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Transcriptomic, proteomic, and metabolomic analyses identify candidate pathways linking maternal cadmium exposure to altered neurodevelopment and behavior

ABSTRACT: Purpose: Identify gene expression changes in whole brains of newborn pups whose mothers were exposed to Cd during pregnancy (50ppm CdCl2) compared to unexposed controls (0ppm CdCl2). Methods: 5- to 7-week-old C57BL/6J (‘B’) and CAST/EiJ (‘C’) females (the F0 generation) were exposed through their drinking water to 0 ppm or 50 ppm Cd in the form of CdCl2 (Sigma-Aldrich, catalog number 202908) for a period of 5 weeks, then mated with a male of the opposite strain. Cd exposure continued throughout pregnancy and was discontinued at litter birth. A hybrid mating scheme (the F1 generation) was employed to enable analyses of allele-specific gene expression and DNA methylation for a separate study (Hudson & Cowley, manuscript in preparation). The genotypes of F1 mice are referred to as ‘BxC’ (B mother x C father). F1 animals were sacrificed and dissected within 24 hours of birth; sample sizes can be found in supplementary tables associated with the publication. F1 mice were dissected within 24 hours of birth. All animal work was approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee, under protocol 16-045-B. All experiments were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology. Frozen whole brains obtained from four 0 ppm and four 50 ppm newborn female BxC pups representing four litters per exposure group were pulverized using a hammer and liquid nitrogen. 7-10 mg of tissue was then used to extract DNA and RNA simultaneously using the AllPrep DNA/RNA/miRNA kit (Qiagen, 80204). RNA was extracted using the recommended protocol for fatty tissues. RNA and DNA were quantified using a Nanodrop 2000. RNA purity and size integrity were determined at the NCSU Genomic Sciences Laboratory (GSL) using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies). Results: We identified 15 significantly differentially expressed genes (DEGs) that aligned to both the B and C genomes. Conclusions: These 15 DEGs (and subsequent confirmatory qRT-PCRs on the 8 samples sequenced here plus an additional Cd dose and samples) suggest altered onset of myelination throughout the whole brain, as well as aberrant Hox gene expression due to maternal Cd exposure.

ORGANISM(S): Mus musculus

PROVIDER: GSE174433 | GEO | 2021/08/18

REPOSITORIES: GEO

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Project description:Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization.InuteroCd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl2 and performed RNA-seq on control and CdCl2 exposed placentae. The top differentially expressed transcript was the Tcl1Upstream Neuron-Associated (Tuna) long non-coding RNA, which was up-regulated over 25-fold in CdCl2 exposed placentae. Tuna has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that Tuna is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated Tuna within the placenta, we used in situ hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of Tuna expression in control samples and determined that Cd-induced Tuna expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that Tunaforms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed Tuna in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl2 exposed cells. We demonstrate significant overlap between genes activated byTunaoverexpression and genes activated by CdCl2 exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that Tuna increases NRF2/NRF2 both at the transcript and protein levels. Tuna drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that Tuna activates oxidative stress response genes through this pathway. This work identifies the lncRNA Tuna as a potential novel player in Cd-induced placental insufficiency.

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Transcriptomic, proteomic, and metabolomic analyses identify candidate pathways linking maternal cadmium exposure to altered neurodevelopment and behavior

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