Carbonyl chloride (phosgene) is a toxic industrial compound widely used in industry for the production of synthetic products, such as polyfoam rubber, plastics, and dyes. Exposure to phosgene results in a latent (1−24 h), potentially life-threatening pulmonary edema and irreversible acute lung injury. A genomic approach was utilized to investigate the molecular mechanism of phosgene-induced lung injury. CD-1 male mice were exposed whole body to either air or a concentration × time amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg × min/m3). Lung tissue was collected from air- or phosgene-exposed mice at 0.5, 1, 4, 8, 12, 24, 48, and 72 h postexposure. RNA was extracted from the lung and used as starting material for the probing of oligonucleotide microarrays to determine changes in gene expression following phosgene exposure. The data were analyzed using principal component analysis to determine the greatest sources of data variability. A three-way analysis of variance based on exposure, time, and sample was performed to identify the genes most significantly changed as a result of phosgene exposure. These genes were rank ordered by p values and categorized based on molecular function and biological process. Some of the most significant changes in gene expression reflect changes in glutathione synthesis and redox regulation of the cell, including upregulation of glutathione S-transferase α-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as γ-glutamyl cysteine synthetase). This is in agreement with previous observations describing changes in redox enzyme activity after phosgene exposure. We are also investigating other pathways that are responsive to phosgene exposure to identify mechanisms of toxicity and potential therapeutic targets.

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Bis-(2-chloroethyl) sulfide (sulfur mustard, SM) is a carcinogenic alkylating agent that has been utilized as a chemical warfare agent. To understand the mechanism of SM-induced lung injury, we analyzed global changes in gene expression in a rat lung SM exposure model. Rats were injected in the femoral vein with liquid SM, which circulates directly to the pulmonary vein and then to the lung. Rats were exposed to 1, 3, or 6 mg/kg of SM, and lungs were harvested at 0.5, 1, 3, 6, and 24 h postinjection. Three biological replicates were used for each time point and dose tested. RNA was extracted from the lungs and used as the starting material for the probing of replicate oligonucleotide microarrays. The gene expression data were analyzed using principal component analysis and two-way analysis of variance to identify the genes most significantly changed across time and dose. These genes were ranked byp value and categorized based on molecular function and biological process. Computer-based data mining algorithms revealed several biological processes affected by SM exposure, including protein catabolism, apoptosis, and glycolysis. Several genes that are significantly upregulated in a dose-dependent fashion have been reported as p53 responsive genes, suggesting that cell cycle regulation and p53 activation are involved in the response to SM exposure in the lung. Thus, SM exposure induces transcriptional changes that reveal the cellular response to this potent alkylating agent. Introductionmodification of DNA by SM has been well-characterized Bis-(2-chloroethyl) sulfide (sulfur mustard, SM)1-3 is due to the use of SM and related molecules as anticancer a carcinogen and chemical warfare agent that was used therapies (1), and covalent modification of proteins by on the battlefield during World War I and has since been SM has also been demonstrated (2-5). A variety of used in several conflicts around the globe. SM exposure molecular targets and pathways have been implicated in usedin eveal cnflctsaroud te gobe.SM xpoure the mechanism of toxicity of SM exposure (1); however, results in cutaneous, pulmonary, and ocular injury. th e mechanism s of cexposuremain;undeDespite much research, an effective medical countermeathe precise mechanisms of cellular injury remain undesure for SM exposure has not been developed because lineated. the molecular mechanism of SM toxicity is not wellThe lung is a primary target of SM vapor. Inhalation understood. SM is a potent alkylating agent capable of exposure causes pulmonary...

Soman (O-pinacolyl methylphosphonofluoridate) is a potent neurotoxicant. Acute exposure to soman causes acetylcholinesterase inhibition, resulting in excessive levels of acetylcholine. Excessive acetylcholine levels cause convulsions, seizures, and respiratory distress. The initial cholinergic crisis can be overcome by rapid anticholinergic therapeutic intervention, resulting in increased survival. However, conventional treatments do not protect the brain from seizure-related damage, and thus, neurodegeneration of soman-sensitive brain areas is a potential postexposure outcome. We performed gene expression profiling of the rat hippocampus following soman exposure to gain greater insight into the molecular pathogenesis of soman-induced neurodegeneration. Male Sprague-Dawley rats were pretreated with the oxime HI-6 (l-(((4-aminocarbonyl)pyridinio)methoxyl)methyl)-2-((hydroxyimino)methyl)-pyridinium dichloride; 125 mg/kg, ip) 30 min prior to challenge with soman (180 microg/kg, sc). One minute after soman challenge, animals were treated with atropine methyl nitrate (2.0 mg/kg, im). Hippocampi were harvested 1, 3, 6, 12, 24, 48, 72, 96, and 168 h after soman exposure and RNA extracted to generate microarray probes for gene expression profiling. Principal component analysis of the microarray data revealed a progressive alteration in gene expression profiles beginning 1 h postexposure and continuing through 24 h postexposure. At 48 h to 168 h postexposure, the gene expression profiles clustered nearer to controls but did not completely return to control profiles. On the basis of the principal component analysis, analysis of variance was used to identify the genes most significantly changed as a result of soman at each postexposure time point. To gain insight into the biological relevance of these gene expression changes, genes were rank ordered by p-value and categorized using gene ontology-based algorithms into biological functions, canonical pathways, and gene networks significantly affected by soman. Numerous signaling and inflammatory pathways were identified as perturbed by soman. These data provide important insights into the molecular pathways involved in soman-induced neuropathology and a basis for generating hypotheses about the mechanism of soman-induced neurodegeneration.

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is a synthetic, high-impact, relatively stable explosive that has been in use since WWII. Exposure to RDX can occur in occupational settings (e.g., during manufacture) or through the inadvertent ingestion of contaminated environmental media such as groundwater. The toxicology of RDX is dominated by acute clonic-tonic seizures at high doses, which remit when exposure is removed and internal RDX levels decrease. Subchronic studies have revealed few other measurable toxic effects. The objective of this study was to examine the acute effects of RDX on the mammalian brain and liver using global gene expression analysis based on a predetermined maximum internal dose. Male Sprague-Dawley rats were given a single, oral, nonseizure-inducing dose of either 3 or 18 mg/kg RDX in a gel capsule. Effects on gene expression in the cerebral cortex and liver were assessed using Affymetrix Rat Genome 230 2.0 whole genome arrays at 0, 3.5, 24, and 48 h postexposure. RDX blood and brain tissue concentrations rapidly increased between 0 and 3.5 h, followed by decreases at 24 h to below the detection limit at 48 h. Pairwise comparison of high and low doses at each time point showed dramatic differential changes in gene expression at 3.5 h, the time of peak RDX in brain and blood. Using Gene Ontology, biological processes that affected neurotransmission were shown to be primarily down-regulated in the brain, the target organ of toxicity, while those that affected metabolism were up-regulated in the liver, the site of metabolism. Overall, these results demonstrate that a single oral dose of RDX is quickly absorbed and transported into the brain where processes related to neurotransmission are negatively affected, consistent with a potential excitotoxic response, whereas in the liver there was a positive effect on biological processes potentially associated with RDX metabolism.

Gene expression profiling is an important tool in the development of medical countermeasures against chemical warfare agents (CWAs). Non-human primates (NHPs), specifically the rhesus macaque (Macaca mulatta), the cynomologus macaque (Macaca fascicularis), and the African green monkey (Chlorocebus aethiops), are vital models in the development of CWA prophylactics, therapeutics, and diagnostics. However, gene expression profiling of these NHPs is complicated by the fact their genomes are not completely sequenced, and that no commercially available oligonucleotide microarrays (genechips) exist. We, therefore, sought to determine whether gene expression profiling of NHPs could be performed using human genechips. Whole blood RNA was isolated from each species and used to generate genechip probes. Hybridization of the NHP samples to human genechips (Affymetrix Human U133 Plus 2.0) resulted in comparable numbers of transcripts detected compared with human samples. Statistical analysis revealed intraspecies reproducibility of genechip quality control metrics; interspecies comparison between NHPs and humans showed little significant difference in the quality and reproducibility of data generated using human genechips. Expression profiles of each species were compared using principal component analysis (PCA) and hierarchical clustering to determine the similarity of the expression profiles within and across the species. The cynomologus group showed the least intraspecies variability, and the human group showed the greatest intraspecies variability. Intraspecies comparison of the expression profiles identified probe sets that were reproducibly detected within each species. Each NHP species was found to be dissimilar to humans; the cynomologus group was the most dissimilar. Interspecies comparison of the expression profiles revealed probe sets that were reproducibly detected in all species examined. These results show that human genechips can be used for expression profiling of NHP samples and provide a foundation for the development of tools for comparing human and NHP gene expression profiles.