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Integrated Omic Analyses Identify Pathways and Regulators Associated with Chemical Alterations of in vitro Neural Network Formation

ABSTRACT: Development of in vitro new approach methodologies (NAMs) has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay (NFA) characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically-induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed (0.1 -10 µM) for 12 days in vitro (DIV) to cytosine arabinoside (CA), 5 fluorouracil (5FU), domoic acid (DA), cypermethrin (CM), deltamethrin (DM), and haloperidol; these exposures targeted specific concentrations that altered network activity in previous studies. RNA-seq from cells and GC/MS of media extracts collected on DIV 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant were analyzed using Ingenuity Pathway Analysis (IPA). All six compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; for example, genes altered by both CA and 5FU exposures are enriched in axonal guidance pathways. Analysis of upstream regulators was heterogeneous across compounds, but identified regulators included CREB1, BDNF, TGFβ1, NTRK2, and PRODH. These results demonstrate that transcriptomic and metabolomic changes following chemical exposure can be determined in the NFA and that different classes of compounds produce differing responses. This approach can enhance information obtained from NAMs and contribute to the identification and development of AOPs associated with DNT.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE174722 | GEO | 2022/04/19

REPOSITORIES: GEO

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Project description: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 (M-bM-^IM-$ 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 20M-CM-^W 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 60 Day 2,6-DNT Exposure, Brain Tissue Investigation (High Laser Intensity Scan): Juvenile male and female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 5, 10, 40, or 60 mg/kg/day). Each treatment group included 12 replicates per sex. Microarray experiments were conducted in a balanced interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the 60-day 2,6-DNT exposure comparing controls and the highest affective 2,6-DNT dose (60 mg/kg/day) in males and females, with all groups including three biological replicates for a total of 24 microarrays. 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.

Project description: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 (M-bM-^IM-$ 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 20M-CM-^W 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 60 Day 2,6-DNT Exposure, Brain Tissue Investigation (Low Laser Intensity Scan): Juvenile male and female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 5, 10, 40, or 60 mg/kg/day). Each treatment group included 12 replicates per sex. Microarray experiments were conducted in a balanced interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the 60-day 2,6-DNT exposure comparing controls and the highest affective 2,6-DNT dose (60 mg/kg/day) in males and females, with all groups including three biological replicates for a total of 24 microarrays. 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 Low Intensity Scan.

Project description: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 (M-bM-^IM-$ 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 20M-CM-^W 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. Female Northern Bobwhite 14 Day 2,6-DNT Exposure, Brain Tissue Investigation (Low Laser Intensity Scan): Juvenile female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 50, 100, 190, or 350 mg/kg/day). Each treatment group included seven birds (at least three of each sex per treatment). Microarray experiments were conducted using an interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the effects of the 14-day 2,6-DNT exposure in females including controls, 60, and 100 mg/kg/day treatments, each including 3 biological replicates. 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 Low Intensity Scan.

Project description: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 (M-bM-^IM-$ 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 20M-CM-^W 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. Female Northern Bobwhite 14 Day 2,6-DNT Exposure, Brain Tissue Investigation (High Laser Intensity Scan): Juvenile female Northern bobwhite (12 weeks of age) were dosed with 2,6-DNT by daily gavage (0, 50, 100, 190, or 350 mg/kg/day). Each treatment group included seven birds (at least three of each sex per treatment). Microarray experiments were conducted using an interwoven-loop design using Cyanine-3 (Cy3) and A647. This experiment investigated the effects of the 14-day 2,6-DNT exposure in females including controls, 60, and 100 mg/kg/day treatments, each including 3 biological replicates. 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.

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Integrated Omic Analyses Identify Pathways and Regulators Associated with Chemical Alterations of in vitro Neural Network Formation

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