Acute inhalation of ozone induces DNA methylation of apelin in lungs of Long-Evans rats

Abstract

Apelin has cardiopulmonary protective properties that promote vasodilation and maintenance of the endothelial barrier. While reductions in apelin have been identified as a contributor to various lung diseases, including pulmonary edema, its role in the effect of air pollutants has not been examined. Thus, in the current study, we sought to investigate if apelin is a downstream target of inhaled ozone and if such change in expression is related to altered DNA methylation in the lung. Male, Long-Evans rats were exposed to filtered air or 1.0 ppm ozone for 4 h. Ventilation changes were assessed using whole-body plethysmography immediately following exposure, and markers of pulmonary edema and inflammation were assessed in the bronchoaveolar lavage (BAL) fluid. The enzymatic regulators of DNA methylation were measured in the lung, along with methylation and hydroxymethylation of the apelin promoter. Data showed that ozone exposure was associated with increased enhanced pause and protein leakage in the BAL fluid. Ozone exposure reduced DNA cytosine-5-methyltransferase (DNMT) activity and Dnmt3a/b gene expression. Exposure-induced upregulation of proliferating cell nuclear antigen, indicative of DNA damage, repair, and maintenance methylation. Increased methylation and reduced hydroxymethylation were measured on the apelin promoter. These epigenetic modifications accompanied ozone-induced reduction of apelin expression and development of pulmonary edema. In conclusion, epigenetic regulation, specifically increased methylation of the apelin promoter downstream of DNA damage, may lead to reductions in protective signaling of the apelinergic system, contributing to the pulmonary edema observed following the exposure to oxidant air pollution.

Keywords: Apelin; DNA methylation; ozone; pulmonary edema.

Fig. 1

Correlation of BAL fluid protein concentration with Penh in rats exposed to 1.0 ppm ozone. Data are presented as individual samples (n=8/group). Abbreviations: bronchoalveolar lavage (BAL), enhanced pause (Penh).

Fig. 2

Expression of common regulators of DNA methylation following 1.0 ppm ozone exposure in the left lung lobe. A: Expression of the Dnmts. B: DNMT activity in nuclear protein isolate. C: Expression of the Tets. D: TET activity in the nuclear protein isolate. Data are presented as mean ± SE following normalization to air control (n=7–8/group; n=1 identified outlier removed from DNMT activity from the ozone group). *p<0.05.

Fig. 3

Quantification of PCNA in the left lung lobe immediately following ozone exposure. Nuclear protein isolate from the lung was assessed for PCNA and was normalized to protein concentration. Data are presented as mean ± SE (n=8/group) following normalization to air control. *p<0.05.

Fig. 4

Regulation of the apelin gene following ozone exposure. A: Apelin (Apln) gene expression. B: For %5mC (methylation) and %5hmC (hydroxymethylation) quantification on the CpG island in the apelin gene, primer sets were developed that spanned a region within the proximal promoter, transcriptional start site, and exon 1. C: qPCR of the apelin gene following 5mC DNA immunoprecipitation. D: qPCR of the apelin gene following 5hmC DNA immunoprecipitation. Data are presented as mean ± SE (n=7–8/group, n=1 identified outlier removed from %5hmC in region 2; %5mC quantification was assessed in n=6/group). **p<0.01, *p<0.05.

Fig. 5

Schematic of potential pathways by which ozone inhalation is associated with acute changes in pulmonary epithelial fluid equilibrium.