mortality associated with sulfur mustard (HD). We have employed an in vivo HD vapor exposure model, bronchoalveolar lavage and histopathology in conjunction with electron paramagnetic resonance (EPR) techniques to provide evidence for HD-induced (free radical/lipid peroxidation associated) lung injury. Anesthetized rats were intratracheally intubated and exposed to 0.35 mg HD vapor over 50 min. Immediately, 1 hr or 24 hr after exposure, lungs were lavaged with the spin trap, alpha-phenyl-t-butyl nitrone (PBN; 0.35 mg/ml). Recovered lavage fluid was assayed by EPR spectroscopy for radical spin adducts. Airway lipid extracts were assayed for thiobarbituric acid reactive products (TBARs); while separate groups of rats were used to evaluate histopathology. EPR results show the presence of an ascorbyl radical at 1 and 24 hr, and a carbon centered PBN spin adduct at 24 hr, both indicative of lipid peroxidation. TBAR (A532nm) formation was also detected at 24 hr. Histopathology revealed multifocal separation of the bronchial epithelium from the submucosa with little or no alveolar involvement at 24 hrs. These studies provide evidence that HD may affect lungs by a free radical mechanism which produces membrane and other tissue damage.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Thu May 13 00:00:00 EDT 1993},
month = {Thu May 13 00:00:00 EDT 1993}
}

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  • Sulfur mustard (HD) is a chemical warfare agent for which there is neither antidote nor adequate therapeutic protection. Animal models are employed to investigate mechanisms of injury and to evaluate protective measures against HD exposure. Researchers whose experiments involve cutaneous application of HD vapor to animals benefit from the detection and quantitation of HD at the exposed site. The ability to detect and quantify HD enables the researcher to follow safe procedures in handling skin samples. We have designed an experimental procedure to measure HD offgassing from animal models. A Minicams(R), which is a portable gas chromatograph (GC) equipped withmore » a flame photometric detector (FPD) and with online sorbent collection and desorption, was used to monitor the HD concentration. Confirming measurements were made using a two-step process that trapped HD on a Tenax sorbent offline and then transferred the sample by means of an ACEM 900 to a OC equipped with either FPD or a mass spectrometer (MS). We collected data from six experiments in which weanling pigs were exposed to saturated HD vapor via vapor caps containing 10 micro of HD. HD concentration was measured in time-weighted-average (TWA) units at a specific HD application site. The currently recommended exposure value for HD is 3 ng/l, 1 TWA unit. In five of the six experiments, Minicams HD concentration values were less than 0.5 TWA, 2 hours postexposure, and in one of the experiments, TWA Minicams concentration was less than 0.5 TWA, 5 hours post-exposure. OCIMS detection was used in three of the experiments to confirm Minicams data and to provide greater sensitivity and selectivity at 0.1 TWA. GC/MS data confirmed that HD concentrations fell below 0.1 TWA in less than S hours for a specific site.« less
  • Ozone is known to induce lipid peroxidation of lung tissue, although no direct evidence of free radical formation has been reported. The authors have used the electron paramagneticresonance (EPR) spin-trapping technique to search for free radicals produced in vivo by ozone exposure. The spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was administered i.p. to male Sprague-Dawley. The rats were then exposed for 2 hrs to either 0, 0.5, 1.0, 1.5 or 2.0 ppm ozone with 8% CO2 to increase their respiratory rate. The concentration of the radical adduct increased as a function of ozone concentration. After administration of 4-POBN, rats were exposed formore » either 0.5, 1.0, 2.0 or 4.0 hrs to either 0 or 2.0 ppm ozone (with CO2). The radical adduct concentration of the ozone-exposed groups at exposure times of 2.0 and 4.0 hrs was significantly different from that of the corresponding air control groups. A correlation was observed between the radical adduct concentration and the lung weight/body weight ratio. These results demonstrate that ozone induces the production of free radicals in rat lungs during inhalation exposure and that radical production may be involved in the induction of lung edema by ozone. This is the first direct evidence for ozone-induced free radical production in vivo.« less
  • Adult rats were exposed to 1 ppm (1.96 mg/cu m) ozone or air for 2 weeks. Animals were sacrificed at 3, 5, 7, or 14 days after the onset of exposure and samples of plasma and lung lavage obtained. Heat inactivated plasma, from animals exposed to ozone for 7 or 14 days, significantly increased DNA synthesis by lung fibroblasts compared with plasma from air exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75, and a molecular weight of 32 +/- 2 Kd. This factor has a dose-dependent effect on lungmore » fibroblasts in culture, but no significant effect on cultured pneumocyte DNA synthesis. The factor is detectable within 72 hours of exposure, and may hold some promise as a marker of early oxidant lung injury.« less
  • Adult rats were exposed to 1 ppm (1.96 mg/cu m) ozone or air for 2 weeks. Animals were sacrificed at 3, 5, 7 or 14 days after the onset of exposure and samples of plasma and lung lavage obtained. Heat inactivated plasma and lavage from animals exposed to ozone, for 5 or 7 days, significantly increased DNA synthesis by lung fibroflasts compared with plasma or lavage from air exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75, and a molecular weight of 38 + or - 3 Kd. This factormore » has a dose-dependent effect on lung pneumocyte DNA synthesis in culture. It has no effect on cultured fibroblast DNA synthesis, and is distinct from a previously described factor in the plasma of these ozone-exposed animals which enhances fibroblast DNA synthesis. The factor is detectable within 5 days of exposure, and may hold some promise as a marker of early oxidant lung injury.« less
  • Ten anesthetized hairless guinea pigs Crl:IAF(HA)BR were exposed to 10 pi of neat sulfur mustard (HD) in a vapor cup on their skin for 7 min. At 24 h postexposure, the guinea pigs were euthanatized and skin sections taken for histologic evaluation. The skin was fixed using either 10% neutral buffered formalin (NBF), McDowell Trump fixative (4CF-IG), Zenker`s formol-saline (Helly`s fluid), or Zenker`s fluid. Fixed skin sections were cut in half: one half was embedded in paraffin and the other half in plastic (glycol methacrylate). Paraffin-embedded tissue was stained with hematoxylin and eosin; plastic-embedded tissue was stained with Lee`s methylenemore » blue basic fuchsin. Skin was also frozen unfixed, sectioned by cryostat, and stained with pinacyanole. HD-exposed skin was evaluated histologically for the presence of epidermal and follicular necrosis, microblister formation, epidermitis, and intracellular edema to determine the optimal fixation and embedding method for lesion preservation. The percentage of histologic sections with lesions varied little between fixatives and was similar for both paraffin and plastic embedding material. Plastic-embedded sections were thinner, allowing better histologic evaluation, but were more difficult to stain. Plastic embedding material did not infiltrate tissue fixed in Zenker`s fluid or Zenker`s formol-saline. Frozen tissue sections were prepared in the least processing time and lesion preservation was comparable to fixed tissue. It was concluded that standard histologic processing using formalin fixation and paraffin embedding is adequate for routine histopathological evaluation of HD skin lesions in the hairless guinea pig.... Sulfur mustard, Vesicating agents, Pathology, Hairless guinea pig model, Fixation.« less