WANG Fei Fei， GENG Chun Mei， HAO Wei Dong， ZHAO Yong Dong3，，LI Qin， WANG Hong Mei， and QIAN Yan

1. Department of Toxicology， School of Public Health， Peking University， Beijing 100191， China； 2. State Key Laboratory of Environmental Criteria and Risk Assessment， Chinese Research Academy of Environmental Sciences，Beijing 100012， China； 3. Department of Toxicology， Baotou Medical College of Public Health， Baotou 014040，Inner Mongolia， China

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Original Article

The Cellular Toxicity of PM2.5Emitted from Coal Combustion in Human Umbilical Vein Endothelial Cells*

WANG Fei Fei1，2， GENG Chun Mei2， HAO Wei Dong1，#， ZHAO Yong Dong3，2，LI Qin2， WANG Hong Mei2， and QIAN Yan2

1. Department of Toxicology， School of Public Health， Peking University， Beijing 100191， China； 2. State Key Laboratory of Environmental Criteria and Risk Assessment， Chinese Research Academy of Environmental Sciences，Beijing 100012， China； 3. Department of Toxicology， Baotou Medical College of Public Health， Baotou 014040，Inner Mongolia， China

Abstract

Objective To explore the relationship between different components of fine particulate matter （PM2.5）emitted from coal combustion and their cytotoxic effect in the vascular endothelial cells.

Methods Coal-fired PM2.5was sampled using a fixed-source dilution channel and flow sampler. The sample components were analyzed by ion chromatography and inductively coupled plasma atomic emission spectroscopy （ICP-AES）. The PM2.5suspension was extracted using an ultrasonic water-bath method and then human umbilical vein endothelial cells （EA.hy926） were treated with various concentrations of the PM2.5suspension. Cell proliferation， oxidative DNA damage， and global DNA methylation levels were used to measure the cellular toxicity of PM2.5emitted from coal combustion.

Results Compared to other types of coal-fired PM2.5preparations， the PM2.5suspension from Yinchuan coal had the highest cytotoxicity. PM2.5suspension from Datong coal had the highest toxic effect while that from Yinchuan coal had the lowest. Exposure to coal-fired PM2.5from Jingxi coal resulted in lower 8-hydroxy-2'-deoxyguanosine （8-OHdG） levels. At the same dose， PM2.5emitted from coal combustion could produce more severe DNA impairment compared to that produced by carbon black. Cell survival rate was negatively correlated with chloride and potassium ions content. The 5-methylcytosine （5-mC）level was positively correlated with Mn and negatively correlated with Zn levels. The 8-OHdG% level was positively correlated with both Mn and Fe.

Conclusion PM2.5emitted from coal combustion can decrease cell viability， increase global DNA methylation， and cause oxidative DNA damage in EA.hy926 cells. Metal components may be important factors that influence cellular toxicity.

PM2.5； Coal combustion； Vascular endothelial cell； Cytotoxicity； DNA methylation

www.besjournal.com （full text） CN: 11-2816/Q Copyright ?2016 by China CDC

INTRODUCTION

In 2004， the American Heart Association （AHA）published its first scientific statement concluding that exposure to particulate matter （PM） air pollution contributes to cardiovascular morbidity and mortality［1］. Increasing evidence has shown that the overall absolute risk for mortality attributable to PM air pollution is much higher for cardiovascular-caused than forrespiratory-caused［2-3］. Heart rate， blood pressure，myocardial infarction， and atherosclerosis are the main health outcome posed by PM exposure［4］. Research has shown that the main pathway by which PM contributes to increased cardiac risk is by promoting atherosclerotic progression via vascular endothelial cell injury［5-10］. Studies have shown that emission from coal burning is mainly responsible for fine particle pollution in China［11-14］. Thus， this study was aimed at exploring the relationship of different components of PM2.5emitted from coal combustion and the cytotoxic effect in the vascular endothelial cells by evaluating cell proliferation， oxidative DNA damage， and global DNA methylation levels.

MATERIALS AND METHODS

Coal Samples and Technical Analysis

Raw coal from four typical coal fields （Yinchuan，Datong， Jingxi， and Zhijin） in China was purchased from state-owned coal mines［15］. Volatile matter， one of the main indexes in the technical analysis of coal，was determined on a dry basis［16］.

Sample Collection of Coal-fired PM2.5

PM2.5emitted from coal combustion was sampled by the dilution tunnel system， which was designed and utilized to measure and analyze the emission status and characteristics of particulate matters emitted from stationary sources［16-17］. The dilution tunnel system had a dynamic dilutor to introduce flue gas into the smog chambers to avoid the loss of particles. Dilution and sampling continued until the combustion finished and the coal samples were broken into pieces， after which they were ignited in the stove.

Emission Characteristics of Coal-fired PM2.5

Particle size distribution and concentration were measured using Model 3090 EEPS? （TSI， USA）. Water-soluble inorganic ions and metal elements were analyzed by ion chromatograph （761 Compact IC， Metrohm， Switzerland） and mass spectrometer （DRC-e， PerkinElmer， USA）.

Sample Extraction

The PM2.5filters were extracted with ultra-pure water in an ultrasonic bath. After ultrasonic elution and freeze-drying， coal-fired PM2.5suspension was prepared and stored at -20 ?C until it was prepared for cell exposure. The effective particle weight after the extraction divided by the total weight of the particles on the filter was determined to calculate the extraction efficiency.

Cell Line and Cell Culture

Human umbilical vein endothelial cell line （EA.hy926） was purchased from ATCC （CRL-2922，Manassas， Virginia， USA）. When 80% confluency was achieved， the cells were washed with phosphate-buffered saline （PBS）， treated with 0.25% parenzyme （Sigma， USA）， and split in a 1:3 ratio of culture to Dulbecco’s Modified Eagle’s Medium （DMEM） plus 10% fetal bovine serum （FBS）. The cells were maintained at 37 °C and 5% CO2［18］.

Experimental Groups

The experimental design had six separate treatments consisting of the following: a solvent control group （PBS）； PM2.5emitted from Yinchuan coal （YC）； PM2.5emitted from Datong coal （DT）； PM2.5emitted from Jingxi coal （JX）； PM2.5emitted from Zhijin coal （ZJ）； and PM2.5carbon black control group （CB， Degussa， Germany）. EA.hy926 cells were treated at concentrations of 0 （PBS）， 10， 25， and 50 μg/mL for 24 h.

Cell Proliferation Assay

Cell viability was determined by MTS assay （CellTiter 96? AQueous One Solution Cell Proliferation Assay kit， Promega， USA） according to the manufacturer's instruction［19］. The MTS reaction was measured using a microplate reader to measure the absorbance at 490 nm.

DNA Extraction

DNA was purified using Cell/Tissue DNA Extraction Kit （A&D Technology Corporation， Beijing，China） according to the manufacturer's instruction. DNA concentration and purity was determined by comparing the ratio of optical density measurements at 260 and 280 nm.

Determination of Global DNA Methylation

Global DNA methylation was determined using the Methyl Flash Methylated DNA Quantification Kit （Epigentek， New York， USA） according to the manufacturer's instruction［20］. The kit measures the methyl-cytosine content as a percentage of total cytosine content.

Quantitative Determination of Oxidative DNA Damage

The percentage of 8-OHdG was measured with an EpiQuik 8-OHdG DNA Damage Quantification Direct Kit （Epigentek， New York， USA） according to the manufacturer's instructions［21］. The results are expressed as quantification （%） relative to a positive control provided in the kit and normalized to the input DNA （ng） by using the formula recommended in the instructions.

Statistical Analysis

Statistical analysis was performed using SPSS version 17.0. Results are expressed as mean±standard deviation of the mean （SD） values. Data analysis was performed using unpaired Student’s t-test and one-way analysis of variance. A stepwise multiple linear regression analysis model was used to check the relationship between variables. A P value less than 0.05 was considered statistically significant.

RESULTS

Volatile Matier of Coal Samples

As shown in Table 1， volatile matier of the coal samples was in the order of DT＞YC＞ZJ＞JX.

Characteristics of Coal-fired PM2.5

During the combustion process， the particle concentration was 0-1.5×106cm-3and decreased atier 1000 s. As shown in Figure 1， the results of ion and metal composition analyses showed that the overall changing trend of content was consistent，although the content of each ion and metal had some differences. SO42-was the most abundant ion emitied from coal combustion with K+and Cl-being the next most abundant ions emitied. Inaddition to common elements （K， Na， Fe， Zn， Ca， and Al） emitied during the coal burning process， trace heavy metals （As， Co， Cr， Ni， and Pb） were also emitied.

Extraction Efficiency

As shown in Table 2， the extraction efficiency of coal-fired PM2.5was in the order of JX＞YC ＞DT＞ZJ.

Cell Proliferation

As shown in Figure 2A-2F， PM2.5emitied from coal combustion and carbon black show a dose-response relationship and could inhibit EA.hy926 cell proliferation. Furthermore， a significant decrease in cell viability was observed in the middle-and high-dose groups. As shown in Figure 2G， PM2.5suspension from Yinchuan coal had the highest cytotoxicity of all the coal-fired PM2.5preparations，and there was no significant difference in the cytotoxicity of PM2.5preparations of coal obtained from other sources. Figure 2H shows that carbon black had higher cytotoxicity than PM2.5emitied from coal combustion in the high-dose group.

Global DNA Methylation

As shown in Figure 3A-3F， in cells treated with carbon black and PM2.5emitied from coal combustion， the global DNA methylation level increased in a dose-dependent manner. Compared with the PBS control group， there were significant differences in all treatment groups. As shown in Figure 3G， the PM2.5suspension from Datong coal had the highest toxic effect and that from Yinchuan coal had the lowest toxic effect in the coal-fired PM2.5groups. At the same dosage， the 5-mC% content in the carbon black group was lower than that in the coal combustion PM2.5groups （Figure 3H）.

Table 1. Volatile Matier and Grades of Coal Samples

Table 2. Extraction Efficiency of Coal-fired PM2.5

Oxidative DNA Damage

All samples of coal-fired PM2.5and carbon black could cause DNA damage in a dose-dependent manner and the corresponding groups showed significant differences compared with the control group （Figure 4A-4F）. As shown in Figure 4G， PM2.5emitted from Jingxi coal induced lower 8-OHdG% levels compared to those in the other treatment groups at high doses. At the same dose， PM2.5emitted from coal combustion could produce more severe DNA impairment compared to that produced by carbon black （Figure 4H）.

Relationships between Composition and Toxic Effects

Stepwise multiple linear regression was performed using cell survival rate， 5-mC%， and 8-OHdG% as the dependent variables and compositions as the independent variables. As shown in Table 3， cell survival rate was negatively correlated with chloride and potassium ions content，and positively correlated with arsenic content. Significant correlations were not found between ions and 5-mC%. Table 3 shows that the 5-mC% level was positively correlated with Mn and negatively correlated with Zn. The 8-OHdG% level had no correlation with ions， but had a positive correlation with Mn and Fe （Table 3）.

DISCUSSION

The volatile matier of coal， correlated with the content of organic matier and coal quality， is the main classification index of coal. Depending on volatile matier， the degree of coalification could be determined. The higher the degree of coal metamorphism and the incombustible mineral content， the lower the volatile matier content［22-23］. The results showed that the volatile matier contents of Datong and Yinchuan coal were higher than those of Jingxi and Zhijin coal. Compared with other kinds of coal-fired PM2.5preparations， PM2.5suspension from Yinchuan coal had the highest cytotoxicity，which was in accordance with the result of volatile matier. Additionally， coal-fired PM2.5from Jingxi coal induced lower 8-OHdG% levels and that from Datong and Yinchuan coal produced more severe DNA impairment， which was in accordance with the volatile matier content. For global DNA methylation，PM2.5suspension from Datong coal had the highest toxic effect， which was also in agreement with the volatile matier content. However， PM2.5emitied from Yinchuan coal had the lowest 5-mC% levels. This result was inconsistent with the results of volatile matier analysis and connected with Mn content， which was positively correlated with the 5-mC% levels in this study. There could be many other reasons for this finding， which need to be further explored.

Several toxicology experiment studies have shown that PM2.5has a dose-dependent inhibitory effect on the growth of vascular endothelial cells［9，24-25］. We also found that PM2.5emitied from coal combustion and carbon black could inhibit EA.hy926 cell proliferation with a dose-response relationship in this study. Studies have shown that respiratory and cardiovascular system diseases caused by fine particle matier could be related to enrichment of trace metals on the surface. Components of PM2.5such as Al， Ca， Fe， Pb， Mn， K，and V have associated with atherogenic index of plasma， and may contribute to the development of atherosclerosis［26］. Specific metal components of PM2.5（Ni， Cu， Se， and As） may be responsible for the elevated levels of systemic inflammatory markers，such as C-reactive protein and interleukin 6. These results suggest that specific metals may be important components responsible for PM2.5-induced cardiovascular effects［27］. The study showed that cell survival rate was negatively correlated with K+and positively correlated with As. Studies on As found that low-dose exposure could induce the hormesis of proliferation in HaCaT and HELF cells with the dose-effect relation of an inverted U curve［28-29］.

Table 3. Regression Analysis of Compositions and Cell Toxic Effects

Particulate matier can influence gene expression by changing the level of DNA methylation，which leads to the development of a disease［30］. In the current study， it is still unclear whether airborne particulate matiers induced down-regulation or up-regulation of the global DNA methylation［31-35］. Theglobal DNA methylation level was measured using MethylFlash Methylated DNA 5-mC Quantification Kit， which is a cost-effective way to accurately measure levels of 5-mC. Using the same method to evaluate the effect of PM10on global DNA methylation in RAW264.7， it was found that exposure to 20 and 50 μg/mL PM10resulted in higher global 5-mC levels compared to those in the control group. However， the global 5-mC levels decreased exposure to 100 μg/mL and increased exposure to 200 μg/mL［36］. Consistent with the results， the global DNA methylation level increased significantly when cells were exposed to 10， 20， and 50 μg/mL PM2.5emitied from coal combustion and carbon black. The results showed that the 5-mC% levels were correlated with Mn and Zn. Similarly， the study suggested that metals can alter the DNA methylation profile［37］.

A 2013 assessment by WHO’s International Agency for Research on Cancer （IARC） concluded that outdoor air pollution is carcinogenic to humans，with the particulate matier component of air pollution most closely associated with increased cancer incidence［38］. Both in vitro and in vivo experimental studies have shown that exposure to particulate matier could induce genotoxicity. The results of this study showed that coal fired PM2.5and carbon black could cause DNA damage in a dose-dependent manner. Additionally， the 8-OHdG% levels were higher than those induced by carbon black and had a positive correlation with Mn and Fe. Research has shown that Polycyclic Aromatic Hydrocarbons （PAHs） and metals were biologically active constituents of PM2.5with regard to the induction of oxidative DNA damage［39-40］. Levels of 8-OHdG were correlated with cadmium and chromium levels［41-42］. However， other studies have investigated the effects of toxic heavy metals （As， Cd，Cr， Ni， and Pb） on oxidative stress and found only As and Ni to show a marginal or a significant positive linear dose-dependent effect on 8-OHdG［40］.

Accepted: December 17， 2015

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Biomed Environ Sci， 2016； 29（2）: 107-116 10.3967/bes2016.012 ISSN: 0895-3988

September 16， 2015；

*This study was supported by the National Science Foundation for Distinguished Young Scholars of China （Grant No.21507122）.

#Correspondence should be addressed to HAO Wei Dong， Tel/Fax: 86-101-82802352， E-mail: whao@bjmu.edu.cn

Biographical note of the first author: WANG Fei Fei， female， born in 1978， PhD， majoring in environmental toxicology.