Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice

Abstract

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.

Keywords: alternative splicing; inhalation; splice junction expression; zinc oxide nanoparticles.

Figure 1.

Size distribution of ZnO NP expressed in number concentration of nanoparticles in the inhalation chamber, with a concentration of 1.93 × 10⁶ ZnO NP/cm³ (mode diameter 7.64 nm, geometric mean diameter 12.7 nm).

Figure 2.

The scanning transmission electron micrograph showing the agglomerates of primary ZnO NP.

Figure 3.

Mean Zn content (± SD) in the lungs of animals exposed to 1.93 × 10⁶ ZnO NP/cm³ for 3 days and 3 months, respectively.

Figure 4.

The transmission electron micrograph of ZnO NP (arrows) in pneumocytes after 3 months exposure to 1.93 × 10⁶ ZnO NP/cm³. Abbreviations: E, erythrocyte; N, nucleus; Pn, pneumocyte.

Figure 5.

A goblet cell hyperplasia at the terminal bronchioli of the lungs (indicated by the arrow) of animals after 3 months exposure to 1.93 × 10⁶ ZnO NP/cm³. This finding, not observed in the control group, may be related to irritation as a consequence of ZnO NP inhalation. Abbreviations: TB, terminal bronchioli; V, vessel.

Figure 6.

Venn diagrams reporting numbers of unique and common deregulated splice junctions (A) and respective genes (B) in the lungs after inhalation exposure to ZnO NP. Three days/low—exposure to 6.46 × 10⁴ NP/cm³ for 3 days; 3 days/high—exposure to 1.93 × 10⁶ NP/cm³ for 3 days; 3 months/low—exposure to 6.46 × 10⁴ NP/cm³ for 3 months; 3 days/high—exposure to 1.93 × 10⁶ NP/cm³ for 3 months.

Figure 7.

mRNA expression levels assessed by RT-qPCR for selected genes. The expression significantly correlated with differential SJ expression data obtained for these genes (R = 0.961, p < .001) indicating that SJ expression could be used as a valid approach to study differential gene expression. Three days/low—exposure to 6.46 × 10⁴ NP/cm³ for 3 days; 3 days/high—exposure to 1.93 × 10⁶ NP/cm³ for 3 days; 3 months/low—exposure to 6.46 × 10⁴ NP/cm³ for 3 months; 3 days/high—exposure to 1.93 × 10⁶ NP/cm³ for 3 months.