Abstract  Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 µM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 µM), Ca2+-ATPase inhibitor thapsigargin (5 µM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

Abstract  The purpose of this research is to determine mechanisms through which hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), environmental degradation product of high energetic munition hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), causes persistent anemia in the rat. We have hypothesized MNX targets hematopoeitic stem cells and, like other myelosuppressive chemicals, will be fibrogenic to the bone marrow. Findings of this period are: 1) additional MNX suppressive effects on peripheral blood cells of myeloid lineage and similar effects of RDX and 2) suppression of bone marrow erythroid and myeloid stem cells of bone marrow by both MNX and RDX. Myeloid development appears more sensitive than erythroid, especially to RDX. These results suggest that MNX- and RDX toxicity in the rat appears to mimic some clinical manifestations of the myeloproliferative disorder, idiopathic myelofibrosis, and thus may offer a model for study of disease progression and intervention strategies. With respect to remediation of RDX-contaminated sites, collectively these data argue that risk of adverse hematological effects from exposure are lessened upon natural remediation to nitro reduced products.

Abstract  Leydig cells are the primary site of androgen biosynthesis in males. Several environmental toxicants target steroidogenesis resulting in both developmental and reproductive effects including testicular dysgenesis syndrome. The aim of this study was to evaluate the effect of several structurally diverse endocrine disrupting compounds (EDC) on steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells possess a fully functional steroidogenic pathway that produces low basal levels of testosterone (T), and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3 and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) elicited concentration- and time- dependent induction of cAMP, progesterone (P) and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3 and Srd5a1 mRNA levels. The evaluation of several structurally diverse male reproductive toxicants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl phthalate (MEHP), glyphosate and RDX in BLTK1 cells suggest different modes of action perturb steroidogenesis. For example, prochloraz and triclosan anti-fungals reduced rhCG-induction of T, consistent with published in vivo data, but did not alter basal T levels. In contrast, atrazine and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T levels, whereas TCDD, glyphosate and RDX had no effect on basal or rhCG-induction of T in BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This model can also be used to elucidate the different mechanisms underlying toxicant-mediated disruption of steroidogeneis.

Abstract  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.

Abstract  The response of olfactory sensory neurons to TNT and RDX as well as to some volatile organic compounds present in the vapors of antipersonnel landmines has been studied both in the pig and in the rat. GC/MS analyses of different plastic components of six different kinds of landmines were performed in order to identify the components of the "perfume" of mines. Studies on rat olfactory mucosa were carried out with electro-olfactogram and calcium imaging techniques, while changes in the cyclic adenosine monophosphate (cAMP) levels following exposure to odorants and explosives were used as a criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. These studies indicate that chemical compounds associated with explosives and explosive devices can activate mammalian olfactory receptors.

Abstract  Overactivation of the N-methyl-D: -aspartate receptor (NMDAR) in postsynaptic neurons leads to glutamate-related excitotoxicity in the central nervous system of mammals. We have built 3-D models of each domain for the universal screening of potential toxicants and their binding mechanisms. Our docking results show that the calculated pK (i) values of glycine and L: -glutamate significantly increase (>1) when the NR1 and NR2A S1S2 domains are closing, respectively. Inversely, D: -cycloserine (DCS) and 5,7-dichlorokynurenic acid (5,7-DCKA) do not show such a dependence on domain closure. Replica exchange molecular dynamics (REMD) confirmed 5 different conformational states of the S1S2 domain along the 308.2 K temperature trajectory. Analysis of residue fluctuations during this temperature trajectory showed that residues in loop 1, loop 2, the amino terminal domain (ATD), and the area linked to ion channel α-helices are involved in this movement. This further implicates the notion that efficacious ligands act through S1S2 lobe movement which can culminate in the opening or closing of the ion channel. We further tested this by docking hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) to the S1S2 domain. Our results predict that these nitramines are not efficacious and thus do not produce excitoxicity when they bind to the S1S2 domain of the NMDAR.

Abstract  Microelectrode arrays (MEAs) can be used to detect drug and chemical induced changes in neuronal network function and have been used for neurotoxicity screening. As a proof-of-concept, the current study assessed the utility of analytical "fingerprinting" using principal components analysis (PCA) and chemical class prediction using support vector machines (SVMs) to classify chemical effects based on MEA data from 16 chemicals. Spontaneous firing rate in primary cortical cultures was increased by bicuculline (BIC), lindane (LND), RDX and picrotoxin (PTX); not changed by nicotine (NIC), acetaminophen (ACE), and glyphosate (GLY); and decreased by muscimol (MUS), verapamil (VER), fipronil (FIP), fluoxetine (FLU), chlorpyrifos oxon (CPO), domoic acid (DA), deltamethrin (DELT) and dimethyl phthalate (DMP). PCA was performed on mean firing rate, bursting parameters and synchrony data for concentrations above each chemical's EC50 for mean firing rate. The first three principal components accounted for 67.5, 19.7, and 6.9% of the data variability and were used to identify separation between chemical classes visually through spatial proximity. In the PCA, there was clear separation of GABAA antagonists BIC, LND, and RDX from other chemicals. For the SVM prediction model, the experiments were classified into the three chemical classes of increasing, decreasing or no change in activity with a mean accuracy of 83.8% under a radial kernel with 10-fold cross-validation. The separation of different chemical classes through PCA and high prediction accuracy in SVM of a small dataset indicates that MEA data may be useful for separating chemicals into effects classes using these or other related approaches.

Abstract  Hexahydro-1,3,5-trinitro-1,3,5-triazine is a synthetic, high-impact, relatively stable compound commonly known as Rapid Detonation Explosive (RDX) and has been used in munitions and formulations since World War II. The primary symptom of acute oral exposure to high doses in humans is transient and reversible seizure, accompanied by nausea, muscle spasms, and disorientation, which are also reported in experimental animals. The occurrence of these preventable effects in humans is often reported in occupational settings that lack standard industrial hygiene practices, resulting in high airborne exposures. Though several studies have correlated measurements of RDX in the brains and plasma of dosed animals with frank seizures, little work has been carried out on the effects of RDX at the molecular level. In this study, we examined the impact of a single orally administered RDX dose on the brain, using a combination of analytical chemistry and genechip microarrays. At nominal doses of 3 and 18 mg/kg in male adult rats, a time course of RDX tissue levels showed peak brain concentrations at 3.5 hours, returning to baseline at 24 and 48 hours, respectively. RNA from dosed and control animals was hybridized to Affymetrix Rat 230 2.0 arrays. Analysis was performed in JMP Genomics with the differentially expressed gene cut-off at p > 0.001. We found sets of genes that were early and late responders to RDX treatment. In addition, pathway analysis indicated effects on the nervious system, cell-to-cell signalling, and cell development, among others. These results represent a preliminary basis for understanding the molecular mechanisms underlying RDX toxicity, and further work is underway.

Abstract  Background: Cell culture systems are useful in studying toxicological effects of chemicals such as Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), however little is known as to how accurately isolated cells reflect responses of intact organs. In this work, we compare transcriptional responses in livers of Sprague-Dawley rats and primary hepatocyte cells after exposure to RDX to determine how faithfully the in vitro model system reflects in vivo responses. Results: Expression patterns were found to be markedly different between liver tissue and primary cell cultures before exposure to RDX. Liver gene expression was enriched in processes important in toxicology such as metabolism of amino acids, lipids, aromatic compounds, and drugs when compared to cells. Transcriptional responses in cells exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours were different from those of livers isolated from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Most of the differentially expressed genes identified across conditions and treatments could be attributed to differences between cells and tissue. Some similarity was observed in RDX effects on gene expression between tissue and cells, but also significant differences that appear to reflect the state of the cell or tissue examined. Conclusion: Liver tissue and primary cells express different suites of genes that suggest they have fundamental differences in their cell physiology. Expression effects related to RDX exposure in cells reflected a fraction of liver responses indicating that care must be taken in extrapolating from primary cells to whole animal organ toxicity effects.

Abstract  The purpose of this research is to determine mechanisms through which hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), environmental degradation product of high energetic munition hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), causes persistent anemia in the rat. We have hypothesized MNX targets hematopoeitic stem cells and, like other myelosuppressive chemicals, will be fibrogenic to the bone marrow. Findings of this period are: 1) the inability of RDX and MNX to directly oxidize hemoglobin ferrous iron to methemoglobin in vitro and of MNX to produce methemoglobin in MNX-treated rats, an alternative mechanism for the observed anemia and 2) nitramine targeting of an early multipotential bone marrow stem cell at earlier times after exposure (7d) and flow cytometric assessment of myeloid and erythroid lineage precursors. Collectively, these results continue to suggest an early erythroid/myeloid lineage precursor and/or to the bone marrow stromal niche supporting hematopoiesis as the target of MNX and RDX. These results suggest that MNX- and RDX toxicity in the rat appears to mimic some clinical manifestations of the myeloproliferative disorder, idiopathic myelofibrosis, and thus may offer a model for study of disease progression and intervention strategies.

Abstract  Rapid, inexpensive, and reliable methods are needed for the toxicological screening of chemical substances with the potential to affect health and the environment. One such method, the neutral red (NR) cytotoxicity assay, is based on incorporation of the supravital dye neutral red into lysosomes of viable cells. The NR uptake assay can be used to detect cytotoxic or cytostatic effects of chemical substances capable of damaging cells, has been adapted to microtiter tissue culture systems, and can be analyzed by means of automated spectrophotometric microplate readers (1,2). Neutral red cytotoxicity assays in continuous rat hepatoma H4IIE cells have been applied to a variety of environ-mentally important munitions and related compounds. The H4IIE cells maintain inducible oxidative microsomal enzymes and were chosen because of their application to detecting other xenobiotics in environmental and biological specimens (6).

Abstract  BACKGROUND: Although microRNAs (miRNAs) have been found to play an important role in many biological and metabolic processes, their functions in animal response to environmental toxicant exposure are largely unknown. OBJECTIVES: We used hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a common environmental contaminant, as a toxicant stressor to investigate toxicant-induced changes in miRNA expression in B6C3F1 mice and the potential mechanism of RDX-induced toxic action. METHODS: B6C3F1 mice were fed diets with or without 5 mg/kg RDX for 28 days. After the feeding trials, we isolated RNAs from both brain and liver tissues and analyzed the expression profiles of 567 known mouse miRNAs using microarray and quantitative real-time polymerase chain reaction technologies. RESULTS: RDX exposure induced significant changes in miRNA expression profiles. A total of 113 miRNAs, belonging to 75 families, showed significantly altered expression patterns after RDX exposure. Of the 113 miRNAs, 10 were significantly up-regulated and 3 were significantly down-regulated (p < 0.01) in both mouse brain and liver. Many miRNAs had tissue-specific responses to RDX exposure. Specifically, expression of seven miRNAs was up-regulated in the brain but down-regulated in the liver or up-regulated in the liver but down-regulated in the brain (p < 0.01). Many aberrantly expressed miRNAs were related to various cancers, toxicant-metabolizing enzymes, and neurotoxicity. We found a significant up-regulation of oncogenic miRNAs and a significant down-regulation of tumor-suppressing miRNAs, which included let-7, miR-17-92, miR-10b, miR-15, miR-16, miR-26, and miR-181. CONCLUSIONS: Environmental toxicant exposure alters the expression of a suite of miRNAs.

Abstract  BACKGROUND: Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy, trinitrated cyclic compound that has been used worldwide since World War II as an explosive in both military and civilian applications. RDX can be released in the environment by way of waste streams generated during the manufacture, use, and disposal of RDX-containing munitions and can leach into groundwater from unexploded munitions found on training ranges. For > 60 years, it has been known that exposure to high doses of RDX causes generalized seizures, but the mechanism has remained unknown.

OBJECTIVE: We investigated the mechanism by which RDX induces seizures.

METHODS AND RESULTS: By screening the affinity of RDX for a number of neurotransmitter receptors, we found that RDX binds exclusively to the picrotoxin convulsant site of the γ-aminobutyric acid type A (GABA(A)) ionophore. Whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents and the amplitude of GABA-evoked postsynaptic currents. In extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges.

CONCLUSIONS: These results suggest that binding to the GABA(A) receptor convulsant site is the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures. Knowledge of the molecular site and the mechanism of RDX action with respect to seizure induction can guide therapeutic strategies, allow more accurate development of safe thresholds for exposures, and help prevent the development of new explosives or other munitions that could pose similar health risks.

Abstract  BACKGROUND: RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. RESULTS: Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. CONCLUSIONS: Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.