Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern bobwhite (Colinus virginianus) (part 7 of 10)

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 (≤ 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 20× 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. Northern Bobwhite 14 Day RDX High Dose Exposure, Brain Tissue Investigation (High Laser Intensity Scan): Juvenile male and female Northern bobwhite (12 weeks of age) were dosed with RDX by daily gavage (0, 20, 80, 125, or 180 mg/kg/day). Each treatment group included seven birds (at least three of each sex per treatment). All RDX doses in the 14-day high-dose experiment elicited seizures within 3 days of experiment initiation (Johnson et al., 2007). Microarray experiments were conducted using a balanced interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the 14-day RDX high-dose exposure. All RDX-dosed quail accumulated ~20 mg/kg RDX in brain tissues (Johnson et al., 2007) and exhibited seizures. Transcript expression was compared among three male and three female controls versus three male and three female RDX-seized quail incorporating two dye swaps per biological replicate totaling 24 microarrays. Hybridizations included four technical replicates and two dye swaps per biological replicate resulting in a total of 24 micorarrays assayed. To broaden signal detection, each microarray was scanned at high and low laser power to resolve low-intensity spots and reduce signal saturation, respectively (Skibbe et al., 2006). This dataset represents the High Intensity Scan.