Original article
Hyperoxia induces epigenetic changes in newborn mice lungs

https://doi.org/10.1016/j.freeradbiomed.2018.04.566Get rights and content

Highlights

  • Hyperoxia increases DNA methylation in mouse lungs.

  • The resulting epigenetic changes lead to expression alteration of TGF-β pathway.

  • Hyperoxia may be a programming factor in newborn mice.

Abstract

Supplemental oxygen exposure is a risk factor for the development of bronchopulmonary dysplasia (BPD). Reactive oxygen species may damage lung tissue, but hyperoxia also has the potential to alter genome activity via changes in DNA methylation. Understanding the epigenetic potential of hyperoxia would enable further improvement of the therapeutic strategies for BPD.

Here we aimed to identify hyperoxia-related alterations in DNA methylation, which could affect the activity of crucial genetic pathways involved in the development of hyperoxic lung injury.

Newborn mice (n = 24) were randomized to hyperoxia (85% O2) or normoxia groups for 14 days, followed by normoxia for the subsequent 14 days. The mice were sacrificed on day 28, and lung tissue was analyzed using microarrays developed for the assessment of genome methylation and expression profiles.

The mean DNA methylation level was higher in the hyperoxia group than the normoxia group. The analysis of specific DNA fragments revealed hypermethylation of > 1000 gene promoters in the hyperoxia group, confirming the presence of the DNA-hypermethylation effect of hyperoxia.

Further analysis showed significant enrichment of the TGF-β signaling pathway (p = 0.0013). The hypermethylated genes included Tgfbr1, Crebbp, and Creb1, which play central roles in the TGF-β signaling pathway and cell cycle regulation.

Genome expression analysis revealed in the hyperoxia group complementary downregulation of genes that are crucial for cell cycle regulation (Crebbp, Smad2, and Smad3).

These results suggest the involvement of the methylation of TGF-β pathway genes in lung tissue reaction to hyperoxia. The data also suggest that hyperoxia may be a programming factor in newborn mice.

Introduction

Supplemental oxygen administered to premature babies might injure the lungs [1] and, consequently, contribute to the development of bronchopulmonary dysplasia (BPD). However, the pathogenesis of BPD, which is a serious complication of prematurity, is complex and includes a range of external risk factors, as well as genetic susceptibility. Functional alterations of genomic pathways related to several genes encoding important growth factors, such as vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and insulin-like growth factor (IGF), are among key factors responsible for such susceptibility [2]. VEGF regulates endothelial cell differentiation and angiogenesis and plays a central role in the formation of embryonic vasculature [3]. TGF-β is involved in inhibition of branching morphogenesis and alveolarization in embryonic lung development [4]. IGF is also involved in growth and injury repair processes in many organs, including the lungs [5].

It is well known that oxidative stress due to reactive oxygen species (ROS) and reactive nitrogen species (RNS) can (chemically) damage lung tissue. However, hyperoxia also has the potential to alter the genome activity in lung cells by inducing DNA modifications. ROS/RNS modify cytosine with the oxidative conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine. Peroxides can also modify cytosine to 5-chlorocytosine, which mimics 5-mC [6], [7]. The above changes induce improper DNA methylation by inhibiting the binding of DNA methyltransferase 1 (Dnmt1) to DNA [8], [9]. The subsequent alteration of the methylation pattern within the CpG sequences can, in turn, result in gene silencing [10]. The above processes might serve as an example of epigenetic regulation of genome activity.

Understanding the epigenetic potential of hyperoxic lung injury might enable further improvement of therapeutic strategies. Therefore, in this study, we aimed at identifying specific, hyperoxia-related alterations of DNA methylation, which could affect the activity of crucial genetic pathways involved in the development of hyperoxic newborn lung injury. To achieve this, we used an established model with newborn mice exposed to long-term hyperoxia [11] and performed whole-genome methylation analysis with complementary expression assessment of specific genes of interest in lung tissue.

Section snippets

Animal experiment

A total of 24 newborn mice (C57Bl/6Tac) were randomized to hyperoxia (85% O2; 12 animals) or normoxia (21% O2; 12 animals) groups for 14 days, followed by normoxia conditions for all animals for the subsequent 14 days. All mice had free access to food and water and were kept under standard conditions in A-Chambers (O2 – monitor ProOX110, CO2 – monitor ProCO2 P120, BioSpherix). Lung tissue was harvested on day 28 after euthanasia with a zolazepam/tiletamine/xylazine/fentanyl cocktail. Tissue

Results

Histological assessment of lung tissue samples confirmed the presence of oxygen-related lung damage in the hyperoxia group. Fig. 1 shows arrested alveolarization with enlarged alveolar spaces and simplified alveolar structure resulting from prolonged hyperoxia.

The mean methylation level of all microarray probes was increased in the hyperoxia group compared to the normoxia group (average values for mean combined Z-scores 0.57 and − 0.31, respectively). This suggests the presence of an overall

Discussion

Hyperoxia-related alterations of cell signaling and genome expression have been thoroughly described and reviewed in the literature [14], [15], [16], [17]. It was shown that the immaturity of the antioxidant defense system, especially in extremely preterm infants [18], [19], may be a predisposing factor to BPD, as the use of high oxygen concentrations, and positive pressure ventilation upon resuscitation and in the neonatal intensive care unit. However, less is known about the influence of

Declaration of interest

None.

Funding

The work was funded by South and Eastern Norway Regional Health Authority; Source number: 6051, Project no.: 39570 and, partially, by the Polish-Norwegian Research Program, operated by the National Centre for Research and Development under the Norwegian Financial Mechanism 2009–2014 in the frame of Project Contract no. Pol-Nor/196065/54/2013.

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    The authors contributed equally to this study.

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