Genotoxic effects and oxidative stress induced by organic extracts of particulate matter (PM10) collected from a subway tunnel in Seoul, Korea

Abstract

Particulate matter (PM) has become an important health risk factor in our society. PM can easily deposit in the bronchi and lungs, causing diverse diseases such as respiratory infections, lung cancers and cardiovascular diseases. In recent days, more and more toxicological studies have been dealing with air particles in distinctive areas including industrial areas, transportation sites, or indoors. Studies on subway PM in particular, have been recognizing PM as an important health risk factor because many people use subways as a major mode of public transportation (4 million people a day in Korea). The main aim of the present study was to evaluate the genotoxic effects of organic extract (OE) of subway PM10 and potential attribution of PAHs to these effects. Particles were collected in the subway tunnel at Kil-eum station (Line 4) for one month and then extracted with Dichloromethane (DCM). Chinese Hamster Ovary cells (CHO-K1) and human normal bronchial cells (BEAS-2B) were exposed to OE, and MN and Comet assays were conducted to analyze the genotoxicity. The results showed that OE increased DNA or chromosome damages in both cell lines. In the modified Comet assay and MN assay with free radical scavengers, we confirmed that the genotoxic effect of OE was partially due to the oxidative damage on DNA. DCFH-DA assay also indicated that OE induced ROS generation in BEAS-2B cells. PAHs [benzo(a)anthracene, benzo(k)fluoranthrene, etc.], the most well-known carcinogens in polluted air, were detected in Kil-eum PM10. In conclusion, our findings confirmed that OE of subway PM10 has genotoxic effects on normal human lung cells, and oxidative stress could be one of the major mechanisms of these genotoxic effects. In addition, some genotoxic and carcinogenic PAHs were detected in OE by GC/MS/MS, even though PAHs level was not enough to increase CYP1A1 gene. Therefore, we suggest that additive or synergistic effects by unidentified chemicals as well as PAHs contained in OE of subway PM10 may induce genotoxic effects and further researches are needed to identify the genotoxic compounds in subway PM.

Introduction

Particulate matter (PM) has been considered as an air pollutant that plays an important role in diverse health effects. Several epidemiological studies have demonstrated that exposure to PM increases the risk of lung cancer, respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD) and arteriosclerosis [1], [2], [3]. Also, daily mortality was reported to increase by 0.6% for each 10 μg/m³ increase in PM10 [4], [5].

Relationships between PM and these biological effects have been assessed in a large number of studies, especially PM in traffic regions [6]. Even though the subway is a popular public transportation system (the Seoul Metro subway system hosts more than 4 million people a day in Korea), relatively little is known about subway particles and their potential health effects compared to street particles. Recently, however, more studies have been conducted regarding subway particles, and some included toxicological experiments to evaluate the biological effects of subway PM10 [7], [8]. PM10, PM with an aerodynamic diameter of less than 10 μm [8], is known to be generated by friction between wheels and rails, brake wear, and vaporization of metals due to sparking in the subway system [9], [10]. Among traffic sites, the health effects of subway PM are now recognized as important evaluation criteria for two reasons. First, there is an unexpectedly high chance of exposure to subway PM. This is not only because more and more people use the subway in many large cities but because the underground air circulation is poor due to the closed space. Second, according to several researchers, subway particles are more genotoxic and induce more oxidative stress than street particles [7], [8], [11]. Some have pointed out that the shape or surface of the subway particles could effect on their toxicity; Karlsson et al. [7] described that subway particles have sharp edges and arrowhead shapes with flat surfaces, which allow them to easily attach to cells and penetrate into the cell membrane. Other studies attributed the toxicity of subway particles to their different constituents compared to street particles, especially the iron concentration. Iron is the most enriched element in subway PM, up to 30–60% higher compared to PM at ground level [12]. Iron catalyzes hydroxyl radical formation from superoxide and hydrogen (Fenton reaction, Haber–Weiss reaction), which can be the reason for radical-mediated injuries [13].

To date, subway PM toxicity has mainly been attributed to metal compounds attached or absorbed to particles in many studies [7], [8], including Fe, Mn, Cr, Ni and Cu as the most enriched metals in subway stations [14], [15], [16], [17]. However, the toxicity of organic compounds contained in subway particles has barely been discussed yet. Polycyclic aromatic hydrocarbons (PAHs) are the most representative substances in organic compounds, some of which are classified as probable human carcinogens, such as benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, and indeno[1,2,3-cd]pyrene, by US Environmental Protection Agency (EPA). Whereas PAHs are generally considered one of the most causative chemicals in the toxicity of airborne PM, there have not been many studies addressing PAH toxicity in subway PM.

The main aim of the present study was to evaluate toxic effects of organic compounds in subway PM10 by in vitro tests. Particles were collected from the subway tunnel at Kil-eum station (Line 4) in Seoul for one month and extracted with Dichloromethane (DCM). Two different cell lines were chosen for this study: CHO-K1 cells to analyze the general toxicity of subway particles and BEAS-2B cells to evaluate the toxic effects of subway particles especially on normal human lung cells. We first examined the cytotoxic effects of subway PM10 in both cell lines by the WST-1 assay. We then carried out CBMN and comet assays in both cell lines to assess chromosome or DNA damage. We also conducted DCFH-DA assays, CBMN assays with scavengers, and modified-comet assays in BEAS-2B cells in order to examine oxidative stress caused by subway PM10. Finally, we used a GC/MS/MS system to identify PAHs as possible toxic compounds in the organic extract derived from subway PM10.