© Crown copyright (2016), Dstl. Published under the OGL.
Article pubs.acs.org/crt
Acute Gene Expression Profile of Lung Tissue Following Sulfur Mustard Inhalation Exposure in Large Anesthetized Swine Bronwen J. A. Jugg,† Heidi Hoard-Fruchey,*,‡ Cristin Rothwell,‡ James F. Dillman,‡ Jonathan David,† John Jenner,† and Alfred M. Sciuto‡ †
CBR Division, Dstl Porton Down, Salisbury, Wiltshire SP4 0JQ, U.K. US Army Medical Research Institute of Chemical Defense, 2900 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010, United States
‡
S Supporting Information *
ABSTRACT: Sulfur mustard (HD) is a vesicating and alkylating agent widely used on the battlefield during World War I and more recently in the Iran-Iraq War. It targets the eyes, skin, and lungs, producing skin burns, conjunctivitis, and compromised respiratory function; early acute effects lead to long-term consequences. However, it is the effects on the lungs that drive morbidity and eventual mortality. The temporal postexposure response to HD within lung tissue raises the question of whether toxicity is driven by the alkylating properties of HD on critical homeostatic pathways. We have established an anesthetized swine model of inhaled HD vapor exposure to investigate the toxic effects of HD 12 h postexposure. Large white female swine were anesthetized and instrumented prior to exposure to air, 60 (sublethal) or 100 μg·kg−1 (∼LD40) doses of HD (10 min). Physiological parameters were continuously assessed. Data indicate that exposure to 100 μg·kg−1 HD lowered arterial blood oxygenation and increased shunt fraction and lavage protein compared with those of airexposed controls and the 60 μg·kg−1 dose of HD. Histopathology showed an increased total pathology score between the 100 μg· kg−1 HD group and air-exposed controls. Principal component analysis of differentially expressed genes demonstrated a distinct and separable response of inhaled HD between air-exposed controls and the 60 and 100 μg·kg−1 doses of HD. Canonical pathway analysis demonstrated changes in acute phase response signaling, aryl hydrocarbon receptor signaling, NRF-2 mediated oxidative stress, and zymosterol biosynthesis in the 60 and 100 μg·kg−1 HD dose group. Transcriptional changes also indicated alterations in immune response, cancer, and cell signaling and metabolism canonical pathways. The 100 μg·kg−1 dose group also showed significant changes in cholesterol biosynthesis. Taken together, exposure to inhaled HD had a significant effect on physiological responses coinciding with acute changes in gene expression and lung histopathology. In addition, transcriptomics support the observed beneficial effects of N-acetyl-L-cysteine for treatment of acute inhalation HD exposure.
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INTRODUCTION Sulfur mustard (bis(2-chloroethyl) sulfide; HD) is a chemical warfare agent that was first used on the battlefield in World War I and in several conflicts around the globe.1,2 HD has been classified as a Class 1 human carcinogen3 on the basis of animal4,5 and epidemiological evidence.6,7 It is known to alkylate a variety of cellular macromolecules by nucleophilic interaction, especially DNA.8 One proposed mechanism is that HD cyclizes and forms the reactive sulfonium ion responsible for the alkylation of DNA that results in the loss of important enzyme cofactors such as glutathione9 and the modification of a number of biochemical processes. These processes cause diverse effects such as the activation of proteases,10,11 loss of energy storage molecules such as ATP12 and NAD,13 activation of apoptosis,14,15 loss of calcium regulation,16 loss of cell cycle regulation,17 perturbation of the cytoskeleton,18 cytokine production,19,20 and modulation of basement membrane components.21 The evidence suggests that many of these This article not subject to U.S. Copyright. Published 2016 by the American Chemical Society
cellular pathways contribute to HD-induced cellular and tissue damage.22 HD exposure can also cause oxidative stress and hence reactive oxygen species-induced tissue damage in the lung,23 liver, and kidneys.24 Depleted intracellular glutathione favors the production of endogenously produced oxygen species with subsequent peroxidation of membrane lipids.25 Reactive oxygen species produced in macrophages and polymorphonuclear leukocytes (PMNs) can lead to an inflammatory response caused by the formation of cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-4, and IL-5, platelet activating factor,26 and the reactive arachidonic acid metabolites.16 Thus, the toxic effects of HD may be the result of direct damage induced by alkylation or by the production of reactive oxygen species from activated inflammatory cells. Received: March 1, 2016 Published: September 12, 2016 1602
DOI: 10.1021/acs.chemrestox.6b00069 Chem. Res. Toxicol. 2016, 29, 1602−1610
Chemical Research in Toxicology
Article
Animals were premedicated with midazolam hydrochloride (“Hypnovel”, Roche Products Ltd., UK) by intramuscular injection (0.6 mg·kg−1) and anesthesia induced by inhalation of 5% isofluorane (“IsoFlo”, Abbott Laboratories, UK) in 70% oxygen and 30% nitrous oxide as previously described.37 Animals were intubated with a size 10 oral endotracheal (ET) tube which had been lined with polytetrafluoroethylene (PTFE) to enable safe and accurate dosing to HD vapor.37 The PTFE lining of the ET tube, which remained in place throughout the study, results in the ET tube being inflexible such that the animals are unable to clear obstructions by coughing. Therefore, from 6 h postexposure the ET tube was suctioned at hourly intervals, with the suction line restricted so that it could not pass the end of the ET tube and suction secretions from the lungs. Surgery was performed under aseptic conditions for the insertion of arterial and venous catheters and a Bonano catheter which was introduced into the bladder via open cystotomy. A Pulse Contour Continuous Cardiac Output (PiCCO) catheter for assessment of hemodynamic parameters was inserted into the right femoral artery and attached to a PiCCO monitor. Other noninvasive physiological monitoring devices were attached to a Propaq 106EL monitor.37 Throughout the study, a maintenance infusion of 0.9% w/v sodium chloride and 4% w/v glucose was delivered (2.5 mL·kg−1·h−1) to replace insensible losses. Following surgery, i.v. anesthesia was established and maintained using 1% propofol (10−12 mg·kg−1·h−1) (Fresenius Propoven , Fresenius Kabi Ltd., UK) and alfentanil hydrochloride (0.5−2.5 μg·kg−1·h−1) (Rapifen Janssen-Cilag Ltd., UK). Exposure to HD. Animals were exposed to HD as described previously.36 Briefly, animals were maintained supine while breathing spontaneously and baseline measurements taken (1 h). They were then connected to the exposure apparatus via the ET tube, a size 2 Fleisch pneumotachograph, and a sample port. HD vapor was generated from two custom Porton vapor generators operated in parallel. HD concentration was measured in real time using a Gasmet FTIR spectrometer (Quantitech, UK). Respiratory parameters were recorded in real time using eDAcq (EMMS Data Acquisition) software (Infodisp, UK) throughout, and subsequent to, the exposure. For all HD exposures, the target inhaled dose was delivered over ∼10 min. Animals were randomly assigned to air (n = 6), HD vapor at 60 μg· kg−1 (n = 3), or HD vapor at 100 μg·kg−1 (n = 7) and monitored for 12 h. Physiological Measurements. Cardiac and pulmonary physiological parameters were recorded every 5 min for the first 30 min following exposure and then every 30 min throughout the experiment (up to 12 h).37 Blood Sample Analysis. Arterial and mixed venous blood samples were taken prior to and immediately after exposure, at 30 min and then hourly. Blood was taken into appropriate anticoagulants and analyzed immediately for arterial and venous blood gas, co-oximetry, hematocrit, electrolytes, glucose, and lactate (GEM Premier 3500 blood-gas analyzer, Instrumentation Laboratories UK Ltd., UK); blood was stored for hematological analysis (4 °C) or centrifuged (3000 rpm, 10 min), and the plasma stored for future analysis (−80 °C). Total urine output was noted hourly and aliquot samples retained (−80 °C). Post-mortem and Histopathology. At 12 h postexposure or when the animal became moribund (defined as periods of asystole with a mixed venous oxygenation [SvO2] of
