In vivo epigenetic effects induced by engineered nanomaterials: A case study of copper oxide and laser printer-emitted engineered nanoparticles

Abstract

Evidence continues to grow on potential environmental health hazards associated with engineered nanomaterials (ENMs). While the geno- and cytotoxic effects of ENMs have been investigated, their potential to target the epigenome remains largely unknown. The aim of this study is two-fold: 1) determining whether or not industry relevant ENMs can affect the epigenome in vivo and 2) validating a recently developed in vitro epigenetic screening platform for inhaled ENMs. Laser printer-emitted engineered nanoparticles (PEPs) released from nano-enabled toners during consumer use and copper oxide (CuO) were chosen since these particles induced significant epigenetic changes in a recent in vitro companion study. In this study, the epigenetic alterations in lung tissue, alveolar macrophages and peripheral blood from intratracheally instilled mice were evaluated. The methylation of global DNA and transposable elements (TEs), the expression of the DNA methylation machinery and TEs, in addition to general toxicological effects in the lung were assessed. CuO exhibited higher cell-damaging potential to the lung, while PEPs showed a greater ability to target the epigenome. Alterations in the methylation status of global DNA and TEs, and expression of TEs and DNA machinery in mouse lung were observed after exposure to CuO and PEPs. Additionally, epigenetic changes were detected in the peripheral blood after PEPs exposure. Altogether, CuO and PEPs can induce epigenetic alterations in a mouse experimental model, which in turn confirms that the recently developed in vitro epigenetic platform using macrophage and epithelial cell lines can be successfully utilized in the epigenetic screening of ENMs.

Keywords: Copper oxide; DNA methylation; epigenetics; printer-emitted engineered nanoparticles; transposable elements.

Figure 1

Overall research strategy and experimental design for the in vitro (Lu et al., 2015) and in vivo epigenetic studies.

Figure 2

Lung injury and oxidative stress induced by ENMs. (A–B) Lactate dehydrogenase release (A) and myeloperoxidase activity (B) in BALF after exposure to CuO and PEPs in vivo. (C–E) 8-oxo-G levels in genomic DNA after exposure to CuO and PEPs in vitro and in vivo; (C) in RAW264.7 cells, (D) in SAEC cells, and (E) in lung tissue isolated from exposed animals. (F–G) The Hmox1 gene expression was measured in the alveolar macrophages (F) and lung tissue (G) isolated from exposed animals. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ANOVA with Dunnett’s test. 8-oxo-G values are not available for alveolar macrophages.

Figure 3

Levels of global and TEs-associated DNA methylation in response to ENMs exposure. (A–B) Global DNA methylation was assessed by measuring 5-mC (A) and 5-hmC (B) levels in whole genome DNA by HPLC. (C–J) The methylation status of the four regions of LINE-1 after exposure to CuO and PEPs; (C–D) The methylation status of the 5′ UTR region of LINE-1 in the alveolar macrophages (C) and lung tissue (D) isolated from exposed animals, (E–F) The methylation status of the open reading frames 1 (ORF1) region of LINE-1 in the alveolar macrophages (E) and lung tissue (F), (G–H) The methylation status of the ORF2 region of LINE-1 in the alveolar macrophages (G) and lung tissue (H) isolated from exposed animals, (I–J) The methylation status of the 3′ UTR region of LINE-1 in the alveolar macrophages (I) and lung tissue (J) isolated from exposed animals. (K–L) The methylation status of SINE B1 in the alveolar macrophages (K) and lung tissue (L) isolated from exposed animals. *p ≤ 0.05, ***p ≤ 0.001, ANOVA with Dunnett’s test. Whole genome values are not available for alveolar macrophages. AM: alveolar macrophages.

Figure 4

Analysis the expression of DNA methylation machinery. (A–B) The expression levels of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b in the alveolar macrophages (A) and lung tissue (B) isolated from the exposed animals of CuO and PEPs treatments. (C–D) The expression levels of methylcytosine-deoxygenase Tet1 in the alveolar macrophages (C) and lung tissue (D) isolated from the exposed animals of CuO and PEPs treatments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ANOVA with Dunnett’s test.

Figure 5

Retrotransposon transcript levels in response to ENMs exposure. (A–B) The expression levels of LINE-1 5′ UTR in the alveolar macrophages (A) and lung tissue (B) isolated from the exposed animals of CuO and PEPs treatments. (C–D) The expression levels of SINE B1 in the alveolar macrophages (C) and lung tissue (D) isolated from the mice of CuO and PEPs exposures. *p ≤ 0.05, ANOVA with Dunnett’s test.

Figure 6

Epigenetic parameters in peripheral blood of ENMs-exposed mice. (A–B) The levels of Dnmt1 (A) and Dnmt3a (B). (C–D) The methylation (C) and expression (D) of the 5′ UTR region of LINE-1 retrotransposon in the peripheral leukocytes of exposed mice. *p ≤ 0.05, ANOVA with Dunnett’s test.