Diesel exhaust particles induce apoptosis via p53 and Mdm2 in J774A.1 macrophage cell line

Abstract

Diesel exhaust particles (DEP) are known to cause cardiopulmonary diseases due to their proinflammatory and cytotoxic effects. Continuous exposure to DEP potentiates chronic inflammatory processes and acute symptomatic responses in the respiratory tract. Recent studies have emphasized that alveolar cell apoptosis is a crucial step in chronic inflammation and lung injury. The phenomenon of apoptosis is a key event that successfully clears damaged cells, and its failure leads to the development of more serious diseases, such as lung cancer. The mechanism and molecular target of DEP-induced apoptosis in the respiratory tract remain unclear.

In this study, J774A.1 macrophage cells were used to investigate the p53-mediated apoptotic pathway induced by DEP exposure. The results showed that murine double minute 2 (Mdm2), a negative regulator of p53, was downregulated at the protein level by DEP exposure. In contrast, the pro-apoptotic protein Bcl-2-associated X protein (Bax), an endogenous target of p53-dependent transcriptional activation, was continuously upregulated at the mRNA and protein levels by DEP exposure. Furthermore, pifithrin-α (p53 inhibitor) blocked DEP-induced apoptosis as well as p53 activation. Taken together, the findings of the present study suggest that DEP trigger apoptosis in J774A.1 macrophage cells via the activation of p53, followed by Bax.

Introduction

With the increasing use of diesel-powered engines, particulate air pollution is gaining recognition as a major public health hazard and contributor to the burden of pulmonary and cardiovascular diseases (Atkinson et al., 2001, Bayram et al., 2006). Diesel exhaust particles (DEP) are composed of particulate matter (PM) and contain porous carbon nuclei, which adsorb vast amounts of organic compounds, such as polyaromatic hydrocarbons (PAH), nitroaromatic hydrocarbons, heterocyclics, quinones, aldehydes, and aliphatic hydrocarbons (Schuetzle and Lewtas, 1986, Draper, 1986), as well as trace amounts of heavy metals such as iron, copper, chromium, and nickel (Vouk and Piver, 1983, Goldsmith et al., 1998). PM is usually classified according to size, and PM_2.5 denotes particles with an aerodynamic diameter of less than 2.5 μm. Due to their relatively small size and large surface area, PM_2.5 are able to gain access to biological sites and cause functional changes in critical targets in the lung (Donaldson et al., 2001, Hetland et al., 2004, Oberdorster and Utell, 2002). DEP derived from diesel-powered engines are a major source of PM_2.5 (Schuetzle and Lewtas, 1986). The Air Resource Board (ARB, 1997) reported that approximately 94% of the particles emitted by diesel engines are less than 2.5 μm in size. The proinflammatory effects of DEP have been linked to the ability of PM to generate reactive oxygen species (ROS) and oxidative stress in macrophages, bronchial epithelial cells, and other types of cells (Gurgueira et al., 2002, Hiura et al., 1999, Kumagai et al., 1997, Nel et al., 2001). Organic chemicals, including PAH, and their oxygenated derivatives (e.g., quinones) are able to initiate a redox cycle and generate ROS in target cell populations, such as macrophages (Kumagai et al., 1997, Squadrito et al., 2001).

Although the effects of DEP have been the subject of intense investigation, the mechanisms underlying the deleterious effects of DEP on respiratory organs remain unclear. There is now increasing evidence that both the production of ROS and secretion of inflammatory cytokines interact to exaggerate cell death processes, including apoptosis (Haddad, 2004, Hetland et al., 2004, Shukla et al., 2000). Apoptosis plays an important role in most lung diseases. Failure to clear unwanted cells by apoptosis results in prolonged inflammation due to the release of their toxic contents and delays repair processes. Unlike accidental cell death or necrosis, apoptotic cells should be quickly recognized and ingested by phagocytes before their toxic contents are released (Nilesh et al., 2006). The molecular mechanisms involved in apoptosis have been partially elucidated and are known to be mediated intracellularly by multiple genes, including p53 and Bcl-2-associated X protein (Bax) (Prives and Hall, 1999, Cartron et al., 2003).

The p53 protein acts as a powerful transcription factor, and it has been extensively studied. The p53 protein binds to as many as 300 different promoter elements in the human genome (Wei et al., 2006), and it broadly alters and regulates the patterns of specific gene expression involved in the cell cycle, DNA repair, and apoptosis (Kho et al., 2004, Schuler and Green, 2005). The p53 protein was reported to mediate the induction of apoptosis by asbestos (Panduri et al., 2006) and PM (Soberanes et al., 2006) in alveolar epithelial cells. The activation of p53 is an important mechanism involved in the cellular response to genotoxic stress, and we have the question whether DEP are involved in the p53 pathway.

Alveolar macrophages (AM) constitute one of the first lines of defense against pulmonary infections, and they are primarily responsible for clearing bacteria and inhaled particles (Bowden, 1984). DEP are known to induce alveolar damage, alter thiol antioxidant levels in AM, and activate AM to produce ROS and proinflammatory cytokines. Excessive ROS production leads to a state of oxidative stress, finally resulting in cellular and DNA damage (Risom et al., 2005, Ohyama et al., 2007). The continuous stimulation of DEP in cells may lead to cytotoxicity and inflammation in macrophages. The main role of the macrophage is to remove necrotic debris and dust from the lungs by phagocytosis. DEP have been shown to generate ROS and induce apoptosis in macrophages (Hiura et al., 1999, Hiura et al., 2000). However, the role of p53 in the DEP-induced apoptosis of macrophages remains unclear.

In the present study, we investigated whether the p53 pathway mediates DEP-induced apoptosis in J774A.1 macrophage cells, as these studies would contribute to the understanding of DEP toxicity.