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.

Reduction in lead exposures with lead-free ammunition in an advanced urban assault course.

The training of soldiers for urban conflict involves marksmanship instruction on outdoor flat ranges and the teaching of close-quarter battle techniques in indoor facilities, referred to as shoot houses, where intense firing exercises can generate high air lead levels from small arms ammunition, flash bang grenades, and explosive devices. Levels of lead and copper in air were evaluated during five training activities of a 45-day training course using both stationary general area and breathing zone sampling over a 2-year period. Individual blood lead values were determined prior to and at course completion. Mean breathing zone lead concentrations for the five training activities ranged from 0.014 on the outdoor flat range to 0.064 mg/m3 inside shoot houses; with a change to lead-free ammunition the values were reduced to a range of 0.006-0.022 mg/m3. Isolated flash bang grenades generated very high general area lead concentrations (2.0 mg/m3), which in training were associated with the highest measured breathing zone concentration (0.16 mg/m3). For copper, mean breathing zone concentrations increased from 0.010 to 0.037 mg/m3 with the change to lead-free frangible ammunition on the outdoor range, but remained below the permissible exposure limit for copper fume. Inside shoot houses, mean breathing zone copper concentrations exceeded the permissible exposure limit with ball and lead-free frangible ammunition, ranging from 0.077-0.13 mg/m3. With the introduction of lead-free ammunition, when comparing the blood lead differences between start and finish of the course, there was a significant reduction in the mean blood lead difference from 13.3 µg/dL to 5.4 µg/dL. Options for mitigation of potentially high exposure areas using improved ventilation designs are discussed. These results advocate for improved designs for shoot house training facilities, stress the importance of removing lead from ammunition and explosive devices for training, and promote the continued need for implementation of controls to mitigate and manage metal exposures during training.

Gene expression in mouse muscle over time after nickel pellet implantation.

The transition metal nickel is used in a wide variety of alloys and medical devices. Nickel can cause a range of toxicities from allergy in humans to tumors when implanted in animals. Several microarray studies have examined nickel toxicity, but so far none have comprehensively profiled expression over an extended period. In this work, male mice were implanted with a single nickel pellet in the muscle of the right leg with the left leg used as a control. At 3 week intervals up to 12 months, nickel concentrations in bioflulids and microarrays of surrounding tissue were used to track gene expression patterns. Pellet biocorrosion resulted in varying levels of systemic nickel over time, with peaks of 600 µg L-1 in serum, while global gene expression was cyclical in nature with immune related genes topping the list of overexpressed genes. IPA and KEGG pathway analyses was used to attribute overall biological function to changes in gene expression levels, supported by GO enrichment analysis. IPA pathways identified sirtuin, mitochondria, and oxidative phosphorylation as top pathways, based predominantly on downregulated genes, whereas immune processes were associated with upregulated genes. Top KEGG pathways identified were lysosome, osteoclast differentiation, and phasgosome. Both pathway approaches identified common immune responses, as well as hypoxia, toll like receptor, and matrix metalloproteinases. Overall, pathway analysis identified a negative impact on energy metabolism, and a positive impact on immune function, in particular the acute phase response. Inside the cell the impacts were on mitochondria and lysosome. New pathways and genes responsive to nickel were identified from the large dataset in this study which represents the first long-term analysis of the effects of chronic nickel exposure on global gene expression.

Neuromuscular anomalies following oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) in a one-generation study with Japanese quail (Coturnix japonica).

Substances used as explosives in munitions by the military often result in environmental releases through manufacturing, testing, training, and combat activities. The toxicity of 3-nitro-1,2,4-triazol-5-one (nitrotriazolone or NTO) was evaluated following oral exposure in Japanese quail (Coturnix japonica) to determine if environmental releases result in unacceptable risks to avian populations. In an acute test at the limit dose (2000 mg/kg), one female was ataxic, exhibited tremors, and showed signs of neurological toxicity approximately 24 h after dosing. In a subsequent one-generation study, parental generation (F0) birds were exposed orally to 1000, 500, 100, or 20mg/kg-day NTO suspended in corn oil. After 5 consecutive days of dosing, 2-week-old birds receiving 1000 mg/kg-day displayed ataxia, convulsions, backward arching of the neck (opisthotonos), and alternated between prostrate inactivity and ataxic wing activity. Birds in the 500 mg/kg-day group exhibited neuromuscular anomalies after 17 days exposure. Ultimately, all of the 1000 mg/kg-day birds and all but one of the 500 mg/kg-day birds met euthanasia criteria and were humanely euthanized prior to behavioral and reproductive evaluation. As such, first-generation (F1) birds were exposed to 100 or 20 mg/kg-day NTO. Mild neuromuscular anomalies occurred in 10% of F1 birds from the 100 mg/kg-day group, but not in birds from 20 mg/kg-day or controls in either generation. Vacuolization of cerebellum and/or the brainstem was observed on histopathologic examination in a dose-dependent manner. Therefore, brain vacuoles and neuromuscular anomalies were identified as critical endpoints in this study. A mean Benchmark Dose (BMD) for brain vacuoles of 62 mg/kg-day was derived for male and female F0-generation quail, which corresponded to a Benchmark Dose Low (BMDL10) of 35 mg/kg-day.

Matching target dose to target organ.

In vitro assays have become a mainstay of modern approaches to toxicology with the promise of replacing or reducing the number of in vivo tests required to establish benchmark doses, as well as increasing mechanistic understanding. However, matching target dose to target organ is an often overlooked aspect of in vitro assays, and the calibration of in vitro exposure against in vivo benchmark doses is often ignored, inadvertently or otherwise.  An example of this was recently published in Environmental Health Perspectives by Wagner et al (2016), where neural stems cells were used to model the molecular toxicity of lead.  On closer examination of the in vitro work, the doses used in media reflected in vivo lead doses that would be at the highest end of lead toxicity, perhaps even lethal.  Here we discuss the doses used and suggest more realistic doses for future work with stem cells or other neuronal cell lines.

Toxicokinetic Model Development for the Insensitive Munitions Component 2,4-Dinitroanisole.

The Armed Forces are developing new explosives that are less susceptible to unintentional detonation (insensitive munitions [IMX]). 2,4-Dinitroanisole (DNAN) is a component of IMX. Toxicokinetic data for DNAN are required to support interpretation of toxicology studies and refinement of dose estimates for human risk assessment. Male Sprague-Dawley rats were dosed by gavage (5, 20, or 80 mg DNAN/kg), and blood and tissue samples were analyzed to determine the levels of DNAN and its metabolite 2,4-dinitrophenol (DNP). These data and data from the literature were used to develop preliminary physiologically based pharmacokinetic (PBPK) models. The model simulations indicated saturable metabolism of DNAN in rats at higher tested doses. The PBPK model was extrapolated to estimate the toxicokinetics of DNAN and DNP in humans, allowing the estimation of human-equivalent no-effect levels of DNAN exposure from no-observed adverse effect levels determined in laboratory animals, which may guide the selection of exposure limits for DNAN.

Toxicokinetic Model Development for the Insensitive Munitions Component 3-Nitro-1,2,4-Triazol-5-One.

3-Nitro-1,2,4-triazol-5-one (NTO) is a component of insensitive munitions that are potential replacements for conventional explosives. Toxicokinetic data can aid in the interpretation of toxicity studies and interspecies extrapolation, but only limited data on the toxicokinetics and metabolism of NTO are available. To supplement these limited data, further in vivo studies of NTO in rats were conducted and blood concentrations were measured, tissue distribution of NTO was estimated using an in silico method, and physiologically based pharmacokinetic models of the disposition of NTO in rats and macaques were developed and extrapolated to humans. The model predictions can be used to extrapolate from designated points of departure identified from rat toxicology studies to provide a scientific basis for estimates of acceptable human exposure levels for NTO.

A rat RNA-Seq transcriptomic BodyMap across 11 organs and 4 developmental stages.

The rat has been used extensively as a model for evaluating chemical toxicities and for understanding drug mechanisms. However, its transcriptome across multiple organs, or developmental stages, has not yet been reported. Here we show, as part of the SEQC consortium efforts, a comprehensive rat transcriptomic BodyMap created by performing RNA-Seq on 320 samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats. We catalogue the expression profiles of 40,064 genes, 65,167 transcripts, 31,909 alternatively spliced transcript variants and 2,367 non-coding genes/non-coding RNAs (ncRNAs) annotated in AceView. We find that organ-enriched, differentially expressed genes reflect the known organ-specific biological activities. A large number of transcripts show organ-specific, age-dependent or sex-specific differential expression patterns. We create a web-based, open-access rat BodyMap database of expression profiles with crosslinks to other widely used databases, anticipating that it will serve as a primary resource for biomedical research using the rat model.

Biomarkers of oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN) in blood and urine of rhesus macaques (Macaca mulatta).

The U.S. Department of Defense is using the chemicals 2,4-dinitroanisole (DNAN) and 3-nitro-1, 2,4-triazol-5-one (NTO) in new munitions development. In a screen for biomarkers of exposure, these compounds were measured in urine and blood of male rhesus monkeys after oral doses. NTO peaked at 4 h, with urinary concentrations at least 100-fold higher than that of blood or serum while 4-dinitrophenol (DNP), a metabolite of DNAN, appeared in blood at concentrations 10- to 20-fold higher than the parent compound. For human exposure monitoring, urine is optimal for NTO while the metabolite DNP in blood is best for DNAN.

Solubility of lead and copper in biochar-amended small arms range soils: influence of soil organic carbon and pH.

Biochar is often considered a strong heavy metal stabilizing agent. However, biochar in some cases had no effects on, or increased the soluble concentrations of, heavy metals in soil. The objective of this study was to determine the factors causing some biochars to stabilize and others to dissolve heavy metals in soil. Seven small arms range soils with known total organic carbon (TOC), cation exchange capacity, pH, and total Pb and Cu contents were first screened for soluble Pb and Cu concentrations. Over 2 weeks successive equilibrations using weak acid (pH 4.5 sulfuric acid) and acetate buffer (0.1 M at pH 4.9), Alaska soil containing disproportionately high (31.6%) TOC had nearly 100% residual (insoluble) Pb and Cu. This soil was then compared with sandy soils from Maryland containing significantly lower (0.5-2.0%) TOC in the presence of 10 wt % (i) plant biochar activated to increase the surface-bound carboxyl and phosphate ligands (PS450A), (ii) manure biochar enriched with soluble P (BL700), and (iii) unactivated plant biochars produced at 350 °C (CH350) and 700 °C (CH500) and by flash carbonization (corn). In weak acid, the pH was set by soil and biochar, and the biochars increasingly stabilized Pb with repeated extractions. In pH 4.9 acetate buffer, PS450A and BL700 stabilized Pb, and only PS450A stabilized Cu. Surface ligands of PS450A likely complexed and stabilized Pb and Cu even under acidic pH in the presence of competing acetate ligand. Oppositely, unactivated plant biochars (CH350, CH500, and corn) mobilized Pb and Cu in sandy soils; the putative mechanism is the formation of soluble complexes with biochar-borne dissolved organic carbon. In summary, unactivated plant biochars can inadvertently increase dissolved Pb and Cu concentrations of sandy, low TOC soils when used to stabilize other contaminants.

Lead retention by broiler litter biochars in small arms range soil: impact of pyrolysis temperature.

Phosphorus-rich manure biochar has a potential for stabilizing Pb and other heavy metal contaminants, as well as serving as a sterile fertilizer. In this study, broiler litter biochars produced at 350 and 650 °C were employed to understand how biochar's elemental composition (P, K, Ca, Mg, Na, Cu, Pb, Sb, and Zn) affects the extent of heavy metal stabilization. Soil incubation experiments were conducted using a sandy, slightly acidic (pH 6.11) Pb-contaminated (19906 mg kg(-1) total Pb primarily as PbCO(3)) small arms range (SAR) soil fraction (<250 µm) amended with 2-20 wt % biochar. The Pb stabilization in pH 4.9 acetate buffer reached maximum at lower (2-10 wt %) biochar amendment rate, and 350 °C biochar containing more soluble P was better able to stabilize Pb than the 650 °C biochar. The 350 °C biochar consistently released greater amounts of P, K, Mg, Na, and Ca than 650 °C biochar in both unbuffered (pH 4.5 sulfuric acid) and buffered (pH 4.9 acetate) systems, despite 1.9-4.5-fold greater total content of the 650 °C biochar. Biochars, however, did not influence the total extractable Pb over three consecutive equilibration periods consisting of (1) 1 week in pH 4.5 sulfuric acid (simulated leaching by rainfall), (2) 1 week in pH 4.9 acetate buffer (standard solution for toxicity characteristic leaching procedure), and (3) 1 h in pH 1.5 glycine at 37 °C (in vitro bioaccessibility procedure). Overall, lower pyrolysis temperature was favorable for stabilizing Pb (major risk driver of SAR soils) and releasing P, K, Ca, and other plant nutrients in a sandy acidic soil.

Retention of heavy metals by carboxyl functional groups of biochars in small arms range soil.

Long-term effectiveness of biochar for heavy metal stabilization depends upon biochar's sorptive property and recalcitrance in soil. To understand the role of carboxyl functional groups on heavy metal stabilization, cottonseed hull biochar and flax shive steam-activated biochar having a low O/C ratio (0.04-0.06) and high fixed carbon content (~80% dry weight basis) were oxidized using concentrated H(2)SO(4)/HNO(3) and 30% HNO(3). Oxidized and unoxidized biochars were characterized for O/C ratio, total acidity, pH, moisture, ash, volatile matter, and fixed carbon contents, Brunauer-Emmett-Teller surface area, and attenuated total reflectance Fourier transform infrared spectral features. Characterized biochars were amended (2%, 5%, 10%, and 20% in grams of biochar per gram of soil) on a sandy, slightly acidic (pH 6.27) heavy metal contaminated small arms range soil fraction (<250 µm) having low total organic carbon (0.518%) and low cation exchange capacity (0.95 cmol(c) kg(-1)). Oxidized biochars rich in carboxyl functional groups exhibited significantly greater Pb, Cu, and Zn stabilization ability compared to unoxidized biochars, especially in pH 4.9 acetate buffer (standard solution for the toxicity characteristic leaching procedure). Oppositely, only oxidized biochars caused desorption of Sb, indicating a counteracting impact of carboxyl functional groups on the solubility of anions and cations. The results suggested that appropriate selection of biochar oxidant will produce recalcitrant biochars rich in carboxyl functional groups for a long-term heavy metal stabilization strategy in contaminated soils.

RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

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.

Lead and copper in pigeons (Columbia livia) exposed to a small arms-range soil.

Small arms-range (SAR) soils can be contaminated with metals from spent copper (Cu)-jacketed bullets. Avian species are particularly at risk because they are exposed to lead (Pb) through ingestion of grit, soil intake from preening, or ingestion of contaminated food near ranges. Examination of the effects of Pb on birds at ranges have mainly focused on intake and toxicity of Pb shot pellets or fragments; however, Pb in soils may be an important pathway of exposure. To evaluate the uptake and effects of Pb from an actual range, the soil fraction (<250 µm) from a contaminated SAR soil was used to dose pigeons (Columbia livia) for 14 days at low (2700 µg Pb and 215 µg Cu/d) and high (5400 µg Pb and 430 µg Cu/d) doses. At the end of the study, blood Pb and erythrocyte protoporphyrin were determined, and tissues were analyzed for Pb and Cu. Results showed that Pb was absorbed in a dose-response manner in blood, tissues, and feathers, and erythrocyte protoporphyrin, a biomarker of early Pb effect, was increased at blood Pb levels >50 µg/dL. Four tissues showed differential retention of Pb, with kidney having the highest concentration followed by liver, brain, and heart, whereas Cu levels were not changed. To examine possible interactions with other metals, amendments of either Cu or tungstate were made to the soil sample. Although these amendments seemed to decrease the absorption of Pb, the results were ambiguous compared with sodium chloride controls. Overall, this study showed that intake of SAR soils contaminated with Pb and Cu causes an increase in Pb body burdens in birds and that the response can be modulated by amending soils with salts of metals.

Evaluation of small arms range soils for metal contamination and lead bioavailability.

Although small arms ranges are known to be contaminated with lead, the full extent of metal contamination has not been described, nor has the oral bioavailability of lead in these soils. In this work, soil samples from ranges with diverse geochemical backgrounds were sieved to <250 microm and analyzed for total metal content. Soils had consistently high levels of lead and copper, ranging from 4549 to 24 484 microg/g and 223 to 2936 microg/g, respectively, while arsenic, antimony, nickel, and zinc concentrations were 100-fold lower. For lead bioavailability measurements, two widely accepted methods were used: an in vivo juvenile swine relative bioavailability method measuring lead absorption from ingested soils relative to equivalent lead acetate concentrations and an in vitro bioaccessibility procedure which measured acid-extractable lead as a percent of total lead in the soil. For eight samples, the mean relative bioavailability and bioaccessibility of lead for the eight soils was about 100% (108 +/- 18% and 95 +/- 6%, respectively) showing good agreement between both methods. Risk assessment and/or remediation of small arms ranges should therefore assume high bioavailability of lead.

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.

Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells.

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller V(max) for iron uptake compared with control cell lines. One clone was further characterized and found to display an approximately 50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.