Neurotoxicogenomic Investigations to Assess Mechanisms of Action of the Munitions Constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus)

Abstract

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.

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

“…Gust et al (2009) hypothesized that the mode of action underlying RDX-induced seizures in Northern bobwhite is the inhibition of neuronal membrane depolarization back to the baseline voltage after an action potential thereby resulting in hyper-excitation. In that study, a variety of molecular mechanisms contributed to the mode of action including impacts on glutamate metabolism, Na + and K + ion transporters, Cl − ion channels, Ca2+ signaling and neurotransmitter release factors.…”

“…Observed effects of RDX exposure in the ecotoxicological model species fathead minnow include lethality, impaired growth and reduced reproduction (Burton et al, 1994;Talmage et al, 1999). Evidence that the central nervous system is the primary toxicological target for RDX include observation of RDX-induced convulsions in rats (Meyer et al, 2005), miniature swine (Schneider et al, 1977), mice (Dilley et al, 1978), Northern bobwhite (Johnson et al, 2007;Quinn et al, 2009;Gust et al, 2009) and humans (Stone et al, 1969;Woody et al, 1986). Neurotoxicogenomic investigations in rat and Northern bobwhite indicate that RDX-induced seizures are likely driven by various impacts on genes and pathways involved in the neurophysiology of brain tissue (Bannon et al, 2009;Gust et al, 2009 Given that RDX may accumulate in surface waters, the potential for impacts on CNS in fish is of concern.…”

“…Evidence that the central nervous system is the primary toxicological target for RDX include observation of RDX-induced convulsions in rats (Meyer et al, 2005), miniature swine (Schneider et al, 1977), mice (Dilley et al, 1978), Northern bobwhite (Johnson et al, 2007;Quinn et al, 2009;Gust et al, 2009) and humans (Stone et al, 1969;Woody et al, 1986). Neurotoxicogenomic investigations in rat and Northern bobwhite indicate that RDX-induced seizures are likely driven by various impacts on genes and pathways involved in the neurophysiology of brain tissue (Bannon et al, 2009;Gust et al, 2009 Given that RDX may accumulate in surface waters, the potential for impacts on CNS in fish is of concern. The purpose of our study was to screen for the effects of RDX exposure on transcript expression in brain tissues of the ecotoxicological fish model (fathead minnow) and develop a comprehensive suite of hypothetical mechanisms of action underlying the observed toxicological impacts caused by RDX.…”

Genomic investigation of year‐long and multigenerational exposures of fathead minnow to the munitions compound RDX

“…Immediately following sacrifice, half of each tissue sample was fixed in RNA Later TM (Ambion) as per the manufacturer's directions while the other half was flash-frozen using liquid nitrogen for analytical chemistry. RNA extraction from tissues and assessment of RNA quality were conducted as described previously [24]. RNA samples were considered of adequate quality for use in microarray analysis if the RNA integrity number from the NanoChip (Agilent) analysis was !8.…”

“…Reductions in survival and reproduction were observed at !5 mg/L for all fish species tested with limited sublethal impacts observed below that concentration. In addition, RDX has been observed to elicit neurotoxic effects [22][23][24][25][26] and hepatotoxicity [22,26] in a wide phylogenetic spectrum of animal species. We therefore focused our investigations on the effects of RDX exposure on transcript expression in fathead minnow brain and liver tissues.…”