PM: generalidades toxicidad (propiedades), genotoxicidad y estrés oxidativo

Mutation Research 749 (2012) 39–47

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Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

Genotoxic effects and oxidative stress induced by organic extracts of particulate

matter (PM10) collected from a subway tunnel in Seoul, Korea
Mi Hyun Jung a , Ha Ryong Kim a , Yong Joo Park a , Duck Shin Park b ,
Kyu Hyuck Chung a,∗,1 , Seung Min Oh c,∗∗,1
a
School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea
b
Eco-transport Research Division, Korea Railroad Research Institute, 176 Woram-dong, Uiwang, Gyeonggi-do 437-757, Republic of Korea
c
Fusion Technology Laboratory, Hoseo University, 165 Sechul-ri, Asan, ChungcheongNam-do 336-795, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Particulate matter (PM) has become an important health risk factor in our society. PM can easily deposit
Received 24 November 2011 in the bronchi and lungs, causing diverse diseases such as respiratory infections, lung cancers and cardio-
Received in revised form 11 July 2012 vascular diseases. In recent days, more and more toxicological studies have been dealing with air particles
Accepted 5 August 2012
in distinctive areas including industrial areas, transportation sites, or indoors. Studies on subway PM in
Available online 13 August 2012
particular, have been recognizing PM as an important health risk factor because many people use sub-
ways as a major mode of public transportation (4 million people a day in Korea). The main aim of the
Keywords:
present study was to evaluate the genotoxic effects of organic extract (OE) of subway PM10 and potential
PM10
Subway
attribution of PAHs to these effects. Particles were collected in the subway tunnel at Kil-eum station
Genotoxicity (Line 4) for one month and then extracted with Dichloromethane (DCM). Chinese Hamster Ovary cells
ROS (CHO-K1) and human normal bronchial cells (BEAS-2B) were exposed to OE, and MN and Comet assays
PAHs 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.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Relationships between PM and these biological effects have

been assessed in a large number of studies, especially PM in traffic
Particulate matter (PM) has been considered as an air pollu- regions [6]. Even though the subway is a popular public transporta-
tant that plays an important role in diverse health effects. Several tion system (the Seoul Metro subway system hosts more than 4
epidemiological studies have demonstrated that exposure to PM million people a day in Korea), relatively little is known about sub-
increases the risk of lung cancer, respiratory disorders such as way particles and their potential health effects compared to street
asthma and chronic obstructive pulmonary disease (COPD) and particles. Recently, however, more studies have been conducted
arteriosclerosis [1–3]. Also, daily mortality was reported to increase regarding subway particles, and some included toxicological exper-
by 0.6% for each 10 ␮g/m3 increase in PM10 [4,5]. iments to evaluate the biological effects of subway PM10 [7,8].
PM10, PM with an aerodynamic diameter of less than 10 ␮m [8], is
known to be generated by friction between wheels and rails, brake
wear, and vaporization of metals due to sparking in the subway
∗ Corresponding author. Tel.: +82 31 290 7714; fax: +82 31 290 7771.
system [9,10]. Among traffic sites, the health effects of subway PM
∗∗ Corresponding author. Tel.: +82 41 540 9697; fax: +82 41 540 9697.
are now recognized as important evaluation criteria for two rea-
E-mail addresses: [email protected] (K.H. Chung),
[email protected] (S.M. Oh). sons. First, there is an unexpectedly high chance of exposure to
1
K.H. Chung and S.M. Oh contributed equally. subway PM. This is not only because more and more people use the

1383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.mrgentox.2012.08.002
40 M.H. Jung et al. / Mutation Research 749 (2012) 39–47

subway in many large cities but because the underground air circu- 2.2. Cell culture
lation is poor due to the closed space. Second, according to several
BEAS-2B cells were obtained from the American Type Culture Collection
researchers, subway particles are more genotoxic and induce more
(ATCC). The BEAS-2B cells were grown in bronchial-epithelial growth medium
oxidative stress than street particles [7,8,11]. Some have pointed (BEGM) supplemented with 0.5 ng/ml recombinant epidermal growth factor (EGF),
out that the shape or surface of the subway particles could effect 500 ng/ml hydrocortisone, 0.005 mg/ml insulin, 0.035 mg/ml bovine pituitary
on their toxicity; Karlsson et al. [7] described that subway particles extract, 500 nM ethanolamine, 500 nM phosphoethanolamine, 0.1 ng/ml transfer-
have sharp edges and arrowhead shapes with flat surfaces, which rin, 6.5 ng/ml 3,3 ,5-triiodothyronine, 500 ng/ml epinephrine, 0.1 ng/ml retinoic acid
and trace elements in a humidified atmosphere at 37 ◦ C and 5% CO2 . CHO-K1 cells
allow them to easily attach to cells and penetrate into the cell mem- were obtained from the Korean cell line bank (KCLB), Seoul, Korea. The CHO-K1
brane. Other studies attributed the toxicity of subway particles to cells were grown in RPMI1640 supplemented with 5% fetal bovine serum (FBS),
their different constituents compared to street particles, especially penicillin (100 units/ml) and streptomycin (100 ␮g/ml) at 37 ◦ C in an atmosphere of
the iron concentration. Iron is the most enriched element in sub- 5% CO2 /95% air under saturating humidity.
way PM, up to 30–60% higher compared to PM at ground level
2.3. Cell viability test
[12]. Iron catalyzes hydroxyl radical formation from superoxide and
hydrogen (Fenton reaction, Haber–Weiss reaction), which can be The cell viability of CHO-K1 and BEAS-2B cells was assessed using the WST-
the reason for radical-mediated injuries [13]. 1 method. CHO-K1 cells (4000 cells/well) and BEAS-2B cells (8000 cells/well) were
To date, subway PM toxicity has mainly been attributed to seeded for 24 h in 96-well plates and treated with both organic extract (OE) of sub-
way PM10 and blank extract at concentrations from 0.8 ␮g/ml to 200 ␮g/ml for 24 h.
metal compounds attached or absorbed to particles in many
According to the manufacturer’s instructions, we added 10 ␮l of Cell Proliferation
studies [7,8], including Fe, Mn, Cr, Ni and Cu as the most enriched Reagent WST-1 to each well and incubated the plates for 4 h at 37 ◦ C and 5% CO2 .
metals in subway stations [14–17]. However, the toxicity of Cell viability was quantified by measuring the absorbance at 440 nm using an ELISA
organic compounds contained in subway particles has barely reader (VERSAmax, Molecular Devices, CA).
been discussed yet. Polycyclic aromatic hydrocarbons (PAHs)
are the most representative substances in organic compounds, 2.4. In vitro cytokinesis-block micronucleus assay

some of which are classified as probable human carcinogens, such To investigate the macro-genotoxic effects of OE, we performed the in vitro
as benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, cytokinesis-block micronucleus (CBMN) assay, which is recognized as a good alter-
benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, and native to the conventional chromosome aberration technique [18]. In the CBMN
indeno[1,2,3-cd]pyrene, by US Environmental Protection Agency assay, we used Chinese hamster ovary cells (CHO-K1) as standard mammalian cells
and human lung epithelial cells (BEAS-2B).
(EPA). Whereas PAHs are generally considered one of the most
CHO-K1 cells were seeded into 8-well chamber slides at a density of
causative chemicals in the toxicity of airborne PM, there have not 1.5 × 104 cells/well and incubated for 24 h until they reached 60–70% confluence.
been many studies addressing PAH toxicity in subway PM. The treatment medium was prepared by adding OE to 5% FBS-RPMI medium in
The main aim of the present study was to evaluate toxic effects the presence or absence of 4% S9-mixture (metabolic activation system; rat liver
of organic compounds in subway PM10 by in vitro tests. Particles homogenates). Also, BEAS-2B cells were seeded into the same 8-well chamber slides
at a density of 5 × 104 cells/well. After 24 h, we replaced the medium with the treat-
were collected from the subway tunnel at Kil-eum station (Line 4) in ment medium prepared by adding OE to BEGM medium. For each independent test,
Seoul for one month and extracted with Dichloromethane (DCM). at least two slides were prepared. In the case of tests with S9 mixture, after 4 h
Two different cell lines were chosen for this study: CHO-K1 cells treatment, the treatment medium was removed by aspiration and the cells were
to analyze the general toxicity of subway particles and BEAS-2B washed once with the same amount of 5% FBS-RPMI medium for CHO-K1 cells and
BEGM for BEAS-2B cells. To restrict the analysis to cells that had passed through their
cells to evaluate the toxic effects of subway particles especially on
first division, the cells were incubated in the growth medium containing 3 ␮g/ml
normal human lung cells. We first examined the cytotoxic effects of cytochalasin B (cytokinesis-block micronucleus assay) [19]. In addition, in order to
subway PM10 in both cell lines by the WST-1 assay. We then carried investigate the genotoxic effects of OE mediated by free radicals, we carried out
out CBMN and comet assays in both cell lines to assess chromosome CBMN assay with combined treatment of various free radical scavengers, such as
or DNA damage. We also conducted DCFH-DA assays, CBMN assays superoxide dismutase (SOD; O2 radical scavenger, 30 units/ml), catalase (CAT; H2 O2
scavengers, 1000 units/ml), mannitol (M; OH scavenger, 0.5 ␮M), and sodium selen-
with scavengers, and modified-comet assays in BEAS-2B cells in ite (SE; H2 O2 scavengers, 1 ␮M), in BEAS-2B cells. One cycle after cytochalasin B was
order to examine oxidative stress caused by subway PM10. Finally, added, the growth medium was removed from the 8-well chamber slide by aspira-
we used a GC/MS/MS system to identify PAHs as possible toxic tion. The slide was washed twice with the same amount of PBS and left for 5 min at
compounds in the organic extract derived from subway PM10. 4 ◦ C after 1% sodium citrate was added. The slide was then placed in fresh fixative
(99:1 = methanol: acetic acid) for 20 min at room temperature, and subsequently
left on a clean bench to air-dry before being placed in ribonuclease A (10 ␮g/ml
2. Methods in 2 × SSC) for 5 min at 30 ◦ C. The slide was then rinsed in 2 × SSC (0.02 M sodium
citrate, 0.3 M NaCl) and left on a clean bench to air-dry. After thorough air dry-
2.1. PM10 sampling and preparation ing, the slide was stained with 5% Giemsa solution for 20 min. According to the
micronucleus scoring criterion, a score of 1000 binucleate cells per independent
PM10 were collected from Kil-eum subway station using a high-volume sam- culture was obtained. Positive mutagenic effects of the samples were defined as a
pler equipped with a cascade impactor using Teflon-coated fiber filters (SIBATA, three-fold or higher increase in the MN frequency over the control in at least one of
10 cm × 10 cm). Subway particles were collected from February 23 to April 9, at the tested doses [20]. To assess the toxicity to cells induced by cytochalasin-B, the
a flow rate of 28.3 l/min. The collection time was approximately 55,600 min and cytokinesis-block proliferation index (CBPI) was measured in BEAS-2B cells exposed
the total volume of air was 1630.1 m3 . The collected PM was 55.7 mg which was to cytochalasin-B and samples. CBPI was defined as [21]

(1 × no. of mononucleated cells) + (2 × no. of binucleated cells) + (3 × no. of tri- and tetranucleated cells)
total number cells scored should be determined in 500 cells

calculated by subtracting blank Teflon filter from PM-collected Teflon filter. Taken 2.5. In vitro single cell gel electrophoresis (In vitro Comet Assay) and modified
together, the concentration of PM10 and PM2.5 determined in our study is 34 ␮g/m3 comet assay
and 4.45 ␮g/m3 , respectively. The collected PM mainly included PM10 and the
weight ratio between PM2.5 (less than 2.5 ␮m) and PM10 (2.5–10 ␮m) was approx- To evaluate the genotoxicity of OE, we performed the comet assay as
imately 0.13. Pieces (1 cm × 1 cm) were cut from the filters, and each was immersed described by Singh et al. [22] and used CHO-K1 and BEAS-2B cells. BEAS-2B cells
in a volumetric flask. 100 ml of DCM was added and the flask was sonicated sev- (40 × 104 cells/well) and CHO-K1 cells (20 × 104 cells/well) were seeded on a 6-well
eral times for 30 min. After more DCM was added, the suspension was evaporated plate and the culture was maintained at 37 ◦ C under 5% CO2 . After 24 h incuba-
on rotary evaporators. The procedures were repeated twice. The particles were sus- tion, the cells were exposed to OE for 24 h. Following this, the treated cells were
pended in DMSO (final concentration, 100 mg/ml) and the impurities were removed trypsinized to produce a single-cell suspension, washed in ice-cold phosphate-
by means of a 0.22 ␮m syringe filter. The solutions were stored at −20 ◦ C prior to buffered saline (PBS) and resuspended in 0.7% low melting point agar (Quantum
cell exposure. Biotechnologies Inc., Canada) in PBS at a concentration of 5 × 104 cells/ml. A 150 ␮l
 M.H. Jung et al. / Mutation Research 749 (2012) 39–47 41

Table 1 120
Primers for PCR analysis.

Genes Primers
100
18s rRNA Forward 5 -TAGAGTGTTCAAAGCAGGCCC-3
Reverse 5 -CCAACAAAATAGAACCGCGGT-3
**

Cell Viability (%)

CYP 1A1 Forward 5 -CACCATCCCCCACAGCAC-3 80
**
Reverse 5 -ACAAAGACACAACGCCCCTT-3

60 **
aliquot of this suspension was pipetted onto precoated glass slides, a cover glass was
placed on top, and the agar was allowed to set at 4 ◦ C. The sample was then placed in
40
lysis solution. For the alkaline assay, cells were lysed in pH 10 lysis solution [2.5 M
BEAS-2B cells
NaCl, 100 mM Na2 EDTA, 10 mM Tris–HCl, 1% Triton X-100, 10% (v/v) DMSO] at 4 ◦ C
CHO cells
for 60 min. In the normal comet assay, the slide was covered with 0.7% low melting
20
point agar and the agar was allowed to set at 4 ◦ C. In the modified comet assay [23],
the slides were washed three times (for 5 min each) with enzyme buffer (40 mM
HEPES, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml BSA; pH 8.0) and covered with 100 ␮l
0
of formamidopyrimidine DNA-glycosylase (FPG) or endonuclease III (ENDOIII). After
incubation for 30 min or 45 min at 37 ◦ C, the lysed cells were rinsed and allowed an
0 1.6 3.1 6.3 12.5 25 50 100
unwinding time of 30 min in electrophoresis buffer (1 mM Na2 EDTA, 300 mM NaOH; OE Concentration (µg/ml)
pH 13). Electrophoresis was then performed for 30 min at 25 V on ice and the cells
were neutralized with 0.4 M Tris–HCl (pH 7.5) for 10 min. The cells were stained Fig. 1. Cell viability in CHO-K1 and BEAS-2B cells exposed to the organic extract
with ethidium bromide (20 ␮g/ml) just prior to the image analysis. DNA migration (OE) of particulate matter (PM10). The cells were incubated with OE (1.6–100 ␮g/ml)
was assessed by fluorescence microscopy (Leica DMLB, excitation filter 515–560 nm for 24 h. WST-1 assays were carried out as described in Section 2. The results are
and a barrier filter of 590 nm) with a digital camera. The images were evaluated by expressed as mean ± S.D. of two separate experiments for each data point. Values
an auto image analysis system (Komet version 5.0; Kinetic Imaging Ltd., UK). The are significantly different from the control (0 ␮g/ml): **p < 0.01.
olive tail moment (% DNA in tail × distance between centers of mass) was used to
quantify the DNA damage based on the random scoring of 100 nuclei per slide. A
minimum of two replicate Comet slides were prepared for each sample. volume of 25 ␮l. The PCR amplification was assayed using a Smart Cycler (Cepheid,
Sunnyvale, California, USA). The fluorescence signals were measured at the end of
2.6. Determination of ROS generation each extension step. The threshold cycle (Ct) was determined for each sample using
the exponential growth phase, and the baseline signal from the fluorescence versus
The methods of Hussain et al. [24] and Fotakis et al. [25] were adopted to the cycle number plots. To ensure that a single product was amplified, melt curve
determine the increase of intracellular reactive oxygen species (ROS) in BEAS-2B analysis was performed on the PCR products at the end of each PCR run.
cells treated with OE. Cells were seeded in 48-well plates at an initial density of
7.5 × 104 cells/well. After 24 h, cells were exposed to OE (1.6–100 ␮g/ml) for 24 h 2.10. GC/MS/MS
and then loaded with 2 ,7 -dichlorohydrofluorescein diacetate (DCFH-DA) (Invit-
rogen) for 1 h as an intracellular ROS indicator. Each well was washed twice with The PAH concentrations in the organic extract from subway PM10 were
PBS, followed by lysis using 0.1 N NaOH. DCF (green fluorescence) production was determined by GC/MS/MS using an Agilent 7890A/7700GC/TQ-MS. The HP-5MS
measured in a fluorometer (PerkinElmer, LS50B) with an excitation wavelength of 30 m × 0.25 mm × 0.25 ␮m column was used. A 1 ␮l aliquot of the derivatized extract
485 nm and an emission wavelength of 535 nm. was injected splitlessly into the column (HP-5MS 30 m × 0.25 mm × 0.25 ␮m) and
the flow of carrier gas (Helium) through the column was 69 ml/min. The column
2.7. Total RNA isolation oven temperature was programmed to keep the initial temperature at 70 ◦ C for
3 min, to rise from 70 ◦ C to 300 ◦ C at 10 ◦ C/min, and to maintain the final tempera-
BEAS-2B cells (60 × 104 cells/well) were seeded on a 6-well plate and the cul- ture, 300 ◦ C, for 10 min. The injector temperature was 270 ◦ C and the MSD transfer
ture was incubated for 24 h at 37 ◦ C. After OE exposure, total RNA was isolated from line temperature was 280 ◦ C.
BEAS-2B cells using Trizol reagent (Invitrogen, USA). After each well was treated
with 1 ml Trizol for 5 min at room temperature to permit the complete dissociation
2.11. Statistical methods
of nucleoprotein complexes, cells were scraped into 1.5 ml Eppendorf tubes and
0.2 ml chloroform was added to each tubes. The samples were mixed vigorously and Sigma Plot 10.0 (Jandel Science Software, San Rafael, CA) and Excel 2007
then centrifuged at 12,000 × g for 15 min at 4 ◦ C. Centrifugation separated the bipha- (Microsoft, Redmond, WA) were used to analyze the data. The data from each assay
sic mixtures into the lower red, phenol-chloroform phase and the upper colorless, were expressed as mean ± standard deviation. Statistical analysis was performed
aqueous phase. The RNA was precipitated from the aqueous phase by mixing with using the SPSS (version 17.0 SPSS Inc., Chicago, IL) program. Differences between
0.5 ml isopropanol. The samples were shaken briefly, incubated at room tempera- groups were tested by t-test following one-way analysis of variance (ANOVA). Sta-
ture for 10 min, and centrifuged at 12,000 × g for 15 min at 4 ◦ C. The supernatant was tistical significance was accepted at **p < 0.01, *p < 0.05.
removed and the RNA pellet was washed twice with 75% ethanol in diethyl pyrocar-
bonate (DEPC)-treated H2 O. The pellet was air dried and dissolved in DEPC-treated
H2 O. In order to determine the concentration and purity of the RNA solution, 1 ␮l of 3. Results
RNA solution was transferred into an RNase-free tube containing 49 ␮l DEPC-treated
H2 O. The RNA was quantitated by A260 measurement.
3.1. Cytotoxic effect of subway PM10
2.8. Reverse transcriptase PCR
CHO-K1 and BEAS-2B cells were exposed to the organic extract
mRNA species were reversely transcribed into cDNA through the enzymatic (OE) of subway PM10 for 24 h at concentrations from 1.6 ␮g/ml
activity of reverse transcriptases (RT) in the presence of dNTPs and an oligo dT
to 100 ␮g/ml. As shown in Fig. 1, no cytotoxicity was observed in
primer. 1 ␮g mRNA isolated from cell was prepared in a reaction tube with 0.5 ␮g/ml
of random primers and 1 ␮l of AMV reverse transcriptase, 4 ␮l of 5× RT buffer, 0.5 ␮l
BEAS-2B cells in WST-1 results. However, cytotoxicity in CHO-K1
of RNase inhibitor, and 1 ␮l of 10 mM dNTPs. The RT conditions were as follows: cells exposed to OE increased significantly (*p < 0.05) in a dose-
room temperature for 10 min, 42 ◦ C for 50 min, and 94 ◦ C for 5 min. The reaction dependent manner.
was performed according to the manufacturer’s instructions.

2.9. Quantitative real-time PCR 3.2. Genotoxic effect of subway PM10 in CHO-K1 cells

cDNA from each of the samples was used. The specific primers were designed In order to investigate the genotoxic effects of OE, we conducted
and shown in Table 1. 18s ribosome levels were used to normalize the results. The the CBMN assay and the comet assay using CHO-K1 cells. In the
real-time fluorescence signal generated by the nonspecific double-stranded DNA
binding dye, SYBR Green I, was analyzed using Smart Cycler software. PCR was per-
CBMN assay (Fig. 2), micronucleus formation increased in CHO-
formed using 0.1 ␮M of the sense and antisense oligonucleotide primers, 12.5 ␮l of K1 cells treated with different concentrations (1.6–100 ␮g/ml) of
2× SYBR Premix Ex Taq (Takara BIOINC, Japan), and 1 ␮l of template DNA in a final OE compared with the control (**p < 0.01). % BN and CBPI were
42 M.H. Jung et al. / Mutation Research 749 (2012) 39–47

50 30
Frequency of MN in 1000 counted cells

S9(-) S9(-)
S9(+) ## S9(+)
40 ##
** 25

** #

Olive Tail Moment

##
20 ## **
30 ## ** **
** **
** 15
20 ##
** ** **
10
**
10
##
**
5 **
**
0
CON 1.6 6.3 25 100
0
CON 1.6 6.3 25 100
OE Concentration (µg/ml)
OE Concentration (µg/ml)
Fig. 2. Micronucleus formation induced by the organic extract (OE) of subway PM10
using the cytokinesis-block micronucleus (CBMN) assay in CHO-K1 cells. MN for- Fig. 3. DNA breakage in CHO-K1 cells exposed to by the organic extract (OE) of sub-
mation was observed in the absence or presence of rat liver homogenates (S9) in way PM10, as determined by the comet assay. Cells were pre-treated with rat liver
CHO-K1 cells. Cells were respectively exposed to OE (1.6–100 ␮g/ml). CHO-K1 cells homogenates (S9) for 4 h or not and then incubated with OE in RPMI supplemented
were pre-treated with S9 for 4 h or not and then incubated with OE in RPMI supple- with 5% FBS. The assays were carried out as described in Section 2. The results are
mented with 5% FBS in the presence of cytochalasin B for 24 h at 37 ◦ C. The assays expressed as mean ± S.D. of three separate experiments for each data point of the
were carried out as described in Section 2. The results are expressed as mean ± S.D. olive tail moment (%DNA in tail × distance between centers of mass). Values are sig-
of four separate experiments for each data point. Binucleated cell number (% BN) and nificantly different from CON (**p < 0.01) or different from data in the absence of S9
cytokinesis blocked proliferative index (CBPI) were approximately 81.6% and 1.92 (## p < 0.01, # p < 0.05).
for S9(−) and 75.8% and 1.83 for S9(+), respectively. Values are significantly differ-
ent from CON (**p < 0.01) or different from data in the absence of S9 (## p < 0.01).
The number of MN induced by methyl methanesulfonate (MMS; 50 ␮g/ml, positive Therefore, the genotoxic potential of OE in human lung cells was
control) was 27.3 for S9(−) and 41.5 for S9(+).
shown by CBMN and comet assays.

81.6% and 1.92 for S9(−) and 75.8% and 1.83 for S9(+), respec- 3.4. Oxidative stress induced by subway PM10
tively. Treatment with OE did not induce significant reductions in
the percent of binucleated cells or CBPI (data not shown), indicat- 3.4.1. ROS generation
ing that OE did not induce cell cycle delay for 24 h. In the CBMN In order to identify oxidative stress induced by OE in BEAS-2B
assay, MN formation at the highest dose (100 ␮g/ml) of OE was cells, we first assessed the involvement of intracellular ROS for-
increased by 2.6-fold without S9 treatment and by 2.9-fold with S9 mation using DCFH-DA method [26]. DCF fluorescence intensity
treatment compared with those in each control group and both was increased dose-dependently by OE (1.6–100 ␮g/ml) after 24 h
were stimulated in a dose-dependent manner (Fig. 2). In addi- of exposure. ROS generation in the cells treated with the highest
tion, cells co-treated with OE and S9 showed more MN formation
than cells treated with OE alone, which was statistically proven
10
(## p < 0.01). In the comet assay (Fig. 3), DNA breakage significantly **
Frequency of MN in 1000 counted cells

increased in CHO-K1 cells treated with different concentrations

(1.6–100 ␮g/ml) of OE compared with the control (**p < 0.01).
8 **
100 ␮g/ml OE caused approximately 500% increase in DNA break-
age with S9 treatment and 400% increase without S9 compared with ** **
the control (**p < 0.01). Furthermore, at three different doses (6.3,
6
25, 100 ␮g/ml), DNA breakage with S9 treatment was statistically
higher than without S9 (# p < 0.05, ## p < 0.01). This was consistent *
with what was shown in the CBMN assay. 4

3.3. Genotoxic effect of subway PM10 in BEAS-2B cells

2
Subway PM10 can easily deposit in human lungs, inducing sev-
eral genotoxic effects. In order to investigate genotoxic effects of
OE, especially in human lungs, we conducted the CBMN assay and 0
the comet assay using BEAS-2B cells. In the CBMN assay (Fig. 4), CON 1.6 6.3 25 100 B(a)P
MN formation was increased in BEAS-2B cells treated with differ- (1µM)
OE Concentration (µg/ml)
ent concentrations (1.6–100 ␮g/ml) of OE, and MN formation at the
highest dose (100 ␮g/ml) was induced 2.8 times higher compared Fig. 4. Micronucleus formation induced by the organic extract (OE) of subway PM10
with the control. Also, significant MN formation was induced 2.05 using cytokinesis-block micronucleus (CBMN) assay in human lung cells (BEAS-2B).
times in cells treated with B(a)P (benzo(a)pyrene, 1 ␮M) compared BEAS-2B cells were incubated with OE (1.6–100 ␮g/ml) in BEGM in the presence of
with the control. In the comet assay (Fig. 5), significant DNA break- cytochalasin B for 24 h at 37 ◦ C. The assays were carried out as described in Section 2.
The results are expressed as mean ± S.D. of four separate experiments for each data
age was observed in BEAS-2B cells in a dose-dependent manner.
point of MN frequency per 1000 cells. Binucleated cell number (% BN) and cytokinesis
100 ␮g/ml OE and 1 ␮M B(a)P respectively caused 300% and 150% blocked proliferative index (CBPI) were approximately 80.0% and 1.86, respectively.
increase in DNA breakage compared with the control (**p < 0.01). Values are significantly different from CON (white bar, **p < 0.01, *p < 0.05).
 M.H. Jung et al. / Mutation Research 749 (2012) 39–47 43

25 14

Frequency of MN in 1000 counted cells

** **
12
20 ## ##
10
## ** **
Olive Tail Moment

*
15 8 ##
**
**
** 6
10

4
5
2

0 0
CON 1.6 6.3 25 100 N.a.N. BaP CON SOD CAT M SE

OE Conc. (µg/ml) (1 µM) + OE (100 g/ml)

Fig. 5. DNA breakage in BEAS-2B cells exposed to by the organic extract (OE) Fig. 7. Effects of oxidant modulators on MN formation by the organic extract (OE)
of subway PM10, as determined by the comet assay. Cells were incubated with of subway PM10 in BEAS-2B cells. The cells were incubated with OE and scavenger
OE (1.6–100 ␮g/ml) in BEGM in for 24 h at 37 ◦ C. The assays were carried out as agents [superoxide dismutase (SOD; O2 radical scavenger, 30 units/ml), catalase
described in Section 2. The results are expressed as mean ± S.D. of three separate (CAT; H2 O2 scavengers, 1000 units/ml), mannitol (M; OH scavenger, 0.5 ␮M), and
experiments for each data point of the olive tail moment (%DNA in tail × distance sodium selenite (SE; H2 O2 scavengers, 1 ␮M)] in BEGM for 24 h. The assays were
between centers of mass). Values are significantly different from the control (CON): carried out as described in Section 2. The results are expressed as mean ± S.D. of
**p < 0.01. four separate experiments for each data point of MN frequency per 1000 cells. Binu-
cleated cell number (% BN) and cytokinesis blocked proliferative index (CBPI) were
approximately 87.8% and 1.93, respectively. Values are significantly different from
concentration of subway PM (100 ␮g/ml) was 1.6-fold higher in CON (**p < 0.01, *p < 0.05) or OE (## p < 0.01).
comparison with the control (*p < 0.05) (Fig. 6). This result con-
firmed that OE exposure enhanced the ROS generation in BEAS-2B DNA-damaging effects of OE compared to ENDOIII control groups
cells. (**p < 0.01). In addition, the differences between the effects in the
absence and in the presence of ENDOIII were statistically significant
3.4.2. Oxidative stress (## p < 0.01) (Fig. 8). DNA damages was not significantly increased
To determine the oxidative stress of OE mediated by free radi- in the presence of FPG (the data is not shown).
cals, CBMN assay combined with treatment of various free radical
scavengers (SOD, CAT, M, and SE) was performed. As shown in Fig. 7, 3.5. Identification of PAHs in OE
MN formation induced by OE was significantly decreased by free
radical scavenger agents (## p < 0.01). Furthermore, we compared 3.5.1. CYP1A1 induction
DNA breakage caused by OE alone and by OE with post-treatment In order to identify PAHs in OE, mRNA expression of CYP1A1,
of endonucleases, such as FPG or ENDOIII, in the modified comet the xenobiotic-metabolizing CYP form inducible by PAHs, was
assay. Post-treatment with ENDOIII significantly increased the quantified using a highly specific, sensitive and rapid SYBR green
quantitative real-time RT-PCR (Fig. 9). Relative mRNA expression
2.0
250
- ENDO III
ROS generation (fold induction)

* + ENDO III ##
**
1.5 200
* ##
Olive tail moment

**
150
1.0

100

0.5 ##
50

0.0 0
CON 1.6 6.3 25 100 CON H2O2 OE
OE Concentration (µg/ml) Fig. 8. The effects of endonuclease III (ENDO III) post-treatment on DNA break-
age induced by the organic extract (OE) of subway PM10 in BEAS-2B cells. The
Fig. 6. Effects of the organic extract (OE) of subway PM10 on ROS generation open and hatched bars indicated tail moments without and with ENDOIII digestion.
in BEAS-2B cells. The cells were seeded in 48-well plates and treated with OE The assays were carried out as described in Section 2. The results are expressed as
(1.6–100 ␮g/ml) for 24 h at 37 ◦ C. The assays were carried out as described in Section means ± S.D. of two separate plates for each data point of the Olive tail moment (%
2. ROS was detected by fluorescence measurement of the reported DCF and results DNA in tail × distance between centers of mass). Values are significantly different
are given as fold induction to the control. The data are expressed as mean ± S.D. and from the vehicle control (CON, 0.1% DMSO, white bar, **p < 0.01) or ENDO III CON
values are significantly different from the control (CON): *p < 0.05. (black bar, ## p < 0.01).
44 M.H. Jung et al. / Mutation Research 749 (2012) 39–47

6 equivalency of complex PAH mixtures are based on benzo(a)pyrene

* [27]. As shown in Table 2, PAH-TEQ value for OE is 26.11 ng-TEQ/g-
PM10.
5
CYP1A1 gene expression

4. Discussion
4
Many people take advantage of public transportation systems,
but the air particles generated from such systems could negatively
3
affect human health. The correlation between inhalation of air par-
ticulate matter and severe adverse health effects is widely accepted
2 [1–3]. Several studies have focused on the concentration of PAHs in
airborne PM [28–30], while others have analyzed the metal com-
pounds contained in PM and the important role they play in PM
1
toxicity [31,32]. Compared with airborne PM toxicological stud-
ies, however, not much has been revealed in terms of the health
0 effects of subway PM. Karlsson et al. [7] demonstrated that there is
CON TCDD 1.6 6.3 25 100 a striking difference between subway and street PM, regarding par-
ticle shapes and toxicity, and they concluded that subway particles
OE Concentration (μg/ml) are more genotoxic than street particles. Similar to particles from
other sources, subway PM contains various elements. To date, iron
Fig. 9. Effects of the organic extract (OE) of subway PM10 on CYP1A1 gene expres-
sion in BEAS-2B cells. BEAS-2B cells were exposed to OE (1.6–100 ␮g/ml) and TCDD is known as the most common element in PM and induces high tox-
(10−9 M) for 24 h to identify CYP1A1 gene induction. All data are expressed as fold icity of PM [12]. Iron particles stimulate the production of hydroxyl
induction to the control. The data are expressed as mean ± S.D. and values are sig- radicals via the Haber–Weiss and Fenton reactions, which leads to
nificantly different from the control (CON): *p < 0.05. the oxidative stress on DNA, proteins or lipids [12,33].
Most toxicological studies have focused on the role of metals in
for CYP1A1 showed a tendency to slightly increase, but it did not water-soluble compounds of subway PM. However, adverse effects
show a statistically significant dose-dependent increase by OE. of subway PM may be induced by many other causative chemicals
since particulate matter is a mixture of a variety of elements [34].
3.5.2. PAHs concentration analyzed by GC/MS/MS and i-TEQ Therefore, the purpose of this study is to evaluate the toxicological
[benzo(a)pyrene equivalent] concentration risk of organic compounds in subway PM.
After analyzing OE by GC/MS/MS for 16 carcinogenic PAHs, As PM10 can easily deposit in the respiratory system, the toxicity
we found 10 of them [benzo(a)anthracene, benzo(b)fluoranthrene, of PM10 from urban streets or subways has been discussed in many
benzo(k)fluoranthrene, anthracene, chrysene, fluoranthrene, fluo- other studies. PM10 obtained from the subway tunnel in our study
rene, phenanthrene, pyrene, acenaphthylene] as listed in Table 2. was 34 ␮g/m3 which is lower than concentrations in other studies
In order to determine the carcinogenic potential of the single PAHs (about 150 ␮g/m3 in interior of subway train) [34,35]. Concentra-
observed, ‘toxic equivalency factors (TEF)’ approach was used, in tions of PM in the subway are generally elevated above street-level
consistency with Nisbet and LaGoy’s report [26]. TEF expresses the concentrations [36]. The PM10 concentration measured in outdoor
toxicity factor of single PAH compounds with regard to the effects air of Seoul was on average 47 ␮g/m3 (Ministry of Environment,
of benzo[a]pyrene. TEQ can be mathematically calculated by the Korea, 2011) and was higher than PM10 obtained from the sub-
following equation: way tunnel. In particular, PM collected in the subway tunnel in
Kil-eum station was mostly PM10 and the ratio (PM2.5/PM10)
TEQi = PAHi × TEFi was 0.13. Our result was different from the results by several
 studies; 0.29–0.31 in Paris underground station platform in 2006
TEQ = (TEQi ) [37] and 0.73–0.77 in Los Angeles subway system in 2010 [38].
Subway PM may be influenced by several factors such as station
where TEQi = toxic equivalency of PAH congener i is based depth, date of construction, sampling location, ventilation rate,
on benzo(a)pyrene, PAHi = concentration of PAH congener i, proportion of frictional to regenerative braking, train frequency
TEFi = toxic equivalency factor for PAH congener i, and TEQ = toxic and wheel type (rubber versus steel) [36]. Park and Ha [35] also

Table 2
Concentration of PAHs in organic extract (OE) of subway PM10.

IARCa classification Genotoxicity Concentration (ng/g) PAH-toxicity equivalency PAH-toxic equivalency

factor (TEF)b conc. (ng-TEQ/g)

Benzo(a)anthracene 2A Positive 38.37 0.1 3.84

Benzo(b)fluoranthrene 2B Positive 35.72 0.1 3.57
Benzo(k)fluoranthrene Positive 168.31 0.1 16.83
Anthracene Negative 50.75 0.01 0.51
Chrysene Positive 117.04 0.01 1.17
Fluoranthrene Positive 47.45 0.001 0.05
3
Fluorene Negative 12.79 0.001 0.01
Phenanthrene Questionable 52.31 0.001 0.05
Pyrene Questionable 28.42 0.001 0.03
Acenaphthylene Questionable 47.81 0.001 0.05

Total concentration 598.96 26.11

PAH concentration analyzed by GC/MS/MS system on a HP-5MS column.

a
IARC classification: Group 1, carcinogenic to humans; Group 2A, probably carcinogenic to humans; Group 2B, possibly carcinogenic to humans; Group 3, not classifiable.
b
US EPA (1996a).
 M.H. Jung et al. / Mutation Research 749 (2012) 39–47 45

reported that concentrations and characteristics of PM can be dif- contained in the particles, and (3) redox cycling processes that
ferent according to the age of the subway and sampling location. We metabolize xenobiotics in particles [49]. The last explanation
obtained the PM10 samples from a tunnel of subway constructed in would be appropriate for ROS generation by OE because some
1985. Sampling locations in most reports were platforms or inside xenobiotics, such as PAHs, are known to be contained in organic
the subway in old subway systems constructed before 1980. There- extracts of airborne particles. PAHs originate from incomplete
fore, it is assumed that the different results might be due to the combustion of carbonaceous materials, are distributed widely
circumstances in different subway stations among cities (ventila- in the air and are especially associated with fine particulate
tion system, station depth, date of construction, sampling location, matter [50]. A large number of studies have identified mutagenic
etc.) although a satisfactory explanation has not been found in this and carcinogenic PAHs in air pollution in urban areas. These
study. PAHs mainly contribute to the carcinogenicity of the extractable
We found that OE of subway PM10 has genotoxic effects. CHO- organic matter from diesel particles [51–54]. As diesel and gaso-
K1 cells were first chosen for in vitro studies here because they have line fuels are generally used in cars, buses, trucks and other
many advantages in cytogenetic studies; they are well character- on-road transportation sources, PAH toxicity evaluations have
ized in many respects, are convenient to handle and have a small been widely conducted with street PM, rather than subway PM.
number of large chromosomes of distinctive morphology [39]. The Meanwhile, Fromme et al. [55] measured high concentrations
OE caused a significant increase in DNA damage and chromoso- of PAHs in subway trains. According to this report, PAHs are
mal damage in CHO-K1 cells with or without metabolic activation. considered to contribute less to subway PM toxicity despite the
MN formation at the highest dose (100 ␮g/ml) of OE increased by higher concentration because PAHs found in high amounts in
almost three-fold compared with the control and was stimulated in the subway have relatively low toxicity factor in TEF approach
a dose-dependent manner with or without S9 (Fig. 2). The results compared with PAHs in the car. In particular, relatively high
implied that OE contains indirect-acting and direct-acting geno- amounts of fluoranthrene (TEF = 0.001) and pyrene (TEF = 0.001)
toxic compounds to induce DNA and chromosomal damage. were observed in the subway train, while dibenzo[a,h]anthracene
The lung is the first target tissue of oxidant-producing com- (TEF = 5) and benzo[a]pyrene (TEF = 1) were contained in the air
pounds, such as cigarette smoke, pollutants, and asbestos fibers of cars. Specifically, ten PAHs were detected in this study: ace-
[40]. When exposed to these compounds, the bronchial epithe- naphthylene, fluorine, anthracene, phenanthrene, fluoranthrene,
lial cells are mainly involved in defense mechanisms and signaling pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthrene,
responses [41]. Thus, it is important to investigate the genotoxic benzo[k]fluoranthrene. Subway PM10 in this study did not
effects in human lung cells, especially when studying human health contain PAHs of relative high toxicity factor (TEF = 1) such as
effects caused by inhaling toxic compounds [42]. In this context, benzo(a)pyrene and dibenzo[a,h]anthracene. Therefore, CYP1A1
we conducted the same toxicological tests (CBMN, comet assay) gene expression, an indicator of exposure to PAHs was not
in BEAS-2B cells, cultured human bronchial epithelial cells. We significantly increased by OE (Fig. 9).
confirmed that OE induced significant DNA breakage and chro- The average concentration of particulate-phase PAH and
mosomal damage in BEAS-2B cells as well (Figs. 4 and 5). The benzo(a)pyrene in outdoor air of Seoul was 26.6 ng/m3 and
reason why BEAS-2B cells were not treated with the S9 mixture 2.92 ng/m3 , respectively [56]. The particulate PAHs concentration
is that BEAS-2B cells possess carcinogen-metabolizing capabilities, at Seoul was lower than those at Beijing [57,58], Gaungzhou [59],
including PAH-metabolizing enzyme systems [43]. Furthermore, Hong Kong [60], and Taiwan [61]. On the other hand, the par-
it is widely accepted that generation of ROS could largely con- ticulate PAHs concentration at Seoul was generally higher than
tribute to the adverse health effects of air pollution particles those at Birmingham in UK [62], São Paulo in Brazil [63], Mel-
[31,44,45]. Production of ROS can cause severe oxidative stress bourne in Australia [64], Algiers in Algeria [65]. PAHs including
within cells through the formation of oxidized cellular macro- benzo(a)pyrene in subway tunnel can be induced by various causes
molecules, including nucleic acids, proteins, and lipids [46,47]. In such as subway ventilation systems, braking systems, wheel type,
particular, subway PM10 is known to induce ROS higher than street air conditioning, and system age. It was also reported that source of
PM10 [7,8,11]. Karlsson et al. [11] had put their effort on the study PAHs in subway trains might be tar pitch used as a preservative for
of mechanisms for genotoxicity caused by subway PM10. They wooden railway ties and the use of diesel engines inside the tunnel
reported that the subway PM10 formed intracellular ROS and also system [55]. Subway trains in Seoul use concrete railway ties, steel
assumed that the genotoxicity is likely to be caused from high wheels and a much improved ventilation system than before. In
reactive surfaces by oxidative stress. We used scavenger agents particular, Ministry of Environment in Korea has carried out policy
to identify clearly mechanisms of genotoxicity. Firstly, the DCFH- for improvement of air quality in subway from 2008 to 2012. These
DA assay showed that OE generated ROS in BEAS-2B cells in a may be the good conditions for the reduction of the PAH level and
dose-dependent manner (Fig. 6). Secondly, MN formation caused possibly explains why T-PAH concentration of PM10 in the subway
by OE was partially decreased by scavenger agents [48], such as tunnel is lower than that of PM in ambient air of Seoul [66,67].
SOD (O2 radical scavenger), CAT, SE (H2 O2 -scavengers) [42], and As shown in Table 2, we calculated T-PAH and T-PAH-TEQ to
M (hydroxyl radical scavenger) (Fig. 7). Furthermore, our findings be 598.96 ng/g and 26.11 ng TEQ/g, respectively and the TEQ of
showed that OE stimulated DNA breakage, especially oxidation of benzo(k)fluoranthrene accounted for the highest percentage. It
pyrimidines as detected in the modified comet assay (Fig. 8). Using could be pointed out that total PAH-TEQ was relatively low; how-
endonucleases such as FPG and ENDO III, which recognize oxi- ever, these findings raise concerns about human health because
dized purines [19,20] and oxidized pyrimidines [21], respectively, some of the contained compounds were classified as possible car-
we found that OE induced more DNA damage with treatment of cinogens [55]. Also, there could be possible synergistic effects
ENDOIII compared to the control. The results directly indicated that among the organic compounds in PM10, as was suggested in other
OE leads to increased oxidative DNA damage including oxidized studies [68]. Other organic compounds such as quinine, or particle
pyrimidines, which play an important role in the genotoxic effects core were known to have influence on genotoxicity or oxidative
of OE. stress of OE [69–71]. It was also reported that there were many
The underlying mechanism of how ROS were produced by synergistic reactions between known mutagens and other uniden-
airborne particles is still uncertain, but there are several possible tified organic compounds in airborne particles [72]. Since PAH-TEQ
explanations; for example, ROS could be induced through (1) value has rarely been discussed, it is hard to directly compare it with
inflammation by particles, (2) Fenton reaction by transition metals other cases. However, the types of PAHs found in this study were
46 M.H. Jung et al. / Mutation Research 749 (2012) 39–47

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