DNA damage response of A549 cells treated with particulate matter (PM10) of urban air pollutants

doi:10.1016/j.canlet.2009.01.010

Cancer Letters

Volume 278, Issue 2, 18 June 2009, Pages 192-200

https://doi.org/10.1016/j.canlet.2009.01.010 Get rights and content

Abstract

We describe the events triggered by a sub-lethal concentration of airborne particulate matter (PM₁₀) in A549 cells, which include the formation DNA double-strand breaks, γH2A.X generation, and 53BP1 recruitment. To protect the genome, cells activated ATM/ATR/Chk1/Chk2/p53 pathway but, after 48 h, cells turned into a senescence-like state. Trolox, an antioxidant, was able to prevent most of the alterations observed after particulate matter exposure, demonstrating the important role of ROS as mediator of PM₁₀-induced genotoxicity and suggesting that DNA damage could be the mechanisms by which particulate matter augment the risk of lung cancer.

Introduction

There is growing concern that exposure to airborne particulate matter (PM) is associated with an increase in morbidity and mortality and that it could augment the risk of lung cancer [1], [2], [3]. PM of an aerodynamic diameter ⩽10 mm (PM₁₀) consists of dust, soot, and other solid, liquid, and aerosol particles, as well as its chemical constituents. Although the mechanisms of PM₁₀-related health effects remain incompletely studied, there is increasing evidence that exposure to PM₁₀ has a major effect in several experimental models [4], [5]. In addition, some of these changes are strongly related to an increase in reactive oxygen species (ROS) [6], [7]. In this regard, one of the most important changes induced by PM₁₀ is DNA damage [8], [9] and the level of this damage has been correlated with mutagenicity and tumor formation in experimental models [10], [11]. The induced DNA damage by PM₁₀ and its constituents involves the formation of DNA double-strand breaks (DSBs) [8], [9], [12], [13]. In response to DSBs caused by other agents like ionizing radiation [14], the histone H2A.X undergoes phosphorylation at Ser139 [14]. The phosphorylated H2A.X protein is defined as γH2A.X and this phosphorylation is mediated by phosphoinositide 3-kinase-related protein kinases (PIKKs), including ataxia telangiectasia mutated (ATM), ATM and Rad-3 related kinase (ATR), and DNA dependent protein kinase (DNA-PKcs) [15]. ATM and ATR, in turn, activate the checkpoint kinases, Chk1 and Chk2, and tumor suppressor 53-binding protein 1 (53BP1). 53BP1 binds to the DNA-binding domain of p53, enhances p53-mediated transcriptional activation, and rapidly colocalizes with γH2A.X in response to DNA damage induced by ionizing radiation [16]. In addition, Chk1 and Chk2 kinases have several effectors, including cell division 25 (Cdc25) family proteins, which have phosphatase activity related to cell cycle regulation, specifically Cdc25A, in response to ionizing radiation [17]. On the other hand, after DNA damage, p53 initiates different transcriptional programs that lead to cell cycle arrest, cellular senescence, or apoptosis in response to γ-radiation through the ATM/ATR and Chk1/Chk2 kinases [18]. To this regard, before p53 induction, a disruption between the complex formed by p53 and MDM2, a p53-specific E3 ubiquitin ligase, is needed for its biological response [19]. In the setting of DNA damage, the cell undergoes cell cycle arrest to provide time to carry out DNA repair [9], [13] or apoptosis, depending on the PM₁₀ concentration used. Based on this information, we decided to investigate if the above cell-signaling pathway (ATM/ATR/Chk1/Chk2/p53) is involved in the response to the DNA damage induced by PM₁₀ exposure in epithelial lung cancer cells even when cells are exposed to a sub-lethal concentration. Since there is an increase in DNA damage correlated to the oxidative stress induced by environmental pollutants [20], [21], we decided to use trolox, a hydroxyl radical scavenger, to investigate if a connection exists between the effect of PM₁₀ exposure on DNA damage and if this effect is related to ROS formation. This study evidences that PM₁₀ activates sensors (53BP1), transducers (ATM/ATR), and effectors (H2A.X, Chk1, Chk2, p53, and MDM2) of DNA damage, pointing toward a strong relation between DNA damage and oxidative stress. Even though, this signal transduction pathway is activated in response to DNA damage after 24 h, a senescence-like phenotype is observed in cells exposed to PM₁₀ after 48 h. These findings provide important clues on the role of PM₁₀ exposure in DNA damage and senescence-like events that are strongly involved in cancer development.

Section snippets

PM₁₀ sampling

PM₁₀ was collected in a commercial zone (with major traffic sources) of Mexico City using a high-volume particle collector with a flux of 1.13 m³/min (GMW model 1200 VFC HVPM10; Sierra Andersen, Smyrna, GA, USA). PM₁₀ was collected on 3.0-μm pore size cellulose nitrate filters (Sartorius AG, Goettingen, Germany), 3 days a week from October 2004–May 2005. Filters were maintained in the dark at 4 °C in a desiccator prior to particle removal. Particles were gently scraped off the membranes with a

PM₁₀ exposure induces DNA damage in A549 cells

To investigate the effect of PM₁₀ in human A549 cells, we first examined the ability of PM₁₀ to induce DNA damage using the comet assay; a well-established method for the DNA damaged detection. In this assay, the damaged DNA migrates out of the nucleus forming a tail, which can be quantified using image analysis. Fig. 1 shows that cells exposed to 10 μg/cm² of PM₁₀ after 24 h have an increased tail length compared to control cells (p < 0.001). Trolox treatment of cells exposed to PM₁₀-induced a

Discussion

A549 cells were exposed to PM₁₀ using a concentration of 10 μg/cm² and the effect of 10 μM trolox, a hydroxyl radical scavenger, was explored to investigate if the DNA damage induced by PM₁₀ is mediated by oxidative stress. One of the most important characteristics of PM₁₀ is their ability to penetrate into the thoracic part of the airways, where they could have adverse effects [33]. The effects observed in this work could be attributed to the components of PM₁₀, including polycyclic aromatic

Acknowledgments

The authors express their gratitude to Yazmín Segura and Raúl Quintana, participants in the field campaign. This work was supported by CONACyT-Mexico Grants 43183-M, AC-2006-52830. Posdoctoral Fellowship to Yolanda I. Chirino from CONACyT (CVU 49870) and a Parker B. Francis Fellowship, USA, to Claudia María García-Cuellar are acknowledged. We thank Ingrid Mascher for revising the English version of the manuscript.

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¹: These authors contributed equally to the work.

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DNA damage response of A549 cells treated with particulate matter (PM10) of urban air pollutants

Abstract

Introduction

Section snippets

PM10 sampling

PM10 exposure induces DNA damage in A549 cells

Discussion

Acknowledgments

J. Biol. Chem.

Cell

Mutat. Res.

Mutat. Res.

Exp. Cell Res.

Anal. Biochem.

Cell

Mutat. Res.

Exp. Gerontol.

J. Biol. Chem.

Cell

Critical review: health effects of fine particulate air pollution: lines that connect

J. Air Waste Manage. Assoc.

Air pollution and cancer: biomarker studies in human populations

Carcinogenesis

Long-term concentrations of ambient air pollutants and incident lung cancer in California adults: results from the AHSMOG study. Adventist Health Study on Smog

Environ. Health Perspect.

Biologic effects induced in vitro by PM10 from three different zones of Mexico City

Environ. Health Perspect.

Induction of the lung myofibroblast PDGF receptor system by urban ambient particles from Mexico City

Am. J. Respir. Cell Mol. Biol.

Personal exposure to ultrafine particles and oxidative DNA damage

Environ. Health Perspect.

Subway particles are more genotoxic than street particles and induce oxidative stress in cultured human lung cells

Chem. Res. Toxicol.

Nanoparticle-driven DNA damage mimics irradiation-related carcinogenesis pathways

Eur. Respir. J.

Particulate matter induces alveolar epithelial cell DNA damage and apoptosis: role of free radicals and the mitochondria

Am. J. Respir. Cell Mol. Biol.

Increase in mutation frequency in lung of Big Blue rat by exposure to diesel exhaust

Carcinogenesis

Lung carcinogenesis and formation of 8-hydroxy-deoxyguanosine in mice by diesel exhaust particles

Carcinogenesis

Nanoparticle carbon black driven DNA damage induces growth arrest and AP-1 and NFkappaB DNA binding in lung epithelial A549 cell line

J. Physiol. Pharmacol.

p53 Mediates particulate matter-induced alveolar epithelial cell mitochondria-regulated apoptosis

Am. J. Respir. Crit. Care Med.

Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

Nat. Cell Biol.

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

BMC Cell Biol.

Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways

J. Cell Biol.

The MDM2-p53 interaction

Mol. Cancer Res.

DNA damage response of A549 cells treated with particulate matter (PM₁₀) of urban air pollutants

PM₁₀ sampling

PM₁₀ exposure induces DNA damage in A549 cells