Abstract 4817: Altered DNA methylation in human lung cell line A549 after treatment with a standard mixture of particulate matter

Abstract

Abstract BACKGROUND: Numerous health studies have shown acute and chronic particulate air pollution (PM, particulate matter) exposures to be associated with lung cancer, as well as with lung growth impairment, chronic obstructive pulmonary disease (COPD), and asthma. Toxicology studies have shown that PM is only a weak mutagen. Whether PM induces DNA methylation alterations, a primary mechanism in cell transformation, has never been studied in lung epithelial cells. In our study, we found a significative modification of P16 DNA methylation after PM treatement. AIM: To investigate PM on 8 cancer and inflammatory genes (TLR-4, CD14, IFNγ, TNFα, RASSF1A, APC, p53, p16) in A549 human alveolar basal epithelial cells. METHODS: A549 cells were treated with a complex standard mixture of particulate matter (NIST-National Institute of Standards and Technology- Standard Reference Material [SRM] 1648a). SRM 1648a treatments were performed with increasing doses of total mixture (15, 31, 62, 125, and 250 μg/ml) dissolved in colture medium. We used untreated cells as a negative control. Only concentrations that did not affect cell viability were considered. The cells were harvested after 2, 6, 12, 24, and 48 hours of continuous exposure, washed with phosphate buffered saline, and stored as cell pellet at -80°C until DNA extraction. DNA methylation was measured by means of bisulfite-Pyrosequencing. All the experiments were conducted in triplicate to confirm repeatability of our results. RESULTS: Using a t-test assuming that the two distributions have the same variance, we found an increase in p16 methylation for a dose of 15 μg/ml (p16 mean(untreated-treated)=0,83; p-value=0,05); an hypomethylation for a dose of 62 μg/ml (p16 mean(untreated-treated)=−0,77; p-value=0,004) as well as for a dose of 250 μg/ml (p16 mean(untreated-treated)=−0,78; p-value=0,01). We observed also an increase in APC methylation for a dose of 250 μg/ml (APC mean(untreated-treated)=2,90; p-value=0,02). CONCLUSION: This finding indicates that different levels of exposure to PM can induce changes in the DNA methylation levels in vitro. Our hypothesis is that exposure to PM can cause changes in the gene-specific DNA methylation levels thus enhancing cancerogenesis. Additional studies are required to better define the epigenetic mechanisms by which PM and similar environmental agents induce changes in DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2011-4817