Effects of prenatal pesticide exposure on the fetal brain and placenta transcriptomes in a rodent model

Abstract

Organophosphate and pyrethroid pesticides are among the most extensively used insecticides worldwide. Prenatal exposures to both classes of pesticides have been linked to a wide range of neurobehavioral deficits in the offspring. The placenta is a neuroendocrine organ and the crucial regulator of the intrauterine environment; early-life toxicant exposures could impact neurobehavior by disrupting placental processes. Female C57BL/6 J mice were exposed via oral gavage to an organophosphate, chlorpyrifos (CPF) at 5 mg/kg, a pyrethroid, deltamethrin (DM), at 3 mg/kg, or vehicle only control (CTL). Exposure began two weeks before breeding and continued every three days until euthanasia at gestational day 17. The transcriptomes of fetal brain (CTL n = 18, CPF n = 6, DM n = 8) and placenta (CTL n = 19, CPF n = 16, DM n = 12) were obtained through RNA sequencing, and resulting data was evaluated using weighted gene co-expression networks, differential expression, and pathway analyses. Fourteen brain gene co-expression modules were identified; CPF exposure disrupted the module related to ribosome and oxidative phosphorylation, whereas DM disrupted the modules related to extracellular matrix and calcium signaling. In the placenta, network analyses revealed 12 gene co-expression modules. While CPF exposure disrupted modules related to endocytosis, Notch and Mapk signaling, DM exposure dysregulated modules linked to spliceosome, lysosome and Mapk signaling pathways. Overall, in both tissues, CPF exposure impacted oxidative phosphorylation, while DM was linked to genes involved in spliceosome and cell cycle. The transcription factor Max involved in cell proliferation was overexpressed by both pesticides in both tissues. In summary, gestational exposure to two different classes of pesticide can induce similar pathway-level transcriptome changes in the placenta and the brain; further studies should investigate if these changes are linked to neurobehavioral impairments.

Keywords: Chlorpyrifos; Deltamethrin; Organophosphate; Pyrethroid; Weighted gene co-expression network analysis (WGCNA).

Fig. 1.

Animal experimental design. * Chlorpyrifos and deltamethrin were dissolved in corn oil vehicle.

Fig. 2.

A. Bar plot of brain co-expression network modules (n=14), x-axis correspond to the number of genes in the module (exact number displayed at the end each bar). Annotations correspond to the top KEGG pathway and the top 4 hub genes for each module. Genes that did not map to a module are in the grey group. B. Forest plot of generalized linear models results between pesticide exposure and each of the brain network co-expression modules. Modules with a fill point have p-values < 5% FDR.

Fig 3.

Volcano plots for the brain differential expression analyses comparing CPF versus CTL (A) and DM vs CTL (B). Each dot corresponds a gene and these are colored according to brain network modules colors. The top 20 most significant genes are labeled with gene symbol.

Fig. 4.

A. Bar plot of the placenta co-expression network modules (n=12), x-axis correspond to the number of genes in the module (exact number displayed at the end each bar). Annotations correspond to the top KEGG pathway and the top 4 hub genes for each module. Genes that did not map to modules are in the grey group. B. Forest plot of generalized linear models results between pesticide exposure and each of the placenta network co-expression modules. Modules with a fill point have p-values < 5% FDR.

Fig. 5.

Volcano plots for the placenta differential expression analyses comparing CPF versus CTL (A) and DM vs CTL (B). Each dot corresponds a gene and these are colored according to placenta network modules colors. The top 20 most significant genes are labeled with gene symbol.

Fig. 6.

A. Venn diagram showing the overlap between DM dysregulated genes (FDR <5 %) in the placenta (PLC) and the brain (BRN). B. Dot plot displaying the result of the KEGG pathway enrichment analyses for 350 genes significantly associated with DM exposure in the brain and the placenta. Only the top 5 pathways are shown. C. Box plots of Max gene expression in the brain stratified by pesticide exposure. D. Box plots of Max gene expression in the placenta stratified by pesticide exposure. P-values of the top correspond to t-tests between CPF-CTL and DM-CTL.