PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells
Introduction
Epidemiological studies have demonstrated that long-term exposure to high concentrations of airborne particulate matter (PM) increases the risk of respiratory diseases, including lung cancer and atherosclerosis (Pope et al., 2002, Knaapen et al., 2004). Among them, chronic respiratory diseases are one of the major problems concerning public health subjects. Generally, the adverse effect of PM on human health is determined by its size, surface area, and chemical composition of the PM. In particular, increased exposure to the fine particles with a mean aerodynamic diameter of <2.5 μm (PM2.5) is associated with an increased risk of cardiovascular and respiratory deaths (Anderson et al., 2012, Ghio et al., 2012). PM2.5 has been listed as an important air pollutant due to its potential to bioaccumulation and oxidative damage to humans (Brunekreef and Holgate, 2002).
PM2.5-induced oxidative stress has been considered as an important molecular mechanism of PM2.5-mediated toxicity. Oxidative stress refers to a critical imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses (Brigelius-Flohé, 2009), and ROS has been identified as signaling molecules in various pathways regulating both cell survival and cell death (Azad et al., 2009, Wu, 2006). PM2.5-induced ROS directly interacts with antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), causing lost of enzymatic activity in vivo and in vitro (Chirino et al., 2010, Pamplona and Costantini, 2011). Moreover, several studies have demonstrated PM2.5-associated inorganic and organic components are responsible for its toxic effects, all of them have described the oxidative stress as common pathway for PM2.5-induced oxidative damage in A549 cells (Billet et al., 2007, Kouassi et al., 2010, Viola et al., 2011, Yi et al., 2012). Excessive levels of ROS, mainly radical form, can cause severe damage to DNA, RNA and proteins (Brigelius-Flohé, 2009). In this respect, subcellular degradation by autophagy may play an essential role in maintaining cell functionality.
Autophagy is the cellular pathway of a self-digestion process that regulates the degradation and recycling of unnecessary intracellular proteins and dysfunctional organelles (Azad et al., 2009, Abounit et al., 2012). During autophay, these cytoplasmic materials are sequestered into double-membraned vesicles (autophagosomes), which fuse with lysosomes to form autolysosomes, and further degrade by lysomal hydrolases (Abounit et al., 2012, Chen et al., 2007). The formation of the autophagosome is involved in various autophagy-related proteins, such as Atg5, Atg12, Beclin1, and microtubule-associated proteins light chain 3 (LC3) (Van Limbergen et al., 2009). Therefore, autophagy has been conventionally considered to be a pathway contributing to cellular homeostasis and adaptation to stress (White and DiPaola, 2009). It has been demonstrated that autophagy also functions as a cytoprotective response against various types of cellular stress by providing the cell with metabolic substrates (Chen et al., 2012). Recently, it was confirmed that ROS can trigger autophagy through several distinct mechanisms involving Atg4, catalase, and the mitochondrial electron transport chain (mETC) (Azad et al., 2009). Although the lung is a primary site of exposure for many inhaled chemical pollutants, the mechanisms underlying PM2.5 related regulation of autophagy has not been fully elucidated.
This work aims to explore the mechanism of PM2.5-related antioxidant defenses and autophagy by employing human lung epithelial A549 cells. Moreover, the function of ROS in this process is also investigated.
Section snippets
Materials
A549 cells were obtained from the Cell Bank of Peking Union Medical College (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl tetrazolium, oiMTT), dimethylsulfoxide (DMSO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Anti-LC3 antibody was obtained from ProteinTech Biotechnology (ProteinTech Ltd. Wuhan, China). Lipofectamine 2000, TRIZOL and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from
PM2.5 physical and chemical characteristics
The morphology of PM2.5 particles by SEM showed a size range of 0.1–1 μm (Fig. 1A). A dynamic light scattering measurement revealed a size range of 0.09–1 μm with a mean of 0.43 μm (Fig. 1B).
The analytical results of PAHs (Table 1) showed that benzo[b]fluoranthene (234.16 ± 21.83 ng/mg), chrysene (222.49 ± 7.09 ng/mg), fluoranthene (228.44 ± 45.70 ng/mg), phenanthrene (196.63 ± 26.37 ng/mg) and pyrene (214.27 ± 41.32 ng/mg) were the dominant PAHs in the PM2.5. The average concentrations of OC and EC in the PM2.5
Discussion
Epidemiological evidences have demonstrated that the greatest health risk of PM is correlated with smaller PM (Levy et al., 2000). PM used in this work was smaller than 2.5 μm, which are respirable and able to penetrate to the alveolar region of the lung to exert their toxicity (Harrison and Yin, 2000).
PM toxicity is mainly related its composition (Harrison and Yin, 2000), including both organic and inorganic compounds (Gualtieri et al., 2010, Yi et al., 2012). Among detected 18 PAHs, relatively
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgements
This work was financially supported by grants from Gong-Yi Program of China Ministry of Environmental Protection (No. 200909016), National Natural Science Foundation of China (No. 10875170), the National Science and Technology Ministry of China (No. 2007BAC27B02-2), and Research Supported by the CAS/SAFEA International Partnership Program for Creative Research Teams. We thank Dr. Yuanxun Zhang and Dr. Junji Cao for valuable technical assistance and discussion.
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