Maternal engineered nanomaterial inhalation during gestation alters the fetal transcriptome

Abstract

Background: The integration of engineered nanomaterials (ENM) is well-established and widespread in clinical, commercial, and domestic applications. Cardiovascular dysfunctions have been reported in adult populations after exposure to a variety of ENM. As the diversity of these exposures continues to increase, the fetal ramifications of maternal exposures have yet to be determined. We, and others, have explored the consequences of ENM inhalation during gestation and identified many cardiovascular and metabolic outcomes in the F1 generation. The purpose of these studies was to identify genetic alterations in the F1 generation of Sprague-Dawley rats that result from maternal ENM inhalation during gestation. Pregnant dams were exposed to nano-titanium dioxide (nano-TiO₂) aerosols (10 ± 0.5 mg/m³) for 7-8 days (calculated, cumulative lung deposition = 217 ± 1 μg) and on GD (gestational day) 20 fetal hearts were isolated. DNA was extracted and immunoprecipitated with modified chromatin marks histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3). Following chromatin immunoprecipitation (ChIP), DNA fragments were sequenced. RNA from fetal hearts was purified and prepared for RNA sequencing and transcriptomic analysis. Ingenuity Pathway Analysis (IPA) was then used to identify pathways most modified by gestational ENM exposure.

Results: The results of the sequencing experiments provide initial evidence that significant epigenetic and transcriptomic changes occur in the cardiac tissue of maternal nano-TiO₂ exposed progeny. The most notable alterations in major biologic systems included immune adaptation and organismal growth. Changes in normal physiology were linked with other tissues, including liver and kidneys.

Conclusions: These results are the first evidence that maternal ENM inhalation impacts the fetal epigenome.

Keywords: Epigenetics; Fetal; Inhalation; Maternal; Nanomaterial; Nanotechnology; Toxicology.

Fig. 1

Evaluating DNA fragmentation and read quality for chromatin immunoprecipitation (ChIP) sequencing. a Using gel electrophoresis, DNA fragments were evaluated to determine size and distribution (average size of fragments = 654.3 bp). Two controls and two maternal nano-TiO₂exposed representative samples are shown. Sample quality was assessed using FastQC for both forward and reverse reads (b) before and (c) after using Trimmomatic. Con = control, Exp = maternal nano-TiO₂ exposed, H3K4me3 and K4 = histone 3 lysine 4 tri-methylation, K27 = histone 3 lysine 27 tri-methylation

Fig. 2

Assessing RNA quality for transcriptomic data. a Gel electrophoresis was implemented to visualize 28S and 18S ribosomal RNA quality. b Cytoplasmic, ribosomal RNA degradation was measured using the Agilent Bioanalyzer 2100. As determined by the RNA Integrity Number (RIN) (left of sample name) the five least degraded samples were chosen for the control (RIN = 5.88 ± 1.22) and exposed (RIN = 6.18 ± 0.92) groups. Exposed = maternal nano-TiO₂ exposed

Fig. 3

Maternal nano-TiO₂ exposure particle characterization for RNA sequencing experiments. a Total aerosol concentration (10 mg/m³) of engineered nano-TiO₂ during maternal exposures. b Nano-TiO₂ size distribution (mobility diameter, 129.4 nm) using a scanning mobility particle sizer (SMPS). c Nano-TiO₂ size distribution (aerodynamic diameter, 143.3 nm) using an electrical low-pressure impactor (ELPI). (D) Transmission electron microscopy image of aerosolized nano-TiO₂ collected via a sampling filter during an exposure

Fig. 4

Chromatin immunoprecipitation (ChIP) sequencing fragment analysis and sample distribution. To measure the distance between subpeaks and find the maximum correlation, the cross-correlation function (CCF) was used to assess a H3K4me3 (248 bp) and (b) H3K27me3 (247 bp). Multi-dimensional scaling (MDS) plots indicate the log fold change (logFC) between samples within the (c) H3K4me3 and (d) H3K27me3 groups, describing sample-to-sample distances. Representative histone peaks are shown for differential binding regions (P ≤ 0.05) for both (e) H3K4me3 and (f) H3K27me3. Con = control, Exp = maternal nano-TiO2 exposed, H3K4me3 = histone 3 lysine 4 tri-methylation, H3K27me3 = histone 3 lysine 27 tri-methylation, Wnt5a = Wnt Family Member 5A, Rn5-8 s = 5.8S ribosomal RNA for Rattus norvegicus

Fig. 5

Assessment of disease and signaling pathways altered epigenetically during maternal nano-TiO₂ exposure. a One of the primary disease pathways (z-score = 9.35 ± 1.89) altered epigenetically during exposure was the increased susceptibility to infection in the H3K4me3 group. Disease and toxicological pathways are constructed from specific, individual canonical signaling pathways. b Depicts the top canonical pathways for H3K4me3 (z-score ≥ ±2.0) that are significantly (P ≤ 0.05) impacted, as indicated by the threshold line. c The top canonical pathways for H3K27me3 (P ≤ 0.05) are also shown following exposure (smaller p-values are associated with increasing red intensity for pathways). d Toxicological functions predicted for genes mapped to H3K4me3 marks

Fig. 6

Sample-to-sample distribution and differential expression analysis for transcriptomic analysis. a Assessment of normalized counts between control vs. control (left) and control vs. maternal nano-TiO₂ exposed (right) using a log2 transformed scale. b Measure of raw count matrices and (c) normalized count matrices to determine variance between samples. d The top differentially regulated gene between groups was determined through the normalized counts for each sample. e The MA-plot reveals the differentially expressed genes (red, P ≤ 0.05) in comparison to genes with non-significant change between groups (grey). The top differentially regulated gene is highlighted (blue). Exposed and Exp = maternal nano-TiO₂ exposed, Car1 = carbonic anhydrase 1

Fig. 7

Assessment of disease and signaling pathways altered transcriptionally during maternal nano-TiO₂ exposure. a Similar to the activation by H3K4me3, transcriptional upregulation of genes associated with increased susceptibility to infection (z-score = 2.02 ± 0.96) was found. b The top canonical pathways (z-score ≥ ±3.45) that are significantly (P ≤ 0.05) impacted transcriptionally, as indicated by the threshold line. The canonical pathways for the RNA sequencing reveal a significant increase in inflammatory and growth signaling. c The top regulator (consistency score = 10.453) determined through pathway analysis of gene expression (arrows = activation, bars = repression). Increasing gene activation (red) and suppression (blue) reveal targeting of multiple cell functions. d Toxicological functions predicted for transcript abundance in the RNA sequencing experiment

Fig. 8

Comparison of epigenetic regulation (H3K4me3 and H3K27me3) and transcriptional changes. a Top canonical pathways, ranked by z-score, which are changed between groups. b Top toxicological functions, ranked by z-score, which are changed between groups. c Top diseases and biological functions, ranked by z-score, which are changed between groups. d Top canonical pathways, ranked by cumulative P-value, which are changed between groups. e Example of one of the top canonical pathways altered during maternal nano-TiO₂ exposure. NF-ĸB signaling changes transcriptionally (right) and epigenetically through H3K4me3 (left) (green = decreased expression, red = increased expression). NF-ĸB = nuclear factor kappa-light-chain-enhancer of activated B cells

Fig. 9

Validation of sequencing and model overview. a The mRNA of Fgfr1, Il-18, and Tgfbr2 were assessed in the sham (green, Sham-Control) and maternal nano-TiO₂ (red, Nano-TiO₂ Exposed) exposed progeny, reference to the RNA sequencing observed change (grey, Sequence). Expression was normalized to the β-Actin reporter gene. b Tgfbr2 was further characterized through ChIP-qPCR of H3K4me3 to measure the binding affinity of the modified histone at the Tgfbr2 promoter loci in the Sham-Control (green) and maternal nano-TiO₂ (red) exposed progeny. Values were normalized to each sample’s input control. Tick marks represent the chromosomal location of each qPCR measurement, ranging from 124,318,034 to 124,319,434 on chromosome 8. c Schematic overview of the experimental model for nano-TiO₂ maternal exposure and examination of the fetal progeny. As an example, the changes in Tgfbr2 are used to illustrate how epigenetic alterations through modification of chromatin can lead to increased expression of the mRNA transcript. Finally, the results of the study suggest that the gestational exposure paradigm impacts the heart, through increased function, while the liver and kidney have a detriment in function. Values are expressed as means ± SE. * = P ≤ 0.05. Fgfr1 = Fibroblast Growth Factor Receptor 1, Il-18 = Interleukin-18, Tgfbr2 = Transforming Growth Factor Beta Receptor 2, H3K4me3 = histone 3 lysine 4 tri-methylation, ChIP = Chromatin Immunoprecipitation