Methylmercury induced toxicogenomic response in C57 and SWV mouse embryos undergoing neural tube closure

Abstract

Methylmercury (MeHg) is a developmental neurotoxicant and teratogen and is hypothesized to perturb a wide range of biological processes, like other metals including arsenic (As) and cadmium (Cd). Common inbred mouse strains including C57 (sensitive) and SWV (resistant) display differences in sensitivity to metals such as As and Cd when exposed during neurulation. In this study, we investigated the impact of MeHg on neurulation, assessing for potential differences in sensitivity and associated toxicogenomic response in C57 and SWV mouse embryos. Parallel with morphological assessments of neural tube closure, we evaluated quantitative differences in MeHg-induced alterations in expression between strains at the gene level and within gene-enriched biological processes. Specifically, we observed differing sensitivities to MeHg-induced impacts on neural tube closure between C57 and SWV embryos in a time-dependent manner. These observations correlated with greater impact on the expression of genes associated with development and environmental stress-related pathways in the C57 compared to the SWV. Additional developmental parameters (e.g. mortality, growth effects) evaluated showed mixed significant effects across the two strains and did not support observations of differential sensitivity to MeHg. This study provides potential insights into MeHg-induced mechanisms of developmental toxicity, alterations associated with increased MeHg sensitivity and common biological processes affected by metals in embryos undergoing neurulation.

Figure 1. The impact of MeHg on closure of the cranial region of the neural tube in C57 and SWV embryos

Embryos isolated GD8.0 + 6, 12, 24h were examined for closure of the anterior neural tube within the hind/brain region. These results were combined with external malformation assessments made on GD8.0 + 240h (GD18), evaluating closure of the cranial region (exencephaly). Data points are based on litter averages (n≥3 for each group). We observed significant effects on neural tube closure in MeHg-exposed C57 and SWV embryos (B_MeHg, p<0.05) and significant differential impacts by MeHg on neural tube closure between strains (B_{MeHg_Strain}, p<0.05).

Figure 2. ANOVA distributions of MeHg-induced gene expression alterations inC57 and SWV embryos

(A) Venn diagrams display the distribution of genes identified to be significantly altered due to selected effects within each one of the three models. (B) Scatter plots display the magnitude of change (MeHg/Con) between MeHg and control in differentially expressed genes in C57 and SWV embryos (12h (A) and 24h (B) post-injection). Genes were identified as statistically significant due to a significant B_MeHg term (p<0.01) in one of our three ANOVA models assessing for genes that are commonly or uniquely altered in the two strains. Genes identified by one of our three ANOVA models are displayed as (A) B_MeHg p<0.01), in both strains (light gray boxes), (B) B_MeHg (p<0.01), C57BL/6J only (dark gray triangles) and (C) B_MeHg (p<0.01), SWV only (black circles).

Figure 3. Quantitative analysis of GO biological processes impacted by MeHg in C57 and SWV embryos

The color-coded diagram represents the absolute mean fold change (MeHg/control) of genes identified to be significantly altered with MeHg in any of the two strains (Models 1–3) within overrepresented GO subsets identified in the C57 and/or SWV. All selected GO subsets were identified as significantly impacted by MeHg in either C57 (*) or SWV (^) using MAPPFinder criteria (Z>2.0, p<0.02, number changed within each GOID>5). The number of genes changed (p<0.01) in either strain is in parentheses next to each GO categorical name. Descriptions on the right represent general description of GO term grouping.

Figure 4. MeHg-induced gene expression alterations associated with development

Cross scatter plots display the magnitude in fold change (MeHg/Con) associated with both C57 (x-axis) and SWV (y-axis) embryos 12h (A) and 24h (B) post injection (GD8.0) for genes that were identified as significantly altered in one of the three models and linked with the GO biological process, System Development (GO:48731).

Figure 5. MeHg-induced gene expression alterations associated with stress response

Cross scatter plot display the magnitude in fold change (MeHg/Con) associated with both C57 (x-axis) and SWV (y-axis) embryos 12h (A) and 24h (B) post injection (GD8.0) for genes that were identified as significantly altered in one of the three models and linked with the specific GO biological process, Response to Stress (GO:6950).

Figure 6. Quantitative analysis of commonly enriched GO biological processes affected by MeHg and Cd

The bar chart represents the absolute mean fold change (treatment/control) of genes identified to be significantly altered with MeHg or Cd [22] within commonly enriched GO biological process. All selected GO subsets were identified as significantly enriched by both MeHg and Cd in the C57 using our cutoff criteria of significance (Z>2.0, p<0.02, number changed within each GOID>3). The total number of genes altered by MeHg and/or Cd (p<0.01) in the C57 strain are displayed to the right of each GO category in parentheses.