N⁶-methyladenosine (M⁶A) in fetal offspring modifies mitochondrial gene expression following gestational nano-TiO₂ inhalation exposure

N-6-methyladenosine (m(6)A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it's role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO2) contributes to cardiac m(6)A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO2 with a target aerosol mass concentration of 12 mg/m(3). At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m(6)A antibodies was performed followed by sequencing of immunoprecipitant (m(6)A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO2 exposed mice (Padj <= 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m(6)A methylation sites (Padj <= 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P <= 0.05). The lack of m(6)A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO2 inhalation exposure.