Kurt A. Gust

Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus)

Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (<or= 17), indicating that 2,6-DNT exposure had relatively little impact on the brain in comparison to RDX. Birds exhibiting RDX-induced seizures accumulated over 20x more RDX in brain tissues in comparison to non-seizing birds even within a common dose. In parallel, expression patterns were unrelated among seizing and non-seizing birds exposed to equivalent RDX doses. In birds experiencing seizures, genes related to neuronal electrophysiology and signal transduction were significantly affected. Comparative toxicology revealed strong similarity in acute exposure effects between RDX and the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) regarding both molecular mechanisms and putative mode of action. In a manner similar to DDT, we hypothesize that RDX elicits seizures by inhibition of neuronal cell repolarization postaction potential leading to heightened neuronal excitability and seizures facilitated by multiple molecular mechanisms.

From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite

The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite (Colinus virginianus) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT.

Subacute toxicity of oral 2,6-dinitrotoluene and 1,3,5-trinitro-1,3,5-triazine (RDX) exposure to the northern bobwhite (Colinus virginianus)

Military activities associated with training have resulted in soil residues of munition compounds and their breakdown products. Often, these areas are valuable habitats used by birds. To evaluate the possibility of adverse effects in birds, the effects from oral exposures of 2,6-dinitrotoluene (2,6-DNT) and 1,3,5-trinitro-1,3,5-triazine (RDX) were tested using a controlled dosing regime in northern bobwhite (Colinus virginianus). Nine groups of birds of mixed sex received either corn oil or 50, 100, 190, or 350 mg 2,6-DNT/kg body weight/d or 20, 80, 125, or 180 mg RDX/kg body weight/d mixed in corn oil via gavage for 14 d. Etiology of disease was markedly different between compounds. Increased RDX exposure caused an inverse relationship to time of death, symptoms including clonic followed by tonic convulsions, and death shortly thereafter. Brain concentrations of RDX postmortem, however, were relatively consistent (mean +/- standard error, 20.5 +/- 2.9 mg/kg tissue). Observations of 2,6-DNT effects included gastrointestinal distress, dehydration, and a reduction in body mass and feed consumption. Together, these data suggest that RDX is more toxic from short, repeated exposures than 2,6-DNT, resulting in central nervous system-related effects, whereas 2,6-DNT causes gastrointestinal disturbances at higher exposures.

Investigations of transcript expression in fathead minnow (Pimephales promelas) brain tissue reveal toxicological impacts of RDX exposure

Production, usage and disposal of the munitions constituent (MC) cyclotrimethylenetrinitramine (RDX) has led to environmental releases on military facilities. The chemical attributes of RDX are conducive for leaching to surface water which may put aquatic organisms at risk of exposure. Because RDX has been observed to cause aberrant neuromuscular effects across a wide range of animal phyla, we assessed the effects of RDX on central nervous system (CNS) functions in the representative aquatic ecotoxicological model species, fathead minnow (Pimephales promelas). We developed a fathead minnow brain-tissue cDNA library enriched for transcripts differentially expressed in response to RDX and trinitrotoluene (TNT) exposure. All 4,128 cDNAs were sequenced, quality filtered and assembled yielding 2230 unique sequences and 945 significant blastx matches (E ≤10(-5)). The cDNA library was leveraged to create custom-spotted microarrays for use in transcript expression assays. The impact of RDX on transcript expression in brain tissue was examined in fathead minnows exposed to RDX at 0.625, 2.5, 5, 10mg/L or an acetone-spike control for 10 days. Overt toxicity of RDX in fathead minnow occurred only at the highest exposure concentration resulting in 50% mortality and weight loss. Conversely, Bayesian analysis of microarray data indicated significant changes in transcript expression at concentrations as low as 0.625 mg/L. In total, 154 cDNAs representing 44 unique transcripts were differentially expressed in RDX exposures, the majority of which were validated by reverse transcriptase-quantitative PCR (RT-qPCR). Investigation of molecular pathways, gene ontology (GO) and individual gene functions affected by RDX exposures indicated changes in metabolic processes involved in: oxygen transport, neurological function, calcium binding/signaling, energy metabolism, cell growth/division, oxidative stress and ubiquitination. In total, our study indicated that RDX exposure affected molecular processes critical to CNS function in fathead minnow.

SUBLETHAL EFFECTS OF SUBACUTE EXPOSURE TO RDX (1,3,5-TRINITRO-1,3,5-TRIAZINE) IN THE NORTHERN BOBWHITE (COLINUS VIRGINIANUS)

Northern bobwhite (Colinus virginianus) were orally exposed via gavage to 0, 0.5, 3, 8, 12, or 17 mg/kg of RDX (1,3,5-trinitro-1,3,5-triazine) in corn oil daily for 14 d to evaluate sublethal effects of this explosive in birds. Mortality occurred at a rates of 100, 67, and 25% for the 17, 12, and 8 mg/kg/d dose groups, respectively. Death was preceded by clonic and tonic convulsions and weight loss caused by gastrointestinal effects. Increases in serum globulin and total leukocytes were observed in the two highest-dose groups. Degeneration of testicular and splenic tissue also was observed. The no-observed-adverse-effects and lowest-observed-adverse-effects levels were determined as 3.0 and 8.0 mg/kg/d, respectively.

Quail Genomics: a knowledgebase for Northern bobwhite.

BackgroundThe Quail Genomics knowledgebase (http://www.quailgenomics.info) has been initiated to share and develop functional genomic data for Northern bobwhite (Colinus virginianus). This web-based platform has been designed to allow researchers to perform analysis and curate genomic information for this non-model species that has little supporting information in GenBank.DescriptionA multi-tissue, normalized cDNA library generated for Northern bobwhite was sequenced using 454 Life Sciences next generation sequencing. The Quail Genomics knowledgebase represents the 478,142 raw ESTs generated from the sequencing effort in addition to assembled nucleotide and protein sequences including 21,980 unigenes annotated with meta-data. A normalized MySQL relational database was established to provide comprehensive search parameters where meta-data can be retrieved using functional and structural information annotation such as gene name, pathways and protein domain. Additionally, blast hit cutoff levels and microarray expression data are available for batch searches. A Gene Ontology (GO) browser from Amigo is locally hosted providing 8,825 unigenes that are putative orthologs to chicken genes. In an effort to address over abundance of Northern bobwhite unigenes (71,384) caused by non-overlapping contigs and singletons, we have built a pipeline that generates scaffolds/supercontigs by aligning partial sequence fragments against the indexed protein database of chicken to build longer sequences that can be visualized in a web browser.ConclusionOur effort provides a central repository for storage and a platform for functional interrogation of the Northern bobwhite sequences providing comprehensive GO annotations, meta-data and a scaffold building pipeline. The Quail Genomics knowledgebase will be integrated with Japanese quail (Coturnix coturnix) data in future builds and incorporate a broader platform for these avian species.

Effects of subchronic exposure to 2,6‐dinitrotoluene in the northern bobwhite (Colinus Virginianus)

Explosives and their breakdown products are commonly found in soils at U.S. military installations. Many areas where these compounds are found represent useful habitat for ground-foraging birds. Because these birds likely are exposed to such compounds, we evaluated the oral toxicity of 2,6-dinitrotoluene (DNT) in a representative ground-foraging species of management concern. Adult male and female northern bobwhite (Colinus virginianus) were exposed to either 0, 5, 10, 40, or 60 mg/kg/d via gavage for 60 d (subchronic) following determination of the median acute lethal dose (320 mg/kg). Circulating levels of red blood cells and hemoglobin were significantly decreased in a dose-dependent manner; however, levels remained within normal ranges. Plasma concentrations of total protein, albumin, globulin, aspartate aminotransferase, and potassium, sodium, and chlorine ions were significantly decreased, and circulating levels of uric acid were significantly increased. Decreased body weight, enlarged gallbladders, edematous gastrointestinal tracts, pale kidneys, pale and fibrous livers, and loose stools were consistent observations. The effects found in the clinical chemistries taken together with histopathological abnormalities observed in sections of hepatic and renal tissue suggest that the liver and kidneys are major targets for 2,6-DNT. Oral exposures to 2,6-DNT appear to affect northern bobwhite in a manner similar to that of the other main DNT isomer, 2,4-DNT, but in more subtle ways, adversely affecting the gastrointestinal system and leading to diarrhea and, ultimately, emaciation. The lowest-observed-adverse-effect level is 40 mg/ kg/d based on hematological measures, and the no-observed-adverse-effect level is 10 mg/kg/d based on the absence of results indicative of adverse effects.

A Systems Toxicology Approach to Elucidate the Mechanisms Involved in RDX Species-Specific Sensitivity

Interspecies uncertainty factors in ecological risk assessment provide conservative estimates of risk where limited or no toxicity data is available. We quantitatively examined the validity of interspecies uncertainty factors by comparing the responses of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) to the energetic compound 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), a known neurotoxicant. Relative toxicity was measured through transcriptional, morphological, and behavioral end points in zebrafish and fathead minnow fry exposed for 96 h to RDX concentrations ranging from 0.9 to 27.7 mg/L. Spinal deformities and lethality occurred at 1.8 and 3.5 mg/L RDX respectively for fathead minnow and at 13.8 and 27.7 mg/L for zebrafish, indicating that zebrafish have an 8-fold greater tolerance for RDX than fathead minnow fry. The number and magnitude of differentially expressed transcripts increased with increasing RDX concentration for both species. Differentially expressed genes were enriched in functions related to neurological disease, oxidative-stress, acute-phase response, vitamin/mineral metabolism and skeletal/muscular disorders. Decreased expression of collagen-coding transcripts were associated with spinal deformity and likely involved in sensitivity to RDX. Our work provides a mechanistic explanation for species-specific sensitivity to RDX where zebrafish responded at lower concentrations with greater numbers of functions related to RDX tolerance than fathead minnow. While the 10-fold interspecies uncertainty factor does provide a reasonable cross-species estimate of toxicity in the present study, the observation that the responses between ZF and FHM are markedly different does initiate a call for concern regarding establishment of broad ecotoxicological conclusions based on model species such as zebrafish.

sCoIn: A scoring algorithm based on complex interactions for reverse engineering regulatory networks

Structural analysis over well studied transcriptional regulatory networks indicates that these complex networks are made up of small set of reoccurring patterns called motifs. While information theoretic approaches have been immensely popular, these approaches rely on inferring the regulatory networks by aggregating pair-wise interactions. In this paper, we propose novel structure based information theoretic approaches to infer transcriptional regulatory networks from the microarray expression data. The core idea is to go beyond pair-wise interactions and consider more complex structures as found in motifs. While this increases the network inference complexity over pair-wise interaction based approaches, it achieves much higher accuracy and yet is scalable to genome-level inference. Detailed performance analyses based on benchmark precision and recall metrics on the known Escherichia colis transcriptional regulatory network indicates that the accuracy of the proposed algorithms is consistently higher in comparison to popular algorithms such as context likelihood of relatedness (CLR), relevance networks (RN) and GEneNetwork Inference with Ensemble of trees (GENIE3). In the proposed approaches the size of structures was limited to three node cases (any node and its two parents). Analysis on a smaller network showed that the performance of the algorithm improved when more complex structures were considered for inference, although such higher level structures may be computationally challenging to infer networks at the genome scale.

Genome scale inference of transcriptional regulatory networks using mutual information on complex interactions

Inferring the genetic network architecture in cells is of great importance to biologists as it can lead to the understanding of cell signaling and metabolic dynamics underlying cellular processes, onset of diseases, and potential discoveries in drug development. The focus today has shifted to genome scale inference approaches using information theoretic metrics such as mutual information over the gene expression data. In this paper, we propose two classes of inference algorithms using scoring schemes on complex interactions which are primarily based on information theoretic metrics. The central idea is to go beyond pair-wise interactions and utilize more complex structures between any node (gene or transcription factor) and its possible multiple regulators (only transcription factors). While this increases the network inference complexity over pair-wise interaction based approaches, it achieves much higher accuracy. We restricted the complex interactions considered in this paper to 3-node structures (any node and its two regulators) to keep our schemes scalable to genome-scale inference and yet achieve higher accuracy than other state of the art approaches. Detailed performance analyses based on benchmark precision and recall metrics over the known Escherichia coli transcriptional regulatory network, indicated that the accuracy of the proposed algorithms (sCoIn, aCoIn and its variants) is consistently higher in comparison to popular algorithms such as context likelihood of relatedness (CLR), relevance networks (RN) and GEneNetwork Inference with Ensemble of trees (GENIE3).