The cytotoxicities induced by PM10 and particle-bound water-soluble species

Science of the Total Environment 354 (2006) 20 – 27 www.elsevier.com/locate/scitotenv

The cytotoxicities induced by PM10 and particle-bound water-soluble species Shui-Jen Chena,*, Shang-Yi Chenga, Meei-Fang Shueb, Kuo-Lin Huanga, Peng-Jy Tsaic, Chih-Chung Lina a

Department of Environmental Engineering and Science, National Pingtung University of Science and Technology, Pingtung 912, Taiwan b Department of Environmental Engineering and Science, Tajen Institute of Technology, Pingtung 907, Taiwan c Department of Environmental and Occupational Health, Medical College, National Cheng Kung University, Tainan 704, Taiwan Received 6 May 2004; accepted 4 November 2004

Abstract A 1-year field sampling of PM10 was performed at a town that usually has the worst air quality in Taiwan to examine if PM10 is a good indicator for pollutant-induced cytotoxicity. The average PM10 concentration in summer was the lowest, while the other three seasons did not show statistical difference in their PM10 means. The pollutant-induced cytotoxicity presented as the cumene-hydroperoxide equivalent concentration (CEC) was found to positively correlate with PM10 concentrations and this study yielded a yearly average of the seasonal CEC 12.F8.54 AM with the magnitudes in sequence for the four seasons as: fallNwinterNspringNsummer. Positive relationship was also found between seasonal PM10 and their corresponding CECs. The exponential regression model obtained from this study shows: CEC=3.305exp(0.0118PM10) (R 2=0.634). The CEC correlates more significantly with NO3 , SO42 , NH4+ and Cl (secondary aerosol species) than with the Na+, K+, Ca2+ and Mg2+ (crustrelated species) in PM10. However, the best multivariable model obtained from this study to relate CEC with the concentrations of PM10-bearing water-soluble species shows: CEC=exp(1.4751+0.0470[SO42 ]+0.0143[NO3 ]) (R 2=0.550). D 2004 Elsevier B.V. All rights reserved. Keywords: Particle-induced cytotoxicity; PM10; Water-soluble inorganic species; Secondary aerosol

1. Introduction The adverse effects of ambient particles on human health have been a concern for decades. Epidemio-

* Corresponding author. Tel.: +886 8 7740263; fax: +886 8 7740364. E-mail address: [email protected] (S.-J. Chen). 0048-9697/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2004.11.012

logical studies have successfully related ambient particulate matter (PM) exposures with the adverse respiratory effects, including chronic obstructive pulmonary disease (COPD) and asthma, and causing an increase in hospital admissions for pneumonia and cardiopulmonary diseases (Dockery et al., 1993; Pope et al., 1995a,b; Tao and Kobzik, 2002). However, it raises an important question, to place the ambient particle sampling on a more health-related basis, if the

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measured PM concentration can be a good indicator for assessing particle-induced cytotoxicity. For the PM exposures, researchers have paid more attention to those fine and ultra-fine ones with aerodynamic diameters less than 10 Am (PM10) and 2.5 Am (PM2.5), respectively. This is mainly because these particles not only can deposit on the extrathoracic airways, but also can penetrate into the lower airways and alveolar region where they may persist for weeks, months or even longer (Lippmann et al., 1980; Brauer et al., 2001). Seaton et al. (1995) found that ultra-fine particles were able to provoke alveolar inflammation, with release of mediators in susceptible individuals, causing exacerbations of lung disease and increasing blood coagulability, leading to the observed increases in cardiovascular deaths associated with urban pollution episodes. On the other hand, PM10 is known to provide the bulk of free radical activity of oxidation to cause an inflammatory response and epithelial injuries in the lungs (Li et al., 1997). Tao et al. (2003) indicated that the exacerbations of pulmonary inflammation in susceptible people (e.g., asthmatics, COPD patients) might be mainly caused by the exertion of toxicity of PM2.5, but 10 AmNPMN2.5 Am could also be toxic. Huang et al. (2002) found that coarse particles (PM10) stimulated higher endotoxin-associated tumor necrosis factor-a production than fine particles (PM2.5) in murine macrophages. Nevertheless, less attention has been paid to examine the association between particle-induced cytotoxicity and PM10 and PM2.5. Recent studies also indicate that particle-induced cytotoxicity is mainly affected by the chemical compositions in PM (Li et al., 1997; Huang et al., 2002; Tao et al., 2003). In addition, secondary inorganic aerosol usually accounts for a substantial fraction (25–50%) of the total mass of PM10 (Redington and Derwent, 2002). It should be noted that the measurement of both PM10 and PM2.5 could be representative to collect the ion species in secondary aerosols. In principle, measuring PM10 is easier and more reliable than measuring PM2.5. PM10 is importantly addressed by Air Quality Standards in several nations (Chen et al., 2003) and it is much more available than PM25 in Taiwan EPA’s database. Accordingly, the present study is set out to examine the relationship between PM10 and particleinduced cytotoxicity. All field samplings were con-

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ducted at Chaochou where is known for the highest PM10 concentrations in Taiwan (Chen et al., 2003). Particularly, it is known that ~40.7% of PM10 collected at Chaochou area were contributed by secondary aerosols such as SO42 , NO3 and NH4+ that are water-soluble (Chen et al., 2003). Therefore, we also examine the cytotoxicity induced by PM10bound water-soluble species (SO42 , NO3 , NH4+ and other five ions). To be more specific in cytotoxicity assessment, in this research, the insoluble components in PM10 were not included, although they might be partially responsible for total PM10-induced cytotoxicity. The results obtained from this study are used to evaluate if PM10 is a good indicator for assessing particle-induced cytotoxicity.

2. Materials and methods 2.1. Sampling strategy A high-volume air sampler (Tisch Environmental TE-6070V, USA) installed with a Teflon filter was used to collect PM10 from a rooftop of a single-story building near a main heavy traffic road at Chaochou, a typical inland urban area located in southern Taiwan. The daily sampling flow rate was specified at 1.13 CMM during the sampling period from June 2002 to May 2003. Hourly wind speeds were recorded simultaneously during sampling period to calculate the mean wind speeds. Particles collected on the filter were measured by using an electrical balance (ANDHM202 with a reading precision of 10 Ag) following pre- and post-weighing procedures to determine the PM10 concentrations. 2.2. Chemical analysis Before and after sampling, the filters were weighed after being conditioned at 25 8C and 40% relative humidity for 24 h. After the post-weighing, the filter was promptly extracted using 100 ml of ultra-pure ` cm). The extract water (specific resistance z18.3 MU was further filtered by a cellulose acetate filter (ADVANTEC MFS, USA cat no.: CO20A025A, pore size: 0.2 Am, diameter: 25 mm) and stored in a plastic vial in a refrigerator at 4 8C until analysis. The concentrations of water-soluble inorganic species

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(Na+, K+, Mg2+, Ca2+, NH4+, Cl , NO3 and SO42 ) were analysed using an ion chromatography (DIONEX 100 with DIONEX CDM-1). The eluents were 1.8 mM Na2CO3/1.7 mM NaHCO3 (2 ml min 1) and 20 mM methane sulfonic acid (1 ml min 1) for anion and cation analyses, respectively. The method detection limit (MDL) in this study were: 0.86 ng m 3 for Na+, 1.48 ng m 3 for K+, 1.6 ng m 3 for Mg2+, 1.29 ng m 3 for Ca2+, 2.21 ng m 3 for NH4+, 5.7 ng m 3 for Cl , 3.5 ng m 3 for NO3 and 7.56 ng m 3 for SO42 , respectively. At least one sample, spiked with a known quantity of ionic species, was analysed per 10 field samples to evaluate recovery efficiencies for quality control purpose. 2.3. Cytotoxicity assay All cytotoxicity assays were carried out on MRC5 cell (female lung epithelial cells) lines purchased from Bioresource Collection and Research Center (BCRC) in Taiwan with the number of BCRC 60023 (Depositor: ATCC number CCL-171). Derived from normal lung tissue of a 14-week-old male fetus, the cells are capable of 42–46 population doublings before the onset of senescence. The cells were grown in Minimum Essential Medium MEM supplemented

with 10% fetal bovine serum (FBS) and maintained in a humidified incubator at 37 8C with 5% CO2 in the air. The particle-induced cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicicty assay (Buttke et al, 1993; Malich et al., 1997; Hayes and Markovic, 2002). For the MTS cytotoxicity assay, 10,000 cells of MRC-5 MEM were seeded in each of 96-well microtitre plates for 24 h. On the next day, cells were fed with the test samples and kept incubated for one day. At the end of the treatments, 20 Al of MTS tetrazolium dye and phenazine methosulfate (PMS) (MTS/PMS 20:1) was added to the wells and incubated for 4 h in a humidified atmosphere. The reaction mixture on each well of the 96-well culture plate was measured using the Fluostar reader (Fluostar Germany) at the wavelength of 492 nm. Each sample was tested in four individual wells. Cumene-hydroperoxide (CH) (12.5 AM) was used as a control positive subject and MEM as a negative control subject. The cytotoxicity of lymphocytes to CH was induced in vitro and the lymphocytes were treated with different doses of CH for 48 h. In the MTS assay, the absorbance of each sample was compared to that exposed to the 12.5-AM CH

Fig. 1. The whole day, nighttime and daytime average PM10 concentration variations in four seasons with S.D. bars (9 sampling days including daytime and nighttime samples in each season: season n=18 and total n=72).

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measured at 492 nm to obtain the sample’s relative effect percentage (REP) (REP=[(sample absorbance DEM absorbance)/(12.5 AM CH absorbance DEM absorbance)]100%). A REP calibration curve was obtained from the controlled exposure at different CH doses. Then, each sample’s cytotoxicity was expressed as cumene-hydroperoxide equivalent concentration (CEC) using the REP calibration curve. Where CEC represents reactive oxygen species generated by CH, a free radical-generating compound as well as a strong oxidizing agent used in industry, may cause membrane damage, cell lysis, organ necrosis and tumor promotion (Sultana et al., 2003). Totally, 72 samples ((6 samples/month)12 months) were tested for the measurement of particle-induced cytotoxicity (as CEC) vs. PM10 concentration.

3. Results and discussion 3.1. PM10 concentrations for different seasons during the sampling period The PM10 concentrations varied with seasons during the 1-year sampling period. The whole day average PM10 concentrations in spring (March to

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May), summer (June to August), fall (September to November) and winter (December to February) were 98F40, 33F5, 115F32 and 113F50 Ag m 3 , respectively (Fig. 1). The fluctuation of PM 10 concentrations appears to be the least during the summer. This is mainly because the summer has the greatest variations in wind speeds (will lead to the reentertainment of particles) and most occurrence of typhoons and afternoon thundershowers (will rainout/rain-off the particulates in air). On the other hand, the highest mean PM10 concentrations were found in autumn and winter. This might be because the prevailing wind blows from northeast in autumn and winter, which led to the PM from upwind Kaohsiung area being transported to the downwind Chaochou area. Here, it should be noted that the Chaochou area is very close to the Chungyang Mountain, which might lead to the accumulation of PM10. Fig. 1 also shows that the difference of PM10 concentrations between daytime and nighttime was smaller in summer but greater in winter. The average concentration of PM10 in daytime (94.6 Ag m 3) is about 10 Ag m 3 higher than that in nighttime (85.9 Ag m 3). This is possible that the sea wind blows from coast to inland and brings the particles from Kaohsiung area to Chaochou in daytime while the wind

Fig. 2. The whole day, nighttime and daytime average PM10-induced cytotoxicity (as CEC) variations in four seasons with S.D. bars (9 sampling days including daytime and nighttime samples in each season: season n=18 and total n=72).

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direction is opposite at night. Nevertheless, the daymean of PM10 is not significantly different from the night-mean of PM10 in the seasons of spring, fall and winter ( pN0.05). Although the p is 0.02 in paired ttest in the PM10 day- and night-mean comparison of summer, the daytime and nighttime PM10 means are still not significantly different at the significant level of 0.01. Therefore, the daytime and nighttime PM10 means were not significantly different in each season, in despite of the different wind directions in daytime and nighttime. 3.2. The relationship between the particle-induced cytotoxicity and PM10 The variation trend of PM10-associated CEC with season (Fig. 2) is similar to that of PM10 vs. season (Fig. 1). This implies a positive relationship between the CEC responses and PM10 concentrations. The yearly average cytotoxicity response of PM10 was 12.2F8.54 AM CEC. The magnitude of CEC values in sequence is: fall (15.9F6.5 AM)Nwinter (14.8F10.0 AM)Nspring (12.7F9.3 AM)Nsummer (5.2F2.0 AM). The variation of PM10 vs. season is completely consistent with that of CEC vs. season in spring, also in summer, including those of daytime and nighttime. However, the CEC was

slightly higher in fall than in winter whereas the PM10 was slightly lower in fall than in winter. In fall and winter, the average daytime CEC was smaller than the average nighttime CEC while this was converse for PM10. Nevertheless, no significant daytime–nighttime difference in CEC means is found for each season ( pN0.05). The seasonal CEC means of spring, fall, and winter are also not statistically different ( pN0.05) but they are different from that of summer at significant levels ( pV0.01). An exponential regression model of CEC= 3.305exp(0.0118PM10) (R 2=0.634) is obtained to correlate the PM10 concentrations with the PM10induced cytotoxicity responses (Fig. 3). This implies that the PM10 concentration is responsible for more than half of the variation of PM10-induced cytotoxicity (in CEC). Although this model yields a moderate dose–response R 2, this value is fairly high for biotoxicity tests (i.e., the R values reported by Huang et al. (2002) for the Na contents in coarse (PM2.5–10) and fine particles (PM2.5) vs. TNF (tumor necrosis factor) production were only 0.26 and 0.10, respectively). A similar approach is also used for the multivariable models to correlate CEC with the concentrations of water-soluble species in PM10 (discussed below).

Fig. 3. Variation of particle-induced cytotoxicity (as CEC) with PM10 concentration (9 sampling days including daytime and nighttime samples in each season: season n=18 and total n=72).

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Fig. 4. The composition of PM10-bearing water-soluble ions in four seasons with S.D. bars (9 sampling days including daytime and nighttime samples in each season: season n=18 and total n=72 for each species).

3.3. The relationship between the concentrations of water-soluble species and particle-induced cytotoxicity Among the eight water-soluble ions in PM10 investigated, NO3 , SO42 , NH4+ and Cl had higher yearly average concentrations (23.1F20.1, 9.6F6.6, 7.1F5.2 and 3.2F2.4 Ag m 3, respectively) than the others (Fig. 4). For the seasonal variation of species average concentration, the NO3 gained higher average concentrations in spring (35.4F18.7 Ag m 3) and winter (32.1F20.7 Ag m 3) but a lower one in

summer (2.3F1.1 Ag m 3). Similarly, NH4+ had a highest average concentration in spring (10.4F4.7 Ag m 3) and a lowest one in summer (1.3F0.6 Ag m 3). SO42 obtained an obviously higher average concentration in fall (15.7F5.4 Ag m 3) than in other seasons, while the highest seasonal average concentration for Cl appeared in winter (4.6F2.4 Ag m 3). In general, Na+, K+, Ca2+ and Mg2+ are the ions of crustal elements, while NO3 , SO42 , NH4+ and Cl are usually found in secondary aerosols although Cl may also be mainly generated form salt sources. Note that

Table 1 Pearson correlation coefficients for the CEC, PM10 and water-soluble species in PM10 CEC PM10 NO3 SO24 Cl NH+4 Na+ K+ Ca2+ Mg2+

CEC

PM10

NO3

SO24

Cl

NH+4

1.00

0.758a 1.00

0.666a 0.871a 1.00

0.627a 0.736a 0.583a 1.00

0.345a 0.515a 0.431a 0.128 1.00

0.521a 0.797a 0.927a 0.568a 0.437a 1.00

CEC: cumene-hydroperoxide equivalent concentration. a a=0.01 (two-tail), significant correlation. b a=0.05 (two-tail), significant correlation.

Na+ 0.041 0.129 0.260b 0.076 0.219 0.338a 1.00

K+

Ca2+

Mg2+

0.161 0.382a 0.347a 0.126 0.562a 0.452a 0.059 1.00

0.256b 0.532a 0.561a 0.437a 0.428a 0.718a 0.439a 0.567a 1.00

0.134 0.333 0.413a 0.152 0.355a 0.523a 0.769a 0.324a 0.745a 1.00

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the correlation coefficient between NO3 and NH4+ is very high in Pearson matrix (Table 1). It is inferred that the predominant ammonium compound is NH4NO3 although other ammonium compounds such as NH4HSO4, (NH4)2SO4 and NH4Cl may also be present (Lee et al., 1996). This reveals that the industrial sources contributed less than the traffic sources to this NO3 generation because the NH4+ in secondary aerosols was mainly due to industrial sources. As can be seen in Table 1, the four crustrelated ions significantly exhibit lower correlation coefficients than the four secondary-aerosols-associated ions, not only in their correlations with PM10 but also with CEC. As a result, the four crust-related ions were not included in the multivariable regression for the CEC vs. the concentrations of water-soluble species in PM10. The Cl was removed from the regression in a stepwise algorithm using SYSTAT (Version 5.0) (Wilkinson et al., 1992) and a partial F ( F p) approach (Kleinbaum et al., 1992) because Cl displayed a p value of 0.062 and the increase of R 2 was not statistically significant (a=0.05) when including Cl . The NH4+ is confounded with NO3 . This is partially supported by their high correlation coefficient (0.927). In addition, NH4+ positively correlates with CEC (R =0.521) but the coefficient of NH 4+ is negative if both NH4+ and NO3 are included in the multivariable regression, so NH4+ is also removed from the model. Consequently, the exponential model obtained for the multivariable regression is CEC=exp(1.4751+0.0470[SO 42 ]+0.0143[NO 3 ]) (R 2=0.550, pV0.0001) using Excel or SYSTAT. The interaction between SO42 and NO3 is not statistically significant.

The yearly average cytotoxicity response of PM10 was 12.2F8.54 AM CEC. The seasonal CEC values were fall (15.9F6.5 AM)Nwinter (14.8F10.0 AM)Nspring (12.7F9.3 AM)Nsummer (5.2F2.0 AM). The variation trend of PM10-associated CEC with season is similar to that of PM10 vs. season, leading to a positive correlation between the CEC responses and PM10 concentrations. The regression model found is CEC=3.305exp(0.0118PM10) (R 2=0.634). All the eight water-soluble ions in PM10 obtained lowest average concentrations in summer than in other seasons. NO3 , SO42 , NH4+ and Cl (secondary aerosol species) had higher yearly average concentrations (23.1F20.1, 9.6F6.6, 7.1F5.2 and 3.2F2.4 Ag m 3, respectively) than the others. Also, the CEC correlation coefficients were significantly higher for NO3 , SO42 , NH4+ and Cl than for the other four ions. The best regression model obtained to correlate CEC with the concentrations of water-soluble species is CEC=exp(1.4751+0.0470[SO42 ]+0.0143[NO3 ]) (R 2=0.550). Therefore, the particle-induced cytotoxicity is mainly attributed to secondary sulfate- and nitrate-associated aerosols in PM10.

Acknowledgment This work was financially supported by Taiwan National Science Council under Contract No. NSC912211-E-020-012. The authors would like to acknowledge the help from Dr. Sheng-Wei Wang at the Environmental and Occupational Health Sciences Institute, Rutgers University.

References 4. Conclusions The PM10 concentrations had seasonal variation in the 1-year sampling period at Chaochou in southern Taiwan. The average PM10 concentration was significantly lower in summer than in the other seasons that had no statistically significant difference in average PM10 concentrations. The average PM10 concentration in winter was approximately 3.5-fold of that in summer. The average daytime and nighttime PM10 were not significantly different in each season.

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