Oxidative DNA damage and inflammatory responses in cultured human cells and in humans exposed to traffic-related particles

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Oxidative DNA damage and inflammatory responses in cultured human cells and in humans exposed to traffic-related particles Udomratana Vattanasit a,b,c , Panida Navasumrit a,b,c,d , Man Bahadur Khadka d , Jantamas Kanitwithayanun a,d , Jeerawan Promvijit a , Herman Autrup e , Mathuros Ruchirawat a,b,c,d,f,∗ a

Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Lak si, Bangkok, Thailand Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand c Inter-University Program in Environmental Toxicology, Technology and Management (Chulabhorn Research Institute, Asian Institute of Technology, Mahidol University), Thailand d Chulabhorn Graduate Institute, Lak si, Bangkok, Thailand e Department of Environmental and Occupational Medicine, Institute of Public Health, University of Aarhus, Aarhus, Denmark f Department of Pharmacology, Faculty of Science, Mahidol University, Phayathai, Bangkok, Thailand b

a r t i c l e

i n f o

Article history: Received 12 November 2012 Received in revised form 2 March 2013 Accepted 5 March 2013 Keywords: Particulate matter DEP ROS Cell cycle Inflammation Biomarker

a b s t r a c t Particulate pollution is a major public health concern because epidemiological studies have demonstrated that exposure to particles is associated with respiratory diseases and lung cancer. Diesel exhaust particles (DEP), which is classified as a human carcinogen (IARC, 2012), are considered a major contributor to traffic-related particulate matter (PM) in urban areas. DEP consists of various compounds, including PAHs and metals which are the principal components that contribute to the toxicity of PM. The present study aimed to investigate effects of PM on induction of oxidative DNA damage and inflammation by using lymphocytes in vitro and in human exposed to PM in the environment. Human lymphoblasts (RPMI 1788) were treated with DEP (SRM 2975) at various concentrations (25–100 ␮g/ml) to compare the extent of responses with alveolar epithelial cells (A549). ROS generation was determined in each cell cycle phase of DEP-treated cells in order to investigate the influence of the cell cycle stage on induction of oxidative stress. The oxidative DNA damage was determined by measurement of 8-hydroxydeoxyguanosine (8-OHdG) whereas the inflammatory responses were determined by mRNA expression of interleukin-6 and -8 (IL-6 and IL-8), Clara cell protein (CC16), and lung surfactant protein-A (SP-A). The results showed that RPMI 1788 and A549 cells had a similar pattern of dose-dependent responses to DEP in terms of particle uptake, ROS generation with highest level found in G2/M phase, 8-OHdG formation, and induction of IL-6 and IL-8 expression. The human study was conducted in 51 healthy subjects residing in traffic-congested areas. The effects of exposure to PM2.5 and particle-bound PAHs and toxic metals on the levels of 8-OHdG in lymphocyte DNA, IL-8 expression in lymphocytes, and serum CC16 were evaluated. 8-OHdG levels correlated with the exposure levels of PM2.5 (P < 0.01) and PAHs (P < 0.05), but this was not the case with IL-8. Serum CC16 showed significantly negative correlations with B[a]P equivalent (P < 0.05) levels, but positive correlation with Pb (P < 0.05). In conclusion, a similar pattern of the dose-dependent responses to DEP in the lymphoblasts and lung cells suggests that circulating lymphocytes could be used as a surrogate for assessing PM-induced oxidative DNA damage and inflammatory responses in the lung. Human exposure to PM leads to oxidative DNA damage whereas PM-induced inflammation was not conclusive and should be further investigated. © 2013 Published by Elsevier GmbH.

Introduction Particulate matter in air pollution has been associated with adverse health effects such as respiratory and cardiovascular

∗ Corresponding author at: Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Lak si, Bangkok 10210, Thailand. Tel.: +66 2 553 8535; fax: +66 2 553 8536. E-mail address: [email protected] (M. Ruchirawat).

diseases as well as lung cancer mortality (Bartoli et al., 2009; Patel et al., 2010; Turner et al., 2011) and is thus of great public health concern. Particulate matter (PM) is a major air pollutant in many big cities, especially where traffic is congested. Vehicle exhaust emissions are mainly responsible for the particulate air pollution in urban areas and diesel exhaust is considered a major contributor. Recently, diesel exhaust has been classified as a human carcinogen (group 1; IARC, 2012). Exposure to diesel exhaust has been well established for its association with an increased risk of lung cancer (Attfield et al., 2012; Garshick et al., 2004; Silverman et al., 2012).

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PM derived from vehicle combustions is mostly in fine and ultrafine fractions. The small size of particles facilitates their ability to get into the respiratory tract and reach the alveoli where the PM are retained resulting in adverse health effects. Fine particles (PM2.5 ) are composed of various carcinogenic compounds including polycyclic aromatic hydrocarbons (PAHs) and metals which are biologically active components resulting in induction of oxidative DNA damages (Wei et al., 2009). The particles or their chemical components induce reactive oxygen species (ROS) which may subsequently lead to cellular oxidative stress (Bonvallot et al., 2001; Knaapen et al., 2004; Kumagai et al., 1997). The oxidative stress can induce oxidative DNA lesions such as 8-hydroxy-deoxyguanosine (8-OHdG) (Hanzalova et al., 2010) and DNA strand breaks (Bonetta et al., 2009; Danielsen et al., 2009). Associations between the levels of 8-OHdG in lymphocyte DNA and in urine and levels of exposure to PM (Sorensen et al., 2003; Svecova et al., 2009; Wei et al., 2009) and to particle-bound PAHs (Ruchirawat et al., 2010) and carcinogenic metals (Zhang et al., 2011) have been reported. 8-OHdG is a marker of oxidative stress to DNA. If left unrepaired, it can lead to a heritable mutation and cancer initiation (Valko et al., 2006). Furthermore, PM-induced ROS has been shown to mediate inflammatory responses through activation of redox-sensitive transcription factors resulting in increased synthesis of proinflammatory cytokines such as interleukin-6 and -8 (IL-6 and IL-8) (Mazzarella et al., 2007; Pourazar et al., 2005). The proinflammatory cytokines are involved in the amplification of inflammatory reactions. Persistent excessive inflammation is important in development of lung damage, vascular dysfunction, and cancer. Clara cell protein 16 kDa (CC16) has been ascribed an anti-inflammatory function to protect the lung from excessive inflammation (Singh and Katyal, 1997). However, evidence on alteration of the lung protein expression as a result of PM exposure and associations between the lung protein and the inflammatory cytokines are still limited. Biomarkers of lung inflammation can be determined in epithelial lining fluid such as bronchoalveolar lavage (BAL) and sputum. However, these biological samples may not be applicable in human biomarker surveys due to invasive technique as well as practical and ethical restrictions in the sampling. Circulating lymphocytes are easily assessable for human biomarker studies. It could be useful as a surrogate for the lung tissue to predict adverse health outcomes from PM exposure. Nevertheless, no study has been conducted to evaluate the similarity in the response of lymphocytes and lung cells. Accordingly, the present study aimed to evaluate whether lymphocytes can be used as a surrogate in assessing the effects of PM on the induction of oxidative DNA damage and inflammation in vitro and in human exposed to traffic-related PM. Human lymphoblast cell line (RPMI 1788) and alveolar epithelial cell line (A549) were treated with a reference DEP (SRM 2975) which has been suggested to be used as a suitable surrogate particle for the study of authentic street particle in respect of oxidative DNA damage (Danielsen et al., 2008a). The extents of oxidative and inflammatory responses were compared. ROS generation was determined in each cell cycle phase of the DEP-treated cells in order to investigate the influence of cell cycle stage on induction of oxidative stress. The oxidative DNA damage was determined by measurement of 8-OHdG formation. The inflammatory responses were assessed by the expression of inflammatory cytokine genes (IL-6 and IL-8) and lung protein genes, CC16 and surfactant protein-A; SP-A. Biological responses to DEP treatment in the lymphocytes and lung cells were studied. In addition, the potential of using lymphocytes as a surrogate for lung target tissue in assessing possible health effects from PM exposure was also investigated in a human study. Assessment of exposure to and health effects, i.e. oxidative and inflammatory response, of traffic-related particles was conducted in 51 healthy subjects

residing in traffic-congested areas in Bangkok. The exposure levels of PM2.5 and its particle-bound PAHs and toxic metals were determined and their correlations with health effects on both oxidative DNA damage and inflammatory responses were evaluated. Materials and methods Cell culture Human lymphoblast (RPMI 1788) and alveolar epithelial adenocarcinoma (A549) cell lines were obtained from the American Type Culture Collection (ATCC). The RPMI 1788 cell line was grown in suspension in RPMI medium 1640 (Gibco, Invitrogen) supplemented with 20% fetal bovine serum and 1% sodium pyruvate. The A549 cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen) supplemented with 10% fetal bovine serum (J.R. Scientific), 100 U penicillin, 100 ␮g/ml streptomycin, and 2 mM l-glutamine. The cell lines were maintained under humidified conditions in a 5% CO2 atmosphere at 37 ◦ C. RPMI 1788 cells were propagated at the time of treatment, whereas A549 cells were seeded in DMEM and cultured for 24 h to allow cells to adhere on the flask and achieve confluence prior to DEP treatment. The effects of DEP on cell viability were assessed using Trypan blue exclusion method. Preparation of DEP suspension National Institute of Standard and Technology (NIST) standard reference material (SRM 2975) was commercially available (Donaldson Company, Inc., Minneapolis, MN). This SRM has been reported to be endotoxin free (Shaw et al., 2010). Stock DEP suspensions of 1 mg/ml were prepared by suspending DEP in the respective serum-free culture medium and subsequently sonicated in an ultrasonic bath for 2 min (Sibata; OGAWA SEIKI) before adding to the serum-free cell cultures at final concentrations of 25, 50, 75, and 100 ␮g/ml. Determination of DEP uptake and intracellular ROS generation in different cell cycle phases Double staining flow cytometry was performed according to the procedure described by Gao et al. (2004), with minor modifications. 2 ,7 -Dichlorodihydrofluorescin diacetate (DCFH-DA; Sigma) and 7amino actinomycin D (7-AAD; Sigma) were used to detect ROS and to identify the cell cycle distribution, respectively; so intracellular ROS could be measured in different cell cycle phases. Briefly, cells were incubated with 10 ␮M DCFH-DA for 10 min and after removing the dye, the stock DEP suspension was added to the cells at final concentration of 25–100 ␮g/ml in serum-free medium. After 3 h of incubation, DEP was removed and the cells were incubated with 20 ␮M 7-AAD in labeling buffer for 5 min. Subsequently, the cells were subjected to flow cytometer for analysis (BD FACScantoTM ; BD Biosciences, USA). The levels of ROS were expressed as DCF levels. Cellular uptake of DEP was simultaneously determined and expressed as side scatter (SSC) intensity. Determination of 8-hydroxydeoxyguanosine (8-OHdG) and deoxyguanosine (dG) The stock suspension of DEP was added to final concentrations of 25–100 ␮g/ml in serum-free medium and incubated with cells for 24 h. The cells were harvested and DNA was isolated using QIAamp® blood Maxi spin column (QIAGEN) according to the manufacturer’s instructions. DNA was enzymatically digested to the deoxynucleoside. Briefly, 100 ␮g of DNA sample was incubated with 8 U of nuclease P1 at 37 ◦ C for 10 min. Next, 5 U of alkaline

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phophatase was added to the mixture and incubated at 37 ◦ C for 2 h. Following the digestion, the hydrolysate was filtered through 0.22 ␮m syringe filter before analysis. The levels of dG and 8OHdG were analyzed by HPLC (Aligent model 1200 series) equipped with a triple quadrupole mass spectrometer (Agilent 6410 Triple Quad LC/MS). The HPLC was connected to a Guanine adduct column (3.0 mm × 150 mm, ESA, Inc., USA) set at 25 ◦ C. A flow rate of 0.5 ml/min was used for the mobile phase of methanol and 0.1% formic acid. The MS/MS system was operated via electrospray source with a positive ion mode. The capillary voltage was 4 kV. The product ion transition of analytes was monitored in the MRM mode at m/z 284.0 → m/z 168.0 for 8-OHdG and m/z 268.0 → m/z 152.0 for dG. The levels of 8-OHdG were expressed as 8-OHdG per 105 dG. Determination of gene expressions by real-time RT-PCR The stock suspension of DEP was added to final concentrations of 25–100 ␮g/ml in the serum-free medium and incubated with cells for 24 h. Lipopolysaccaride (LPS; L9143, Sigma) was used as positive control at final concentration of 10 ␮g/ml. Total RNA was isolated from DEP-treated RPMI 1788 and A549 cells using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s recommendations. The obtained total RNA samples were diluted to the concentration of 10 ng/␮l and subsequently determined for IL-6 and IL-8 relative expression by LightCycler real time RT-PCR (Roche Diagnostic). QuantiTect SYBR® Green RT-PCR Kit (QIAGEN) with the primers of IL-6 (Miranda et al., 2008), IL-8 (Kanazawa et al., 2003), and housekeeping genes (CYPA (Miranda et al., 2008) and ˇ-actin (Strassburg et al., 1997), respectively) were employed. For CC16 and SP-A, 1 ␮g of the total RNA was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) on a PCR system (PerkinElmer) according to the manufacturer’s instructions. Relative expression of CC16 was analyzed by ABI-Step One Plus real time RT-PCR (Applied Biosystems). CC16 and GAPDH primers with TaqMan® probes (Van Haute et al., 2009) and master mix (Applied Biosystems) were used. The cDNA templates were also analyzed for SP-A relative expression by LightCycler real time RT-PCR (Roche Diagnostic). QuantiTect SYBR Green RT-PCR Kit (QIAGEN) with the primers of SP-A (Sun et al., 2006) and a housekeeping gene (CYPA) were employed in the RT-PCR reaction. The mRNA expression levels were normalized to the expression levels of the respective housekeeping genes and the mean expression levels of untreated cells by using the CT method. Study locations and subjects The study was conducted in two areas in Bangkok along Dindaeng and Pradipat roads where the traffic is heavily congested. A total of 51 residents (mean age 45.2; range 18–58 years) were recruited to the study. The study subjects were healthy nonsmokers male (31.4%) and female (68.6%) living or working in the study sites located within 500 m from the main road. Questionnaires were distributed to all participants to obtain personal information (i.e. age and accommodation), occupational information (i.e. workplace and personal protective equipment), and lifestyles (i.e. environmental tobacco smoke exposure, types of diet, etc.). This study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The purpose of the study was explained to all subjects and they have signed the written informed consent to participate in this study.

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conditions in Bangkok during the sampling period were provided by the Thai meteorological department, mean temperature of 31.1 ◦ C (27.6–33.8 ◦ C), relative humidity of 62.3% (54.0–76.0%), and surface wind speed of 24.4 km/h (15.4–32.1 km/h). A preliminary study (unpublished data) demonstrated that there was no significant difference in the level of PM2.5 between weekdays. Ambient and individual exposure levels were determined by collecting the PM2.5 from the ambient air and at breathing zone of the study subjects, respectively. The air particulate was collected for 8 h (7 a.m.–3 p.m.) on a glass filter paper which is inside a PM2.5 size segregated impactor using a personal air sampler attached to a battery-operated SKC air check sampler. The flow rate was set at 2 l/min. Each filter was dried overnight in a desiccator and weighed before and after sample collection to determine the net mass gain due to the particles. The PM2.5 levels in ␮g/m3 were calculated from the net mass gain, air flow rate, and sampling time. After sampling, the filter was wrapped in an aluminium foil and kept at −80 ◦ C until analysis. Biological samples were collected from the subjects at the end of PM2.5 sampling. Whole-blood samples (20 ml) were collected in two tubes containing 4% EDTA in normal saline or heparin solution (1000 i.u. of heparin per 1 ml of whole blood) and in another tube containing no anticoagulant. EDTA blood was used to isolate genomic DNA from leukocytes for analysis of 8-OHdG. Heparinized blood was used to isolate lymphocytes by buoyant density using Ficoll-Paque Plus (Amersham) according to the manufacturer’s instructions within 4–6 h after collection. The isolated lymphocytes were resuspended in cold-freezing medium at concentration of 5 × 106 cells/ml. 1 ml of the cell suspension was transferred to cryovials and then submerged into cryo-freezing container and immediately placed in −80 ◦ C freezer for storage. Blood sample without anticoagulant was centrifuged at 2000 rpm for 10 min and the serum obtained was kept at −80 ◦ C until analysis. Determination of PAHs PAHs were extracted from PM2.5 samples according to a slight modification of the procedure described by Garivait (1999). Briefly, the filter samples were cut into small pieces and extracted by ultrasonication with 10 ml of dichloromethane. The extracts were added with DMSO and mixed well before they were concentrated under a gentle stream of nitrogen. The residues were dissolved with acetonitrile and analyzed by HPLC (Hewlett Packard Series 1100) coupled with a fluorescence detector. Ten PAHs were measured with different wavelength fluorescence detection: fluoranthene (FA), excitation 237 nm, emission 460 nm; pyrene (PY), excitation 234 nm, emission 387 nm; benzo[a]anthracene (B[a]A), excitation 278 nm, emission 392 nm; chrysene (CHR), excitation 262 nm, emission 379 nm; benzo[b]fluoranthene (B[b]F), excitation 256 nm, emission 437 nm; benzo[k]fluoranthene (B[k]F), excitation 240 nm, emission 417 nm; benzo[a]pyrene (B[a]P), excitation 263 nm, emission 410 nm; dibenzo[a,h]anthracene (DB[ah]A), excitation 291 nm, emission 400 nm; benzo[g,h,i]perylene (B[ghi]P), excitation 288 nm, emission 415 nm; indeno[1,2,3-cd]pyrene (IP), excitation 300 nm, emission 500 nm. The concentration of each PAH was quantified by its peak area. B[a]P equivalent was determined by multiplying each individual PAH concentration with its corresponding toxic equivalent factor (TEF) (Nisbet and LaGoy, 1992) and the concentration of total PAHs expressed as B[a]P equivalent was determined. Determination of metals

Sample collection Samples were collected in the dry season (January–March, 2009) when exposure to PM was expected to be high. The meteorological

Analysis of metals was carried out according to a standard procedure for trace metal analysis of ambient air particulate samples using ICP-MS with modification (US EPA, 2007). Briefly, the filter

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samples were cut into small pieces and digested with mixed-acid solution of suprapure nitric acid and hydrochloric acid (9:3) in a microwave oven (Milestone ETHOS). The digested solution was filtered through 0.45 ␮m filter membrane and diluted with deionized water prior to analysis of dissolved metal concentration using Octapole ICP-MS (Agilent 7500C). The sample introduction system of the ICP-MS consisted of a Babington-type nebulizer and spray chamber cooled by a Peltier system. Determination of 8-OHdG in peripheral blood Genomic DNA was isolated from whole-blood by using QIAamp® blood Maxi spin column (QIAGEN) according to the manufacturer’s instructions. DNA samples were converted into single nucleoside and analyzed for 8-OHdG and dG as already described. Determination of IL-8 expression in circulating lymphocytes The isolated lymphocytes were thawed and cultured in RPMI 1640 medium under humidified conditions in a 5% CO2 atmosphere at 37 ◦ C for 3 days. After the incubation time, the cells were washed in PBS and resuspended in the culture medium to the concentration of 2 × 106 cells/ml. RNA was isolated from the cells and purified using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. The concentrations and purity of the RNA samples were determined using NanoDrop® . The RNA samples were stored at −20 ◦ C until analysis. The RNA samples were diluted to the concentration of 10 ng/␮l and subsequently determined for IL-8 relative expression by the LightCycler real time RT-PCR as already described. Determination of CC16 in serum Serum samples were analyzed for CC16 using ELISA Kit (DIAMED) according to the manufacturer’s instructions. Statistical analysis Differences in the biological responses between control and DEP-treated cells were analyzed with two-way independent sample t-test. Pearson correlation was used to determine associations between the biological responses in DEP-treated cells, and between DEP concentrations and the responses. Spearman rank correlation was used to determine associations between the levels of exposure and the levels of biological effects on oxidative DNA damage and inflammatory responses in the study subjects. A P-value less than 0.05 was considered to represent statistical significance. Results In vitro effects of DEP on ROS generation, oxidative DNA damage and inflammatory responses: human lymphoblastoid cells (RPMI 1788) versus human alveolar epithelial adenocarcinoma cells (A549) Human lymphoblastoid cells (RPMI 1788) DEP treatments (25–100 ␮g/ml) for 24 h did not affect viability of RPMI 1788 cells as measured by trypan blue exclusion assay (data not shown). Following DEP treatment, RPMI 1788 cells exhibited a substantial uptake of DEP as evident by a large increase in the side scatter intensity (SSC) in a dose dependent manner as shown in Fig. 1A. In order to examine the influence of cell cycle stage on oxidative damage, ROS generation in each cell cycle phase of DEP-treated cells was determined by using double-staining flow cytometry. RPMI 1788 cells in sub G1, G1/S, S, and G2/M phases showed no significant difference in the DEP uptake. The double

staining flow cytometric analysis also revealed that background levels of ROS in untreated RPMI 1788 cells were higher in G2/M phase than other cell cycle phases. DEP treatment increased ROS generation in RPMI 1788 cells in G2/M phase in a dose-dependent manner and a significant increase in ROS generation in DEP-treated RPMI 1788 cells was observed at concentration 100 ␮g/ml of DEP (Fig. 1B). In accordance with DEP-induced ROS generation, the level of oxidized DNA bases measured as 8-OHdG in RPMI 1788 cells was significantly increased following DEP treatment in a dosedependent manner as shown in Fig. 1C. Dose-dependent increased 8-OHdG in DEP-treated RPMI 1788 cells were approximately 2.1-, 3.4-, 3.5-, and 4.6-fold at 25, 50, 75, and 100 ␮g/ml, respectively. It can be observed that the level of 8-OHdG formation in DEP-treated RPMI 1788 cells was associated with a similar to DEP-induced ROS generation. In order to investigate the effects of DEP on respiratory inflammation, inflammatory cytokines and lung proteins markers of respiratory diseases were examined. As shown in Fig. 1D, expression of IL-6 and IL-8 by DEP treatment in RPMI 1788 cells was increased in a dose-dependent manner. A significant increase in SP-A expression was found when the RPMI 1788 cells were treated with 25 ␮g/ml of DEP (P < 0.05). However, CC16 expression in DEP-treated RPMI 1788 cells was not significantly different from untreated cells at all DEP concentrations. Moreover, the positive control (LPS) significantly increased mRNA expression of IL-6 (P < 0.05) and IL-8 (P < 0.01) but had no effect on CC16 and SP-A mRNA expression (data not shown). Expression of CC16 and SP-A in DEP-treated RPMI 1788 cells did not show significant correlation with IL-6 and IL-8. DEP concentrations and expression of IL-6 and IL-8 in DEP-treated RPMI 1788 cells showed positive correlations with correlation coefficients of 0.790 and 0.873, respectively. Human alveolar epithelial adenocarcinoma cells (A549) In order to evaluate whether circulating lymphocytes could be used as a surrogate cell for the lung tissue, the oxidative and inflammatory responses were also measured in A549 cells which represented the lung target cells, and this cell line is frequently used in particle toxicity studies. A dose-dependent increase in particle uptake was also observed in A549 cells treated with DEP (25–100 ␮g/ml), however, there was no significant difference in the uptake in sub G1, G1/S, S, and G2/M phases (Fig. 2A). The levels of ROS in untreated and DEP-treated A549 cells were higher in the G2/M phase than in other cell cycle phases. Following DEP treatment, only G2/M phase of A549 cells showed a slight dosedependent increase in ROS generation which a significant increase were at concentrations 50–100 ␮g/ml (Fig. 2B). DEP significantly increased the level of 8-OHdG in A549 cells in a dose-dependent manner as shown in Fig. 2C. Furthermore, DEP treatment induced IL-6 and IL-8 expression in A549 cells in a dose-dependent manner (Fig. 2D). A significant reduction of CC16 expression by approximately 50% in A549 cells treated with DEP was observed at 25 ␮g/ml and then remained relatively constant towards 100 ␮g/ml. Significant increase in SP-A mRNA expression was found only in the A549 cells treated with 25 ␮g/ml of DEP (P < 0.01). The positive control (LPS) significantly increased mRNA expression of IL-6 (P < 0.01), IL-8 (P < 0.05), and SP-A (P < 0.05) but had no effect on CC16 mRNA expression (data not shown). CC16 expression was inversely correlated with expression of IL-6 (r = −0.728; P = 0.002) and IL-8 (r = −0.668; P = 0.006) whereas SPA expression did not show significant correlation with IL-6 and IL-8. DEP concentrations and expression of IL-6 and IL-8 in DEPtreated A549 cells showed positive correlations with correlation coefficients of 0.921 and 0.937, respectively. From the results, RPMI 1788 and A549 cells showed similar responses to DEP in terms of DEP uptake, increased intracellular

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A) DEP uptake

B) ROS generation 2000

SubG1 G1/S S G2/M

100

DCF intensity

SSC intensity (% increase)

150

50

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0

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75

1500 1000 500 0

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C) 8-OHdG formation **

3 2 1 0

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IL-6 IL-8 CC16 SP-A

4 3

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D) Inflammatory gene expression

* Relative expression (Fold change)

8-OHdG/105dG (Fold change)

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Fig. 1. DEP uptake quantified by an increase of side scatter (SSC) intensity (A), intracellular ROS generation expressed as DCF intensity (B) in sub G1 ( ), G1/S ( ), S ( ), and G2/M ( ) phases, oxidative DNA damage measured by formation of 8-OHdG (C) and expression of inflammatory cytokine genes (IL-6 and IL-8 ) and lung secretory protein genes (CC16 and SP-A ) (D) in RPMI 1788 cells treated with 25–100 ␮g/ml of DEP were demonstrated. Each point represents the mean ± SE from 3 to 6 independent experiments. *, **, and *** represent statistically significant difference from control at P < 0.05, 0.01, and 0.001, respectively.

ROS generation in G2/M phase cells, and increased 8-OHdG formation in a dose-dependent manner. In addition, up-regulation of the pro-inflammatory cytokines gene expression, IL-6 and IL8, was observed in both cell types but down-regulation of the

anti-inflammatory protein gene expression (CC16) was observed only in A549 cells. However, IL-6 expression was higher than IL-8 expression in A549 cells and IL-8 expression was higher than IL-6 expression in RPMI 1788 cells.

Fig. 2. DEP uptake quantified by an increase of side scatter (SSC) intensity (A), intracellular ROS generation expressed as DCF intensity (B) in sub G1 ( ), G1/S ( ), S ( ), and G2/M ( ) phases, oxidative DNA damage measured by formation of 8-OHdG (C) and expression of inflammatory cytokine genes (IL-6 and IL-8 ) and lung secretory protein genes (CC16 and SP-A ) (D) in A549 cells treated with 25–100 ␮g/ml of DEP were demonstrated. Each point represents the mean ± SE from 3 to 6 independent experiments. *, **, and *** represent statistically significant difference from control at P < 0.05, 0.01, and 0.001, respectively.

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Table 1 Ambient concentrations of PM2.5 and the particle-bound PAHs in air samples collected during 8-h sampling period. Study parameters

Ambient concentrations (n = 5)

PM2.5 (␮g/m3 )

142.77 ± 51.08a 92.62 (63.57–344.30)b

PAHs (ng/m3 ) Total PAHs

4.13 ± 0.30 4.17 (3.49–5.20) 1.78 ± 0.20 1.83 (1.28–2.46)

Total B[a]P equivalent

n represents the total number of samples collected once daily for 5 days. a The values are expressed as mean ± SE. b The values are expressed as median (min–max).

Exposure to PM2.5 and biological effects on oxidative DNA damage and inflammatory responses in study population Since the lymphoblasts showed responses similar to alveolar epithelial cells in terms of the oxidative and inflammatory responses to DEP, a human exposure study was conducted to further investigate the effects of DEP by using circulating lymphocytes of subjects exposed to traffic-related PM and evaluate their associations with the exposure levels. In order to assess exposure to traffic-related PM, the concentrations of PM2.5 and carcinogenic compounds (PAHs and toxic metals) bound to the particles in ambient air and individual exposure levels were measured. The results are shown in Tables 1 and 2, respectively. The mean ambient concentration of PM2.5 in the study locations (142.77 ␮g/m3 ) was higher than the national ambient air standard for PM2.5 as 24 h-average of 50 ␮g/m3 provided by pollution control department (PCD) of Thailand (PCD, 2004) and of 25 and 35 ␮g/m3 provided by WHO and US EPA, respectively (WHO, 2005; US EPA, 2006). The concentrations of PM2.5 and the particlebound PAHs in ambient air were in the same range as the individual exposure concentrations. The concentrations of total PAHs were the summation of 10 PAHs, including fluoranthene (FA), pyrene (PY), benzo[a]anthracene (B[a]A), chrysene (CHR), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[ah]A), benzo[g,h,i]perylene (B[ghi]P), and indeno[1,2,3-cd]pyrene (IP). The individual exposure concentrations of PM2.5 were not

Fig. 3. The mean concentrations of 10 PAHs: fluoranthene (FA ), pyrene (PY ), benzo[a]anthracene (B[a]A ), chrysene (CHR ), benzo[b]fluoranthene ), benzo[k]fluoranthene (B[k]FA ), benzo[a]pyrene (B[a]P (B[b]FA ), dibenzo[a,h]anthracene (DB[ah]A ), benzo[g,h,i]perylene (B[ghi]P ), indeno[c,d]pyrene (IP ) were determined in ambient and individual PM2.5 collected for a period of 8 h in traffic-congested areas in Bangkok.

significantly correlated with total PAHs (r = 0.138; P = 0.333) and B[a]P equivalent (r = 0.014; P = 0.923). Among 10 individual PAHs, IP was the predominant PAH that contributed to approximately 34.9% and 31.3% of the total PAHs in ambient air and in individual exposure samples, respectively (Fig. 3). Qualitative source identification for PAHs was performed by calculating PAH diagnostic ratios. The ratios of B[a]A/CHR, B[a]A/(B[a]A + CHR), B[k]FA/IP, and B[b]FA/B[k]FA were 0.53, 0.34, 0.40, and 0.57, respectively. The ambient and individual exposure concentrations of toxic metals, including As, Cd, Cr, Mn, Ni, and Pb in PM2.5 are presented in Fig. 4. Arsenic (3.11–347.74 ng/m3 ) was found in all ambient air and individual exposure PM2.5 samples whereas other metals were detected in some samples. Cr, Mn, Ni, Cd, and Pb were detected at range 60.42–411.29, 0.19–144.30, 1.73–483.60, 0.81–40.97, and 2.36–5.70 ng/m3 in approximately 13.7%, 23.5%, 43.1%, 47.1%, and 88.2% of the total number of individual samples, respectively. The majority of study subjects were exposed to As, Cd, Ni, and Pb in the concentration range found in the ambient air. The ambient concentrations of Cr and Mn were non-detectable but some individuals were exposed to higher concentrations of the metals in ambient air.

Table 2 Individual exposure concentrations of PM2.5 and the particle-bound PAHs and levels of biomarkers of oxidative DNA damage and inflammatory responses. Study parameters Individual exposure PM2.5 (␮g/m3 )

PAH (ng/m3 ) Total PAHs Total B[a]P equivalent Biomarkers of effects 8-OHdG/105 dG Inflammatory responses IL-8 expression (relative to ␤-actin) Serum CC16 (ng/ml) a b

Number of subjects

Levels

51

183.33 ± 37.17a 113.94 (18.94–1664.48)b

51

7.47 ± 0.98 5.02 (1.48–42.80) 5.78 ± 1.09 2.88 (0.40–46.04)

51

50

0.067 ± 0.004 0.061 (0.031–0.184)

31

0.36 ± 0.03 0.34 (0.11–0.82) 29.36 ± 1.10 27.43 (13.46–44.02)

46

The values are expressed as mean ± SE. The values are expressed as median (min–max).

Fig. 4. Box plots of concentrations of toxic metals bound to PM2.5 in ambient and in individuals. The concentrations of 6 toxic metals were determined in PM2.5 collected from ambient air () and in individuals (䊉) for a period of 8 h in traffic-congested areas in Bangkok.

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Fig. 5. Correlations between individual exposure and biomarkers of effects. The correlations between exposure levels of PM2.5 (×10; 䊉), the particle-bound PAHs (), B[a]Peq () (Panel 5.1), and toxic metals including As () and Pb (♦) (Panel 5.2) and the levels of biomarkers of effects including 8-OHdG (A), IL-8 expression (B), and serum CC16 (C) were demonstrated.

Correlations between the levels of exposure and biomarkers In human exposure study, only 8-OHdG, IL-8 expression, and serum CC16 were selected to assess oxidative DNA damage, inflammatory responses, and lung inflammation. This is because IL-8 showed higher sensitivity over IL-6 to DEP treatment in the in vitro study as shown by higher correlation coefficients. CC16 expression was selected because it was down-regulated in A549 cells while SP-A expression was increased. Moreover, CC16 expression was inversely correlated with the expression of both IL-6 and IL-8 while SP-A expression did not show any correlation. The levels of biomarkers of effects, including 8-OHdG, IL-8 expression, and serum CC16, were presented in Table 2. Correlations between the levels of exposure and biomarkers were demonstrated

in Fig. 5. 8-OHdG in leukocytes of the subjects was significantly correlated with the individual exposure concentrations of PM2.5 at P < 0.01 and PAHs (total PAHs and B[a]P equivalent) at P < 0.05 (Fig. 5.1A) but not with As and Pb which were the most predominant toxic metals in this study (Fig. 5.2A). IL-8 expression was not significantly correlated with PM2.5 and the particle-bound PAHs (Fig. 5.1B) and the toxic metals (Fig. 5.2B). In the case of serum CC16, it can be observed that CC16 was significantly negatively correlated with B[a]P equivalent (P < 0.05) as shown in Fig. 5.1C and PAHs including benzo[k]fluoranthene (B[k]FA) (r = −0.302; P = 0.044), benzo[a]pyrene (B[a]P) (r = −0.328; P = 0.028) and dibenzo[a,h]anthracene (DB[ah]A) (r = −0.359; P = 0.015) (data not shown). However, the serum CC16 was significantly correlated with Pb (P < 0.05) as shown in Fig. 5.2C.

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Discussion In vitro effects of DEP treatment on oxidative and inflammatory responses in lymphoblasts in comparison with alveolar epithelial cells A number of studies have demonstrated that PM can cause adverse health effects in the lung which is the primary target of PM exposure. Induction of oxidative stress and inflammation are important mechanisms for health effects caused by inhaled particles (Mazzoli-Rocha et al., 2010; van Berlo et al., 2010). Therefore, oxidative and inflammatory responses have been extensively studied in the lung epithelial cells as it is the target for the inhaled particles. In the present study, DEP uptake, intracellular ROS generation, oxidative DNA damage, and expression of inflammatory response genes in lymphoblasts (RPMI 1788) and in human alveolar epithelial cell line (A549) which represented the lung target cells were compared in order to evaluate whether circulating lymphocytes could be used as a surrogate for the lung tissue. DEP uptake was determined by flow cytometric light scatter analysis which has been used to measure cellular uptake of nanoparticles (Suzuki et al., 2007; Zucker et al., 2010). Side scatter (SSC) intensity provides information on internal structure and organelles (Zucker et al., 2010). The untreated RPMI 1788 and A549 cells showed minimal SSC intensity which can be related to the organelles inside the cells. Following DEP exposure, substantial increase in SSC intensity was observed in both cell lines suggesting that DEP was markedly internalized by the cells. In addition, RPMI 1788 and A549 cells in different phases of the cell cycle were not different in ability to uptake the particles. This is in line with a recent study in synchronized cells showing that cells in different phases of the cell cycle could internalize nanoparticles at similar rates (Kim et al., 2012). DEP induced ROS in both RPMI 1788 and A549 cells. This is in agreement with other studies showing that DEP increases intracellular ROS in various types of cells (Baulig et al., 2003; FrikkeSchmidt et al., 2011; Okayama et al., 2006). The ROS mediated by DEP can be produced through indirect generation of ROS via metabolic activation of organic compounds (Bonvallot et al., 2001) and alteration of mitochondrial functions (Knaapen et al., 2004). Endogenous ROS has been shown to increase progressively through the cell cycle (Havens et al., 2006). A possible reason that could be suggested for the highest inherent level of ROS in G2/M phase in the present study is that G2/M cells have a higher mitochondrial content than cells in G1 and S phases (Yamamori et al., 2012). The mitochondria are a significant endogenous source of ROS production and a major subcellular target for PM toxicity (Xia et al., 2007). The ROS production is a consequence of interference in mitochondrial electron transfer and opening of permeability transition pores by pro-oxidative PM components such as PAHs and quinones associated with DEP (Xia et al., 2004). In the present study, DEP was internalized by different phase cells at a similar rate but ROS was preferentially generated in G2/M phase cells in both RPMI 1788 and A549 cells. This observation suggests a higher sensitivity of G2/M phase cells to generate ROS in DEP-treated cells. Our results were in line with a study reporting that G2/M transition exhibits the highest levels of ROS and the most susceptibility to arsenic trioxide-induced apoptosis in NB4 promyelocytic leukemia cells (Gao et al., 2004) suggesting that ROS played an important role in determining the cell cycle related apoptosis susceptibility. Accordingly, higher level of ROS in G2/M phase in the present study might determine higher susceptibility to apoptosis induction by DEP of the G2/M cells. Moreover, it is also important to note that the significant increase of ROS in the G2/M cells by DEP may activate G2 checkpoint to cope with the oxidative DNA damages. This was supported by gene expression profiles of airway epithelial cells

induced by coarse, fine, and ultrafine particles showing that G2/M DNA damage checkpoint regulation was one of the top three pathways altered by the particles (Huang et al., 2011). After the G2/M checkpoint, the cells will directly pass to mitosis. If the checkpoint does not work properly, the cells will undergo apoptosis or the unresolved DNA damages will be presented to the daughter cells and carcinogenesis might be initiated. Therefore, the higher sensitivity of G2/M cells to generate ROS in DEP-treated cells observed in the present study provides a mechanism of cancer development from DEP exposure. 8-OHdG is one of the major DNA base modifications by ROS. The DEP employed in the present study has been suggested to be used as a suitable surrogate particle for the study of authentic street particle in respect of oxidative DNA damage (Danielsen et al., 2008a). The mechanism of DEP-induced 8-OHdG formation involves the oxidative modification of DNA by ROS. The increased formation of 8-OHdG in our in vitro study was in accordance with previous studies on DEP-induced oxidative DNA damages in vivo which showed an increase in 8-OHdG formation in lungs of rodents by DEP (Danielsen et al., 2008b; Ichinose et al., 1997). Also, similar pattern of 8-OHdG and ROS generations in both cell lines showed a close relation between oxidative stress and oxidative DNA damage. DEP-induced ROS has been shown to mediate inflammatory responses through activation of redox-sensitive transcription factors resulting in increased synthesis of pro-inflammatory cytokines (Pourazar et al., 2005). IL-6 and IL-8 are cytokines well recognized as markers of inflammation. A study showed that IL-6 and IL-8 have been reported as the two most up-regulated genes by DEP exposure among 20 inflammatory-related genes in human bronchial epithelial cells (Totlandsdal et al., 2010). IL-6 is a multifunctional cytokine known to activate leukocyte produced by many types of cells, usually at sites of tissue inflammation (Yu et al., 2002). Interestingly, we noticed that IL-6 was preferentially up-regulated in A549 cells while IL-8 was preferentially up-regulated in RPMI 1788 cells. Comparing expression of IL-6 and IL-8, the results in the present study support the previous studies that IL-6 is relatively highly expressed by local tissues of inflammation and is more relevant to local activation of leukocytes than IL-8. Moreover, the results also suggest that IL-8 is more likely to be involved in systemic inflammation. Beside the inflammatory cytokines, lung secretory proteins such as Clara cell protein 16 kDa (CC16) and hydrophilic surfactant protein-A (SP-A) have been demonstrated to play a role in inflammation in the lungs. CC16 and SP-A are mainly synthesized by Clara cells and alveolar type II epithelial cells, respectively. CC16 is encoded for an anti-inflammatory protein that also contributed to host defense. The main function of CC16 is to protect the respiratory tract from excessive inflammation (Singh and Katyal, 1997). As an anti-inflammatory molecule, CC16 inhibits infiltration of inflammatory cells (Johansson et al., 2009). In this study, we found that DEP obviously down-regulated CC16 in A549 cells. This is in agreement with other studies that CC16 expression is reduced in the respiratory tract of rodents after exposure to cigarette smoke (Van Miert et al., 2005) and diesel exhaust (Gowdy et al., 2008). Significant negative correlations of gene expression between CC16 and the pro-inflammatory cytokines (IL-6 and IL-8) in DEPtreated A549 cells support the anti-inflammatory role of CC16. In addition, DEP and LPS induced expression of IL-6 and IL-8 but there was only DEP that significantly suppressed expression of CC16 in A549 cells. Even though there were IL-6 and IL-8 produced in LPS-treated cells, there was no significant alteration in CC16 expression. Therefore, the results suggest that the particulate and/or chemical constituents of DEP may directly suppress CC16 expression rather than indirectly suppress it through induction of the inflammatory cytokines. Additionally, the present study showed that treatment with DEP did not change the expression of CC16 in leukocytes.

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SP-A has been considered to be important in alveolar innate immune defense by either direct killing of microorganism or enhancing uptake of pathogens by macrophages (Kuroki et al., 2007). In the present study, we found that DEP significantly induced SP-A mRNA expression in both cell lines only at the lowest dose. At higher doses of DEP, the expression was not significantly different from untreated cells. Low levels of SP-A are associated with increased inflammation in the lung and association between decreased production of SP-A and increased levels of IL-6 has been observed in A549 cells (Doumanov et al., 2011). It may be implied that increased IL-6 at higher doses of DEP could inhibit expression of SP-A. Nevertheless, no significant negative correlation between SP-A and IL-6 expression was observed in the present study. The exact mechanism underlying alteration of the SP-A expression by DEP is still unknown. Studies in mice showed that exposure to DEP caused reduction of SP-A both in mRNA and protein levels in the lung tissue (Ciencewicki et al., 2007; Gowdy et al., 2008). The reduction of the SP-A may be due to two possible causes. Firstly, moderate to high levels of DEP exposure in mice caused lung damage and subsequently reduced production of the protein. Secondly, DEP indirectly suppressed nuclear factor of activated T cell (NFAT) responsible for regulation of SP-A expression resulting in decrease in transcriptional activation of the surfactant protein (Ciencewicki et al., 2007). Our in vitro study was not consistent with the studies in vivo (Ciencewicki et al., 2007; Gowdy et al., 2008). This might be due to differences in the levels of DEP exposure used in the studies and differences between in vivo and in vitro system, or related to lung damage that cannot be repeated in an in vitro system. The results from the in vitro study showed that DEP uptake and DEP-induced ROS generation, 8-OHdG formation, and expression of the pro-inflammatory cytokines (IL-6 and IL-8) in RPMI 1788 cells were dose-dependent in a similar manner to A549 cells. The obtained results suggest that lymphocyte could be a suitable surrogate cell to assess oxidative and inflammatory response in the lung tissue. We therefore further investigated the responses in circulating lymphocytes of human exposed to traffic-related PM and determined their associations with exposure levels of the particles in order to eventually evaluate the potential of using the lymphocytes as a surrogate to assess possible health effects from PM exposure. Evaluation of human exposure to and effects of traffic-related particles on DNA damage and inflammatory responses Apart from the particles themselves, ambient and individual exposure concentrations of PAHs and carcinogenic metals which are major chemical components of PM were also determined. Ambient PAHs in Bangkok have been determined in total suspended particles (TSP) collected from various roadsides in Bangkok in which the concentrations were in the range of 7.10–83.04 ng/m3 (Ruchirawat et al., 2005) and 4.63–99.95 ng/m3 (Tuntawiroon et al., 2007). The concentrations of total PAHs in the ambient PM2.5 in the present study were in the ranges that have been reported in the two previous studies. There are a few studies presenting relationships between concentrations of PM2.5 and PAHs, and the available data show that the relationships were inconsistent. One study measured ambient concentration of traffic-related air toxics and reported that the concentration of PM2.5 was negatively correlated with B[a]P but was positively correlated with total PAHs (Wilhelm et al., 2011). However, another study conducted in an urban environment showed that ambient PM2.5 concentrations were significantly correlated with concentrations of B[a]P but not total PAHs (Mohanraj et al., 2012). In the present study, there was no significant correlation between the concentrations of PM2.5 and the concentrations of either total PAHs or B[a]P equivalent.

9

Predominant PAHs found in different studies varied. It has been reported that the most predominant PAH in total suspended particles (TSP) collected at roadside areas in Bangkok was benzo[ghi]perylene (B[ghi]P), which represented 24–37% of the total 10 PAHs (Tuntawiroon et al., 2007). Another study conducted at heavy traffic roadside areas in Bangkok showed that indeno[c,d]pyrene (IP), B[ghi]P, and benzo[e]pyrene (B[e]P) were the major compounds among 20 PAHs associated with different sizes of particles of less than 10 ␮m (Thongsanit et al., 2003). In this study, IP was the predominant PAH found in the PM2.5 samples. This might be taken as an indication that IP was the predominant PAH in the respirable fraction of particulate matter. IP is classified as a possible human carcinogen while B[ghi]P and B[e]P are not classifiable for it carcinogenicity (IARC, 1984). Therefore, people in the study locations are being exposed to the carcinogenic PAH. IP has been reported as one of the three tracers of automotive emission (which included benzo[k]fluoranthene (B[k]F), B[ghi]P and IP) in receptor modeling (Miguel and Pereira, 1989), therefore our result confirmed that traffic was a major source of the particles. An attempt to link IP and other PAHs to traffic emission was made by calculating PAH diagnostic ratios as qualitative source identification. B[a]A/CHR and B[a]A/(B[a]A + CHR) ratios in the ranges of 0.28–1.2 and 0.22–0.5, respectively, indicate gasoline exhaust emission (Rogge et al., 1993; Simcik et al., 1997). In addition, a B[k]FA/IP ratio of 0.5 and a B[b]FA/B[k]FA ratio of more than 0.5 indicate diesel emission (Li and Kamens, 1993; Park et al., 2002). The ratios of B[a]A/CHR (0.53), B[a]A/(B[a]A + CHR) (0.34), B[k]FA/IP (0.40), and B[b]FA/B[k]FA (0.57) found in the present study suggest contribution of gasoline and diesel emission to the PM2.5 . Most of the metals were detected in some PM2.5 samples but As and Pb were detected in most of the samples. Arsenic is a contaminant in fossil fuels which is emitted during the combustion. In addition, most Pb emissions in the past have been from combustion of gasoline containing the antiknock additive, namely tetraethyl lead. However, after out-phasing of leaded gasoline, there are other sources in urban environment that have been considered as potential sources of Pb such as the wear of brake and tyres as well as road bitumen (Kennedy and Gadd, 2000). Arsenic (As) is carcinogenic to human (IARC, 2004) whereas Pb has been shown to damage the nervous system, kidney, and reproductive system (ATSDR, 2005). So, this population may possibly be at a certain risk of the adverse health effects that may result from exposure to As and Pb. However, biomarkers of exposure and effect for these metals may need to be carefully determined to assess health risk from the metals in the study locations. The ambient concentrations of Cr and Mn were non-detectable but some individuals were exposed to higher levels of the metals suggesting that personal activities may influence the exposure. In the present study, we found that 8-OHdG in leukocytes correlated with exposure levels of PM2.5 and the particle-bound PAHs. This finding was in accordance with the present in vitro study and previous studies showing that 8-oxodG in lymphocyte DNA correlated with personal exposure to PM2.5 (Sorensen et al., 2003) and particle-bound PAHs (Ruchirawat et al., 2010). The results suggested that exposure to PM2.5 can induce oxidative DNA damage and therefore constitutes an increased cancer risk in the human population. However, IL-8 expression in lymphocytes of the subjects did not correlate with exposure levels of either PM2.5 or its PAHs and metal components. The in vitro study showed that DEP caused induction of IL-8 expression in lymphoblast but the induction was not seen in lymphocytes of the human subjects exposed to PM2.5 . In addition, the in vitro study showed that a significant increase of mRNA expression of the inflammatory cytokine genes was found only at high doses. Therefore, IL-8 expression in the lymphocyte of people in occupational settings that are exposed to relatively high levels of particulate matter is interesting and should

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be investigated. According to the in vitro results showing that DEP significantly induced expression of IL-6 and IL-8 in A549 cells at lower doses compared with RPMI 1788 cells, the lung cells are more sensitive to the particles than the lymphoblast. Hence, increased IL-8 expression in the circulating lymphocytes may reflect a more severe inflammatory response in the lung. Concentrations of CC16 in serum of the study subjects were also determined to examine alteration of CC16 production and its association with PM2.5 exposure. Serum CC16 has been widely used as a peripheral biomarker to assess integrity and permeability of the lung epithelium. Concentration of CC16 in the vascular compartment has been shown to decrease with Clara cell toxicity from cumulative smoking (Robin et al., 2002) and increase with lung injury that could lead to increased CC16 leakiness from disease state such as sarcoidosis (Hermans et al., 2001) and from occupational and environmental exposure such as fire smoke and ozone (Bernard et al., 1997; Broeckaert et al., 2000). The positive correlation between the concentration of serum CC16 and Pb suggests that higher exposure to Pb may cause lung damage resulting in increased level of CC16 in serum. Moreover, the present study suggests that increase exposure to the particle-bound PAHs may cause decrease of CC16 production in the lung and subsequent decrease of serum CC16 in healthy people. A decrease in the host defense molecule may result in higher susceptibility to respiratory inflammation. In conclusion, the in vitro study demonstrated that DEP can induce ROS generation, oxidative DNA damage, and expression of pro-inflammatory cytokines (IL-6 and IL-8) in lymphocytes with a similar pattern to the lung target cells. Lymphocytes could be used as a surrogate to assess PM-induced oxidative DNA damage and inflammatory responses in the lung. Interestingly, the most prominent increase in ROS generation of the cells in G2/M phase revealed a novel finding that DEP-induced oxidative stress was dependent on cell cycle stage. The susceptibility of G2/M cells to DEP in terms of oxidative stress is most likely reflected in the severity of DNA damages and possibility of the unrepaired DNA lesions in the daughter cells. Furthermore, the human study showed that increased oxidative DNA damage can be detected in circulating lymphocytes of people exposed to traffic-related PM. However, IL-8 expression in people exposed to relatively high levels of PM should be further investigated. Moreover, people residing in traffic-congested areas are possibly at risk of health effects from PM exposure. Preventive measures to reduce the particulate air pollution in ambient air are needed to mitigate the health effects. Acknowledgement This research work is supported in part by the grant from Center of Excellence on Environmental Health and Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education. References ATSDR, 2005. Toxicological Profile for Lead. Agency for Toxic Substances and Disease Registry, Atlanta, GA. Attfield, M.D., Schleiff, P.L., Lubin, J.H., Blair, A., Stewart, P.A., Vermeulen, R., Coble, J.B., Silverman, D.T., 2012. The diesel exhaust in miners study: a cohort mortality study with emphasis on lung cancer. J. Natl. Cancer Inst. 104 (11), 869–883. Bartoli, C.R., Wellenius, G.A., Diaz, E.A., Lawrence, J., Coull, B.A., Akiyama, I., Lee, L.M., Okabe, K., Verrier, R.L., Godleski, J.J., 2009. Mechanisms of inhaled fine particulate air pollution-induced arterial blood pressure changes. Environ. Health Perspect. 117 (3), 361–366. Baulig, A., Garlatti, M., Bonvallot, V., Marchand, A., Barouki, R., Marano, F., BaezaSquiban, A., 2003. Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 285 (3), L671–L679. Bernard, A., Hermans, C., van Houte, G., 1997. Transient increase of serum CC16 protein after exposure to smoke. Occup. Environ. Med. 54, 63–65.

Bonetta, S., Gianotti, V., Gosetti, F., Oddone, M., Gennaro, M.C., Carraro, E., 2009. DNA damage in A549 cells exposed to different extracts of PM (2.5) from industrial, urban and highway sites. Chemosphere 77 (7), 1030–1034. Bonvallot, V., Baeza-Squiban, A., Baulig, A., Brulant, S., Boland, S., Muzeau, F., Barouki, R., Marano, F., 2001. Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression. Am. J. Respir. Cell Mol. Biol. 25 (4), 515–521. Broeckaert, F., Arsalane, K., Hermans, C., Bergamaschi, E., Brustolin, A., Mutti, A., Bernard, A., 2000. Serum clara cell protein: a sensitive biomarker of increased lung epithelium permeability caused by ambient ozone. Environ. Health Perspect. 108 (6), 533–537. Ciencewicki, J., Gowdy, K., Krantz, Q.T., Linak, W.P., Brighton, L., Gilmour, M.I., Jaspers, I., 2007. Diesel exhaust enhanced susceptibility to influenza infection is associated with decreased surfactant protein expression. Inhal. Toxicol. 19 (14), 1121–1133. Danielsen, P.H., Loft, S., Moller, P., 2008a. DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles. Part. Fibre Toxicol. 5 (6.), http://dx.doi.org/10.1186/1743-8977-5-6. Danielsen, P.H., Risom, L., Wallin, H., Autrup, H., Vogel, U., Loft, S., Moller, P., 2008b. DNA damage in rats after a single oral exposure to diesel exhaust particles. Mutat. Res. 637 (1–2), 49–55. Danielsen, P.H., Loft, S., Kocbach, A., Schwarze, P.E., Moller, P., 2009. Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines. Mutat. Res.-Gen. Tox. Environ. 674 (1–2), 116–122. Doumanov, J., Jordanova, A., Zlatkov, K., Moskova-Doumanova, V., Lalchev, Z., 2011. Investigation of IL-6 effects on SP-A expression in A549 lung cell line. Biotechnology EQ. 26/2012/SE, Special Edition/on-line, pp. 96–99. http://dx.doi.org/10.5504/50YRTIMB.2011.0018 Frikke-Schmidt, H., Roursgaard, M., Lykkesfeldt, J., Loft, S., Nojgaard, J.K., Moller, P., 2011. Effect of vitamin C and iron chelation on diesel exhaust particle and carbon black induced oxidative damage and cell adhesion molecule expression in human endothelial cells. Toxicol. Lett. 203 (3), 181–189. Gao, F., Yi, J., Yuan, J.Q., Shi, G.Y., Tang, X.M., 2004. The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species. Cell Res. 14 (1), 81–85. Garivait, H., 1999. A study on air pollution by airborne polycyclic aromatic hydrocarbons (PAHs) in Bangkok urban atmosphere. Ph.D. Thesis, Dissertation EV-99-1, Asian Institute of Technology, Pathumthani, Thailand. Garshick, E., Laden, F., Hart, J.E., Rosner, B., Smith, T.J., Dockery, D.W., Speizer, F.E., 2004. Lung cancer in railroad workers exposed to diesel exhaust. Environ. Health Perspect. 112 (15), 1539–1543. Gowdy, K., Krantz, Q.T., Daniels, M., Linak, W.P., Jaspers, I., Gilmour, M.I., 2008. Modulation of pulmonary inflammatory responses and antimicrobial defenses in mice exposed to diesel exhaust. Toxicol. Appl. Pharmacol. 229 (3), 310–319. Hanzalova, K., Rossner Jr., P., Sram, R.J., 2010. Oxidative damage induced by carcinogenic polycyclic aromatic hydrocarbons and organic extracts from urban air particulate matter. Mutat. Res. 696 (2), 114–121. Havens, C.G., Ho, A., Yoshioka, N., Dowdy, S.F., 2006. Regulation of late G1/S phase transition and APC Cdh1 by reactive oxygen species. Mol. Cell. Biol. 26 (12), 4701–4711. Hermans, C., Petrek, M., Kolek, V., Weynand, B., Pieters, T., Lambert, M., Bernard, A., 2001. Serum Clara cell protein (CC16), a marker of the integrity of the air–blood barrier in sarcoidosis. Eur. Respir. J. 18 (3), 507–514. Huang, Y.C., Karoly, E.D., Dailey, L.A., Schmitt, M.T., Silbajoris, R., Graff, D.W., Devlin, R.B., 2011. Comparison of gene expression profiles induced by coarse, fine, and ultrafine particulate matter. J. Toxicol. Environ. Health A 74 (5), 296–312. IARC, 1984. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Polynuclear Aromatic Compounds. International Agency for Research on Cancer. IARC, 2004. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs. International Agency for Research on Cancer. IARC, 2012. IARC Monographs on Evaluation of Carcinogenic Risks to Humans. Volume 105: Diesel and Gasoline Engine Exhausts and Some Nitroarenes, Available from: http://www.iarc.fr/en/media-centre/iarcnews/2012/mono105-info.php Ichinose, T., Yajima, Y., Nagashima, M., Takenoshita, S., Nagamachi, Y., Sagai, M., 1997. Lung carcinogenesis and formation of 8-hydroxy-deoxyguanosine in mice by diesel exhaust particles. Carcinogenesis 18 (1), 185–192. Johansson, S., Andersson, K., Wennergren, G., Wenneras, C., Rudin, A., 2009. CC16 inhibits the migration of eosinophils towards the formyl peptide fMLF but not towards PGD2. Inflammation 32 (2), 65–69. Kanazawa, Y., Isomoto, H., Wen, C.Y., Wang, A.P., Saenko, V.A., Ohtsuru, A., Takeshima, F., Omagari, K., Mizuta, Y., Murata, I., 2003. Impact of endoscopically minimal involvement on IL-8 mRNA expression in esophageal mucosa of patients with non-erosive reflux disease. World J. Gastroenterol. 9 (12), 2801–2804. Kennedy, P., Gadd, J., 2000. Preliminary Examination of Inorganic Compounds Present in Tyres, Brake Pads and Road Bitumen in New Zealand. Kingett Mitchell Ltd for Ministry of Transport (Revised on October 2003). Kim, J.A., Aberg, C., Salvati, A., Dawson, K.A., 2012. Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population. Nat. Nanotechnol. 7 (1), 62–68. Knaapen, A.M., Borm, P.J., Albrecht, C., Schins, R.P., 2004. Inhaled particles and lung cancer. Part A. Mechanisms. Int. J. Cancer 109 (6), 799–809.

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Kumagai, Y., Arimoto, T., Shinyashiki, M., Shimojo, N., Nakai, Y., Yoshikawa, T., Sagai, M., 1997. Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage. Free Radic. Biol. Med. 22 (3), 479–487. Kuroki, Y., Takahashi, M., Nishitani, C., 2007. Pulmonary collectins in innate immunity of the lung. Cell. Microbiol. 9 (8), 1871–1879. Li, C.K., Kamens, R.M., 1993. The use of polycyclic aromatic hydrocarbons as source signatures in receptor modeling. Atmos. Environ. 27A, 523–532. Mazzarella, G., Ferraraccio, F., Prati, M.V., Annunziata, S., Bianco, A., Mezzogiorno, A., Liguori, G., Angelillo, I.F., Cazzola, M., 2007. Effects of diesel exhaust particles on human lung epithelial cells: an in vitro study. Respir. Med. 101 (6), 1155–1162. Mazzoli-Rocha, F., Fernandes, S., Einicker-Lamas, M., Zin, W.A., 2010. Roles of oxidative stress in signaling and inflammation induced by particulate matter. Cell Biol. Toxicol. 26 (5), 481–498. Miguel, A.H., Pereira, P.A.P., 1989. Benzo (k) fluoranthene, benzo (ghi) perylene, and indeno (1, 2, 3-cd) pyrene: new tracers of automotive emissions in receptor modeling. Aerosol Sci. Technol. 10 (2), 292–295. Miranda, M., Chacon, M.R., Gutierrez, C., Vilarrasa, N., Gomez, J.M., Caubet, E., Megia, A., Vendrell, J., 2008. LMNA mRNA expression is altered in human obesity and type 2 diabetes. Obesity 16 (8), 1742–1748. Mohanraj, R., Dhanakumar, S., Solaraj, G., 2012. Polycyclic aromatic hydrocarbons bound to PM2.5 in Urban Coimbatore, India with emphasis on source apportionment. Sci. World J., http://dx.doi.org/10.1100/2012/980843 (Article ID 980843). Nisbet, I.C., LaGoy, P.K., 1992. Toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs). Regul. Toxicol. Pharmacol. 16 (3), 290–300. Okayama, Y., Kuwahara, M., Suzuki, A.K., Tsubone, H., 2006. Role of reactive oxygen species on diesel exhaust particle-induced cytotoxicity in rat cardiac myocytes. J. Toxicol. Environ. Health A 69 (18), 1699–1710. Park, S.S., Kim, Y.J., Kang, C.H., 2002. Atmospheric polycyclic aromatic hydrocarbons in Seoul, Korea. Atmos. Environ. 36, 2917–2924. Patel, M.M., Chillrud, S.N., Correa, J.C., Hazi, Y., Feinberg, M., Kc, D., Prakash, S., Ross, J.M., Levy, D., Kinney, P.L., 2010. Traffic-related particulate matter and acute respiratory symptoms among New York city area adolescents. Environ. Health Perspect. 118 (9), 1338–1343. PCD, 2004. Thailand State of Environment: The Decade of 1990s. Pollution Control Department, Ministry of Natural Resources and Environment, Bangkok. Pourazar, J., Mudway, I.S., Samet, J.M., Helleday, R., Blomberg, A., Wilson, S.J., Frew, A.J., Kelly, F.J., Sandstrom, T., 2005. Diesel exhaust activates redox-sensitive transcription factors and kinases in human airways. Am. J. Physiol. Lung Cell. Mol. Physiol. 289 (5), L724–L730. Robin, M., Dong, P., Hermans, C., Bernard, A., Bersten, A.D., Doyle, I.R., 2002. Serum levels of CC16, SP-A and SP-B reflect tobacco-smoke exposure in asymptomatic subjects. Eur. Respir. J. 20 (5), 1152–1161. Rogge, W.F., Hildemann, L.M., Mazurek, M.A., Cass, G.R., Simoneit, B.R.T., 1993. Sources of fine organic aerosol, 2. Noncatalyst and catalystequipped automobiles and heavy-duty diesel trucks. Environ. Sci. Technol. 27, 636–651. Ruchirawat, M., Navasumrit, P., Settachan, D., Tuntaviroon, J., Buthbumrung, N., Sharma, S., 2005. Measurement of genotoxic air pollutant exposures in street vendors and school children in and near Bangkok. Toxicol. Appl. Pharmacol. 206 (2), 207–214. Ruchirawat, M., Navasumrit, P., Settachan, D., 2010. Exposure to benzene in various susceptible populations: co-exposures to 1,3-butadiene and PAHs and implications for carcinogenic risk. Chem. Biol. Interact. 184 (1–2), 67–76. Shaw, C.A., Robertson, S., Miller, M.R., Duffin, R., Tabor, C.M., Donaldson, K., Newby, D.E., Hadoke, P.W., 2010. Diesel Particulate-Exposed Macrophages Cause Marked Endothelial Cell Activation. Am. J. Respir. Cell Mol. Biol. 44 (6), 840–851. Silverman, D.T., Samanic, C.M., Lubin, J.H., Blair, A.E., Stewart, P.A., Vermeulen, R., Coble, J.B., Rothman, N., Schleiff, P.L., Travis, W.D., Ziegler, R.G., Wacholder, S., Attfield, M.D., 2012. The diesel exhaust in miners study: a nested case–control study of lung cancer and diesel exhaust. J. Natl. Cancer Inst. 104 (11), 855–868. Singh, G., Katyal, S.L., 1997. Clara cells and Clara cell 10 kD protein (CC10). Am. J. Respir. Cell Mol. Biol. 17 (2), 141–143. Simcik, M.F., Zhang, H., Eisenreich, S.J., Franz, T.P., 1997. Urban contamination of the Chicago/Coastal Lake Michigan atmosphere by PCBs and PAHs during AEOLOS. Environ. Sci. Technol. 31 (7), 2141–2147. Sorensen, M., Autrup, H., Hertel, O., Wallin, H., Knudsen, L.E., Loft, S., 2003. Personal exposure to PM2.5 and biomarkers of DNA damage. Cancer Epidemiol. Biomarkers Prev. 12 (3), 191–196. Strassburg, C.P., Oldhafer, K., Manns, M.P., Tukey, R.H., 1997. Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue. Mol. Pharmacol. 52 (2), 212–220.

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Sun, K., Brockman, D., Campos, B., Pitzer, B., Myatt, L., 2006. Induction of surfactant protein A expression by cortisol facilitates prostaglandin synthesis in human chorionic trophoblasts. J. Clin. Endocrinol. Metab. 91 (12), 4988–4994. Suzuki, H., Toyooka, T., Ibuki, Y., 2007. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ. Sci. Technol. 41 (8), 3018–3024. Svecova, V., Rossner Jr., P., Dostal, M., Topinka, J., Solansky, I., Sram, R.J., 2009. Urinary 8-oxodeoxyguanosine levels in children exposed to air pollutants. Mutat. Res. 662 (1–2), 37–43. Thongsanit, P., Jinsart, W., Hooper, B., Hooper, M., Limpaseni, W., 2003. Atmospheric particulate matter and polycyclic aromatic hydrocarbons for PM10 and sizesegregated samples in Bangkok. J. Air Waste Manage. Assoc. 53 (12), 1490–1498. Totlandsdal, A.I., Cassee, F.R., Schwarze, P., Refsnes, M., Lag, M., 2010. Diesel exhaust particles induce CYP1A1 and pro-inflammatory responses via differential pathways in human bronchial epithelial cells. Part. Fibre Toxicol. 7, 41, http://dx.doi.org/10.1186/1743-8977-7-41. Tuntawiroon, J., Mahidol, C., Navasumrit, P., Autrup, H., Ruchirawat, M., 2007. Increased health risk in Bangkok children exposed to polycyclic aromatic hydrocarbons from traffic-related sources. Carcinogenesis 28 (4), 816–822. Turner, M.C., Krewski, D., Pope 3rd, C.A., Chen, Y., Gapstur, S.M., Thun, M.J., 2011. Long-term ambient fine particulate matter air pollution and lung cancer in a large cohort of never-smokers. Am. J. Respir. Crit. Care Med. 184 (12), 1374–1381. US EPA, 2006. Final Revisions to the National Ambient Air Quality Standards for Particulate Pollution. United State Environmental Protection Agency, Available from: http://www.epa.gov/pm/pdfs/20060921 factsheet.pdf US EPA, 2007. Standard Operating Procedure (SOP MLD 061) for the Trace Metal Analysis of Ambient Air Particulate Samples using Inductive Coupled PlasmaMass Spectrometry (ICP-MS). US Environmental Protection Agency, California, Available from: http://www.arb.ca.gov/aaqm/sop/mld061 fin.pdf Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160 (1), 1–40. van Berlo, D., Knaapen, A.M., van Schooten, F.J., Schins, R.P., Albrecht, C., 2010. NF-kappaB dependent and independent mechanisms of quartz-induced proinflammatory activation of lung epithelial cells. Part. Fibre Toxicol. 7, 13, http://dx.doi.org/10.1186/1743-8977-7-13. Van Haute, L., De Block, G., Liebaers, I., Sermon, K., De Rycke, M., 2009. Generation of lung epithelial-like tissue from human embryonic stem cells. Respir. Res. 10 (1), 105, http://dx.doi.org/10.1186/1465-9921-10-105. Van Miert, E., Dumont, X., Bernard, A., 2005. CC16 as a marker of lung epithelial hyperpermeability in an acute model of rats exposed to mainstream cigarette smoke. Toxicol. Lett. 159 (2), 115–123. Wei, Y., Han, I.K., Shao, M., Hu, M., Zhang, J., Tang, X., 2009. PM2.5 constituents and oxidative DNA damage in humans. Environ. Sci. Technol. 43 (13), 4757–4762. WHO, 2005. WHO air quality guidelines global update 2005, Report on a working group meeting. World Health Organization, Bonn, Germany. Wilhelm, M., Ghosh, J.K., Su, J., Cockburn, M., Jerrett, M., Ritz, B., 2011. Traffic-related air toxics and preterm birth: a population-based casecontrol study in Los Angeles County, California. Environ. Health 10, 89, http://dx.doi.org/10.1186/1476-069X-10-89. Xia, T., Korge, P., Weiss, J.N., Li, N., Venkatesen, M.I., Sioutas, C., Nel, A., 2004. Quinones and aromatic chemical compounds in particulate matter induce mitochondrial dysfunction: implications for ultrafine particle toxicity. Environ. Health Perspect. 112 (14), 1347–1358. Xia, T., Kovochich, M., Nel, A.E., 2007. Impairment of mitochondrial function by particulate matter (PM) and their toxic components: implications for PM-induced cardiovascular and lung disease. Front. Biosci. 12, 1238–1246. Yamamori, T., Yasui, H., Yamazumi, M., Wada, Y., Nakamura, Y., Nakamura, H., Inanami, O., 2012. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint. Free Radic. Biol. Med. 53 (2), 260–270. Yu, M., Zheng, X., Witschi, H., Pinkerton, K.E., 2002. The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants. Toxicol. Sci. 68 (2), 488–497. Zhang, X.H., Zhang, X., Wang, X.C., Jin, L.F., Yang, Z.P., Jiang, C.X., Chen, Q., Ren, X.B., Cao, J.Z., Wang, Q., Zhu, Y.M., 2011. Chronic occupational exposure to hexavalent chromium causes DNA damage in electroplating workers. BMC Public Health 11, 224, http://dx.doi.org/10.1186/1471-2458-11-224. Zucker, R.M., Massaro, E.J., Sanders, K.M., Degn, L.L., Boyes, W.K., 2010. Detection of TiO2 nanoparticles in cells by flow cytometry. Cytometry A 77 (7), 677–685.

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