Effects of Prenatal Exposure to Titanium Dioxide Nanoparticles on DNA Methylation and Gene Expression Profile in the Mouse Brain

Abstract

Background and Objectives: Titanium dioxide nanoparticles (TiO₂-NP) are important materials used in commercial practice. Reportedly, TiO₂-NP exposure during pregnancy can affect the development of the central nervous system in mouse offspring; however, the underlying mechanism remains unknown. In the present study, we investigated the impact of prenatal TiO₂-NP exposure on global DNA methylation and mRNA expression patterns in the brains of neonatal mice. Materials and Methods: Pregnant C57BL/6J mice were intratracheally administered a TiO₂-NP suspension (100 μg/mouse) on gestational day 10.5, and brains were collected from male and female offspring at day 1 postpartum. After extraction of methylated DNA by immunoprecipitation, the DNA methylation profile was analyzed using a mouse CpG island microarray. Total RNA was obtained, and mRNA expression profiles were comprehensively assessed using microarray analysis. Results: Among genes in the CpG island microarray, DNA methylation was increased in 614 and 2,924 genes and decreased in 6,220 and 6,477 genes in male and female offspring, respectively. Combined with mRNA microarray analysis, 88 and 89 genes were upregulated (≥1.5-fold) accompanied by demethylation of CpG islands, whereas 13 and 33 genes were downregulated (≤0.67-fold) accompanied by methylation of CpG islands in male and female offspring mice, respectively. Gene Set Enrichment Analysis (GSEA) revealed that these genes were enriched in gene ontology terms related to the regulation of transcription factors, cell proliferation, and organism development. Additionally, MeSH terms related to stem cells and morphogenesis were enriched. Conclusion: Prenatal TiO₂-NP exposure induced genome-wide alterations in DNA methylation and mRNA expression in the brains of male and female offspring. Based on GSEA findings, it can be speculated that prenatal TiO₂-NP exposure causes adverse effects on brain functions by altering the DNA methylation state of the fetal brain, especially neural stem cells, resulting in the subsequent abnormal regulation of transcription factors that modulate development and differentiation.

Keywords: DNA methylation; brain; gene expression; prenatal exposure; titanium dioxide nanoparticle.

FIGURE 1

Characterization of titanium dioxide nanoparticles (TiO₂-NPs). Transmission electron microscopy images of TiO₂-NPs in the intratracheally administered suspension (A). Size distribution of TiO₂-NP in the suspension determined by dynamic light scattering (B).

FIGURE 2

The number of genes that show altered DNA methylation states following prenatal TiO₂-NP exposure. Number of genes presenting altered DNA methylation states between Sham and TiO₂-H groups in the brains of 1-day-old offspring mice [(A): male, (B): female]. Black and gray bars indicate the number of genes with increased and decreased DNA methylation levels, respectively, in the TiO₂-H groups when compared with the Sham group. The x-axis shows chromosome numbers.

FIGURE 3

The number of differentially expressed genes with altered DNA methylation following TiO₂-NP exposure.—Venn diagram analysis whether differentially expressed genes are accompanied by alteration of DNA methylation. Venn diagram showing the number of differentially expressed genes with altered DNA methylation following TiO₂-NP (Sham vs. TiO₂-H) in the brains of 1-day-old offspring mice [(A): male, (B): female]. The DNA methylation state and expression level of genes were identified by Mouse CpG Island Microarray and SurePrint G3 Mouse GE 8 × 60 K Microarray, respectively.

FIGURE 4

Scatter plots of brain DNA methylation levels of 1-day-old offspring mice from TiO₂-H and Sham groups determined by Enrichment of Methylated DNA Fragments Followed by qPCR. Methylated DNA fragments were enriched from genomic DNA obtained from each sample {two offspring were randomly selected from each dam [Sham (S): n = 5, TiO₂-H (T): n = 6]} using Methyl Collector Ultra Kit (Active Motif). Then, methylated DNA fragments were subjected to qPCR analysis. Two-way ANOVA with exposure and sex as factors, followed by post-hoc Tukey test. *p < 0.05: main effect of TiO₂-NP exposure on relative DNA methylation; ^# p < 0.05: main effect of sex on relative DNA methylation (Sham vs. Exposure).

FIGURE 5

Scatter plots of brain mRNA expression levels of 1-day-old offspring mice from TiO₂-H and Sham groups determined by qRT-PCR. Total RNA obtained from each sample {two offspring were randomly selected from each dam [Sham (S): n = 5, TiO₂-H (T): n = 6]} was transcribed to cDNA and subjected to qRT-PCR analysis. Two-way ANOVA with exposure and sex as factors, followed by post-hoc Tukey test. *p < 0.05: main effect of TiO₂-NP exposure on expression (Sham vs. Exposure).