Gene Expression and Pathway Analysis in Rat Brain and Liver After Exposure to Royal Demolition Explosive (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine).

The nitramine explosive, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is associated with acute and chronic toxicity in mammals and targets both the central nervous system and liver. After a single oral dose of RDX in male rats, the systemic distribution of RDX and the toxicodynamic response was measured using clinical chemistry and Affymetrix Rat Genome® 230 2.0 gene expression arrays, respectively. Nominal doses of 0, 9 and 36 mg/kg pure RDX were administered to animals followed by liver, cerebral cortex, and hippocampus gene expression analysis at 0, 3.5, 24, and 48 hours. RDX quickly entered the liver and brain, increasing up to 24 hours. For the 36 mg/kg dose, RDX was still measurable in liver and brain at 48 hours, but was non-detectible for the 9 mg/kg dose. At 3.5 hours, the time within which most convulsions reportedly occur after RDX ingestion, the hippocampus displayed the highest response for both gene expression and pathways, while the cortex was relatively non-responsive. The top 2 impacted pathways, primarily involved in neurotransmission, were the GABAergic and glutamatergic pathways. High numbers of genes also responded to RDX in the liver with P450 metabolism pathways significantly involved. Compared to the liver, the hippocampus displayed more consistent biological effects across dose and time with neurotransmission pathways predominating. Overall, based on gene expression data, RDX responses were high in both the hippocampus and liver, but were minimal in the cerebral cortex. These results identify the hippocampus as an important target for RDX based on gene expression.

Assessment of zebrafish embryo photomotor response sensitivity and phase-specific patterns following acute- and long-duration exposure to neurotoxic chemicals and chemical weapon precursors.

Zebrafish are an attractive model for chemical screening due to their adaptability to high-throughput platforms and ability to display complex phenotypes in response to chemical exposure. The photomotor response (PMR) is an established and reproducible phenotype of the zebrafish embryo, observed 24 h post-fertilization in response to a predefined sequence of light stimuli. In an effort to evaluate the sensitivity and effectiveness of the zebrafish embryo PMR assay for toxicity screening, we analyzed chemicals known to cause both neurological effects and developmental abnormalities, following both short (1 h) and long (16 h+) duration exposures. These include chemicals that inhibit aerobic respiration (eg, cyanide), acetyl cholinesterase inhibitors (organophosphates pesticides) and several chemical weapon precursor compounds with variable toxicity profiles and poorly understood mechanisms of toxicity. We observed notable concentration-responsive, phase-specific effects in the PMR after exposure to chemicals with a known mechanism of action. Chemicals with a more general toxicity profile (toxic chemical weapon precursors) appeared to reduce all phases of the PMR without a notable phase-specific effect. Overall, 10 of 20 chemicals evaluated elicited an effect on the PMR response and eight of those 10 chemicals were picked up in both the short- and long-duration assays. In addition, the patterns of response uniquely differentiated chemical weapon precursor effects from those elicited by inhibitors of aerobic respiration and organophosphates. By providing a rapid screening test for neurobehavioral effects, the zebrafish PMR test could help identify potential mechanisms of action and target compounds for more detailed follow-on toxicological evaluations. Approved for public release: distribution unlimited.

Gene expression profiling of rat hippocampus following exposure to the acetylcholinesterase inhibitor soman.

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.

Global gene expression in rat brain and liver after oral exposure to the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

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.

Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide: gene expression profiles correlate with treatment efficacy and an established clinical endpoint.

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent. Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide, and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears after exposure to SM in the presence or absence of treatment compounds. Mouse ears were topically exposed to SM alone or were pretreated for 15 min with a treatment compound and then exposed to SM. Ear tissue was harvested 24 h after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone. Correlation coefficients between gene expression level and ear weight revealed genes involved in mediating responses to both SM exposure and treatment compounds. These data provide a basis for elucidating the mechanisms of response to SM and drug treatment and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.

Genomic analysis of murine pulmonary tissue following carbonyl chloride inhalation.

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 x time amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg x 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 alpha-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as gamma-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.

Comparison of non-human primate and human whole blood tissue gene expression profiles.

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.

Genomic analysis of rodent pulmonary tissue following bis-(2-chloroethyl) sulfide exposure.

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 by p 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.