- Email: [email protected]
Genotoxicity of PM10 and extracted organics collected in an industrial, urban and rural area in Flanders, Belgium
ARTICLE IN PRESS
Environmental Research 96 (2004) 109–118
Genotoxicity of PM10 and extracted organics collected in an industrial, urban and rural area in Flanders, Belgium$ Ethel Brits,* Greet Schoeters, and Luc Verschaeve Vito (Flemish Institute for Technological Research), Department of Environmental Toxicology, Boeretang 200, 2400 Mol, Belgium Received 9 July 2003; received in revised form 11 March 2004; accepted 22 March 2004
Abstract The variation in the genotoxic potency of PM10 in vitro in relation to the particle source type was investigated. Particles were collected at one urban, one rural, and one industrial site in Flanders. Genotoxicity was assessed using four different in vitro test systems exposed to PM10 in suspension and to the organic extracts of PM10. Two of these systems were bacterial assays: the Salmonella mutagenicity test and the Vitotox test. In addition, the Comet assay and Micronucleus test were performed using human blood cells. Results show that exposure to PM10 and the organic extracts from both urban and industrial areas causes significant genetic damage. The Salmonella mutagenicity test was most suitable for the screening of PM10 and the organic extracts; the Micronucleus test was most suitable only for the screening of organic extracts, and original particles were toxic for the exposed lymphocytes. Clear dose–response curves were not established in the Comet and Vitotox assay, and organic extracts were apparently toxic in the latter. The total polycyclic aromatic hydrocarbon content of the organic extracts, as measured with GC/MS, ranged between 1 and 6 ng/m3. Results obtained in this study suggest that PM10 causes DNA damage and mutations. The use of biological tests for the screening of air samples is useful to complement air quality control by chemical measurements. r 2004 Elsevier Inc. All rights reserved. Keywords: PM10; PAH; Genotoxicity; Mutagenicity; In vitro
1. Introduction Airborne particulate matter, currently considered a major air pollutant, causes adverse health effects (Brunekreef and Holgate, 2002). Epidemiological studies in US and European cities have demonstrated an association between PM10 pollution episodes and increased acute and chronic respiratory morbidity (Goldsmith and Kobzik, 1999; Takafuji and Nakagawa, 2000), increased mortality (Dockery et al., 1993; Samet et al., 2000; Goldberg et al., 2001), and increased lung cancer risk (Katsouyanni and Pershagen, 1997; Cohen, 2000; Nyberg et al., 2000; Pope et al., 2002). Adverse health effects are mainly ascribed to inhalable particles with an aerodynamic diameter of less than 10 mM (PM10) (Hornberg et al., 1998; Salvi and Holgate, 1999; Hsiao et al., 2000). Differences in response have $ This study was performed in order of and supported by the VMM, Flemish Environment Agency. *Corresponding author. Fax: +32-14-58-26-57. E-mail address: [email protected] (E. Brits).
0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.03.006
been observed among cities; however, epidemiological studies are not sensitive enough to differentiate in composition of the particles. To explore the variation in toxic potency of the particles in relation to their composition and origin, additional toxicological studies are needed. A better understanding of the underlying toxic mechanisms of PM10 will allow more effective measurement to reduce adverse health effects associated with particulate matter (Health Effects Institute, 2002). PM10 proved to be genotoxic in diverse test systems (De Martinis et al., 1999; Cˇerna´ et al., 2000; Buschini et al., 2001; Binkova´ et al., 2003). This may be partly explained by organic substances adsorbed on the particle surface, such as polycyclic aromatic hydrocarbons (PAHs), nitro-polycyclic aromatic compounds, dioxins, and metals. The relative contribution of these pollutants to PM10 may depend on the particle sources and meteorological conditions. Therefore, the spatial and temporal monitoring of PM10 as a genotoxic pollutant is considered important for the evaluation of health risks for populations in polluted areas. This hazard may depend on local factors such as industrial
ARTICLE IN PRESS 110
E. Brits et al. / Environmental Research 96 (2004) 109–118
emissions, traffic exhaust, and domestic and agricultural emissions. Until now, PM10 monitoring in Flanders was restricted to the measurement of particle concentrations and sporadically PM10 is chemically characterized (Milieumaatschappij, 2002). This approach is insufficient for hazard identification, considering the complexity of PM10. In this study, PM10 air samples and organic extracts of PM10 were characterized for their genotoxic potency. The particles were collected at three monitoring sites: Antwerp, an urban site characterized by high traffic pollution and relatively moderate industrial pollution; Berendrecht, a site with high industrial pollution and moderate traffic pollution; and Peer, a rural site with moderate overall pollution. PM10 and the organic extracts were evaluated for genotoxicity using a battery of four different bioassays, including two bacterial tests and two assays using human leukocytes as target cells. The bioassays used for measuring the genotoxic activity of PM10 and the derived organic extracts were selected to evaluate different, but complementary genotoxic endpoints: SOS repair in response to DNA damage (Vitotox test); DNA damage (Alkaline single-cell gel electrophoresis or Comet assay); gene mutations (Salmonella mutagenicity test); chromosome/genome mutations (Micronucleus test). The Ames Salmonella typhimurium assay is a widely accepted bacterial assay, appropriate to detect mutagens in airborne particulates (Monarca et al., 1997; Bronzetti et al., 1997; De Martinis et al., 1999; Cˇerna´ et al., 2000). Recently, new bacterial tests have been developed for the detection of DNA-damaging agents, including the Vitotox test, a short-term assay used in this study. The Vitotox test measures DNA damage as a result of SOS induction and has shown to be very sensitive at detecting genotoxic activity of pure compounds, but not yet at detecting complex environmental mixtures (Verschaeve et al., 1999). The alkaline Comet assay, applied on human leukocytes, measures DNA strand breaks in alkali-labile sites of individual cells and is often used for environmental monitoring (Don Porto Carero et al., 2001; Buschini et al., 2001; Knaapen et al., 2002). The Micronucleus test on human lymphocytes detects structural and numerical chromosomal aberrations; it has been included in this study as an easier and more rapid alternative to the conventional chromosomal aberration test (Fenech, 2000). The organic extracts of PM10 samples were chemically analyzed for their PAH content using gas chromatography and mass spectrometry (GC/MS). The aim of the study was to compare the genotoxic potency of PM10 from different sites. Additionally, the organic extracts derived from PM10 were tested in comparison with the original particles to evaluate whether the genotoxicity is attributed to the particles
and/or the adsorbed organic compounds. The predictive value of chemical PAH analysis for genotoxic characterization of airborne particles was examined. Based on the whole evaluation, the suitability of the selected bioassays for the screening of PM10 was assessed.
2. Materials and methods 2.1. Air sampling and sample preparation PM10 samples were collected at three sample sites in Flanders. The selected sites are included in the PM10 monitoring network of the Flemish Environmental Agency (VMM), and epidemiological data concerning the prevalence of asthma were available (Wieringa et al., 1998). At the urban site (Borgerhout, the city center of Antwerp), the sampling point was located at a distance of 15 m from a street with high traffic density. The industrial site (Berendrecht) is located at the North of Antwerp in close proximity to a chemical industry and the harbor. The sampling site was located near the motorway in a green environment, yet within a 2-km radius of the industry. The rural site (Peer) is a small, green village in the Province of Limburg. The sampling site was located in a sports park, 2 km outside the center of the village. Particles were collected on preweighed Teflon-coated glass fiber filters (Emfab, Pallflex, Gelman) using a Pourbaix low-volume sampler provided by the Flemish Environment Agency (VMM), during 2 weeks in September 2000. The sampling flow was 0.9 m3/h. The volume of filtered air was 358 m3. Gravimetric analysis of the particulates was standardized by conditioning the filters for 48 h at 25.5 C and 50% humidity before and after PM10 collection. PM10-containing filters were cut into four equal parts, and pooled per fourth and per site. The particle suspension was made by shaking two parts (2/4) of the filters overnight at 37 C in sterile, bidistilled water supplemented with 0.1% Triton X-100 (Sigma). Subsequently, filters were removed from the suspension and sterile bidistilled water supplemented with 0.1% Triton X-100 was added to obtain a suspension of particles per milliliter from 20 m3-filtered air. Another one-fourth was extracted with Dionex ASE 200 Accelerated Solvent Extraction in tetrahydrofurane/n-hexane (20/80) at a pressure of 140 bar and a temperature of 100 C. The solvent was evaporated under N2. The residual matter was redissolved in 10% dimethyl sulfoxide (DMSO) in water to obtain 20 m3 of air volume equivalents per 1 mL of extract volume. Another one-fourth was identically extracted with ASE, but the samples were spiked with 120 mL internal standard. These samples were chemically analyzed with GC/MS as described below.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
2.2. Chemical analysis The PAH concentrations were determined by GC/MS in the extracts. The analysis was carried out using a Carlo Erba MFC 500 capillary GC, Carlo Erba A200S autosampler, and Fisons QMD 1000 low-resolution mass spectrometer. The GC/MS system was equipped with a 30 m 0.25 mm 0.25 mm DB-5MS fused silica GC column. Helium carrier gas was used at a flow rate of 1 mL/min. The temperature program consisted of an isothermal hold at 125 C for 1 min, an increase of 20 C/ min to 205 C and a second increase of 10 C/min to 305 C followed by a 15-min isothermal hold. 2.3. Genotoxicity assays 2.3.1. Salmonella mutagenicity test The Salmonella plate incorporation test was performed according to Maron and Ames (1983). S. typhimurium strain TA98 was kept at 80 C until use. One-hundred microliters of the TA98 strain was added to a 50-mL Erlenmeyer, containing 20 mL of Nutrient Broth growth medium (25 g/L Nutrient Broth No. 2 (Oxoid) in sterile distilled water). Two mg/mL 4-NQO and 20 mg/mL benzo(a)pyrene in the presence of a metabolic activation system were used as positive control. As a metabolic system, S9 was used, and it was stored at 80 C until use. Prior to use, the S9 was combined with an Ames mutagenicity test tablet (Boehringer) dissolved in 18 mL sterile distilled water containing the necessary cofactors such as NADP and G-6-P. The S9 fraction was 10% of the S9-mix volume. The test was performed in plates containing two layers of agar, the bottom agar (minimal glucose plate, Oxoid) for providing a suitable support media and the top agar (0.6% agar, Difco and 0.5% NaCl) for applying the test chemical, metabolic activation reagent if required, and the tester strain. Two milliliters of the top agar with histidine/biotin (Sigma) (0.05 mM), S9 mix, or phosphate buffer (0.5 mL), test chemicals (100 mL), and the desired tester strain (100 mL) was poured over a minimal glucose agar plate. Per dose, three replicate plates were made, five replicates for the negative control. Plates were incubated at 37 C for 48 h. Colonies on each plate were counted. Results were expressed as the number of revertants in function of the mass of PM10 and volume of air sampled. The results were evaluated using one-way ANOVA combined with the Tukey post hoc test (Statistica, Statsoft). The level of significance was set at Po0:05: 2.3.2. Vitotox test The Vitotox test was performed according to Verschaeve et al. (1999). The test is based on S. typhimurium TA104 recN2-4 bacteria that contain
111
the lux operon of Vibrio fischeri under transcriptional control of the recN gene, which is part of the SOS system. After incubation of the bacteria with a genotoxic compound, the recN promoter was derepressed, resulting in expression of the lux operon, and consequently, the expression of light production in function of genotoxicity. As an internal control system, the S. typhimurium strain TA104pr1 strain was used. This is a constitutive light-producing strain with a lux operon under control of the strong promoter pr1. S. typhimurium strains TA104recN2-4 and TA104pr1 were kept at 80 C until use. Twenty microliters of each strain (TA104recN2-4 and TA104pr1) was added to 5 mL of a normal bacterial growth medium. Bacterial cultures were then incubated overnight in an Innova 4000 (New Brunswick Scientific) rotative incubator shaker at 250 rpm and at 37 C. Cultures were then diluted 10-fold. Fifty microliters of each strain (TA104recN2-4 and TA104pr1) of the overnight culture was added to 2.5-mL growth medium. Bacterial cultures were then incubated on an Innova 4000 environmental shaker at 250 rpm and at 37 C (1 h) to obtain log phase growth. 4-NQO was used as positive control for the test without metabolic activation in a final concentration of 4 pg/mL and benzo(a)pyrene in the presence of a metabolic activation system in a final concentration of 8 mg/mL. The S9 was stored at 80 C until use. Prior to use, the S9 was combined with NADP and G-6-P (Sigma). The S9 fraction was 10% of the S9-mix volume. In the final measurement plate, this was again 10-fold diluted to a 1% final solution. The samples without S9mix were provided with phosphate buffer to keep the number of bacteria the same. In each well of the microtiter plate, 90 mL of the 1 h culture was mixed with 10 mL of the test compound. Luminescence was measured with a Microlumat LB96B luminometer (EG&G Berthold) or with a Luminoskan Ascent (Labsystems) including the following parameters: 1 s/well; cycle time=5 min; 60 cycles; incubation temperature=30 C. The signal-to-noise ratio (S/N), being the light production of exposed cells divided by the light production of nonexposed cells, was calculated for each measurement. A test compound was considered genotoxic when the max S/N (recN2-4)/max S/N (pr1)41.5, a clear dose–response curve was generated, and the signal was not generated in the first 20 min.
2.3.3. Comet assay The Comet assay or single-cell gel electrophoresis assay using human leukocytes was performed as described by Singh et al. (1988), with slight modifications. Human whole blood was exposed to three dilutions of the organic extract for 24 h at 37 C in a rotative shaker, with and without S9.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
112
Microscopic slides were coated with 80 mL of 1% normal melting point agarose (Sigma). When this layer had solidified, a second layer containing 5-mL whole blood sample mixed with 300 mL of 0.8% low melting point agarose (Sigma) was placed on the slides. After 5 min of solidification on ice, slides were immersed for minimum 1 h in ice-cold freshly prepared lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 1.2% Tris) with 1% Triton X-100 and 10% DMSO added fresh to lyse cells and to allow DNA unfolding. The slides were then placed on a horizontal gel electrophoresis tank. The unit was filled with electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, 0.1% 8-hydroxyquinoline, 2% DMSO, 17 C) and the slides were placed in this alkaline buffer for 40 min to allow DNA denaturation. Electrophoresis was carried out for 20 min at 1 V/cm (300 mA). After electrophoresis, the slides were rinsed gently three times with neutralization buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 mg/mL) and covered with a coverslip. Slides were stored at 4 C in sealed boxes until analysis. A total of 50 randomly captured comets from each slide were examined at 400 magnification using an epifluorescence microscope (Zeiss). Measurements were performed with a computerized image analysis system (Komet 3.1, Kinetic Imaging Ltd., Liverpool, UK). To quantify DNA damage, the percentage of DNA content in the comet tail was measured. The results were evaluated using the nonparametric Median test (Statistica, Statsoft). The level of significance was set at Po0:05: 2.3.4. Micronucleus test The protocol was used as described in Fenech and Morley (1985) with slight modifications. Supplemented medium was prepared by adding 15% fetal calf serum (Greier), 2% phytohemagglutinin (Sigma), 1% glutamine (Sigma), and 1% penicillin–streptomycin (Gibco) to culture medium RPMI 1640 (Gibco). The supplemented medium was filtered through a 0.22-mm filter. Whole human blood (0.5 mL) was added to 5 mL of the medium and 50 mL of the organic extract (in three dilutions) and cultivated at 37 C for 44 h. To stop the cell cycle in the binuclear stage, 100 mL of cytochalasine B (Sigma) (0.6 mg/mL) was added. The lymphocytes were further cultivated at 37 C for 28 h. After centrifugation (1200 rpm for 10 min), the pellet was resuspended
in 5 mL hypotonic solution 0.075 M KCl. The cell suspension was centrifuged again at 1200 rpm for 10 min, the cells in the pellet were fixed in 5 mL methanol and acetic acid 3:1, and three drops of 37% formaldehyde were added. Following centrifugation, the pellet was resuspended in 5 mL and fixed in three steps. Finally, the pellet was resuspended in 4 mL of fixator, this cell suspension was stored at 20 C for at least 16 h. Then, the cells were sedimented at 1200 rpm for 10 min, supernatant was discarded, and the pellet was resuspended in 2 mL fixator, and sedimented again. Microscopic slides were prepared in duplicate from this final cell suspension, air-dried, and stained with 50% May–Grunwald (Sigma), following a 10% Giemsa (Sigma) solution. One thousand binucleated cells were counted on each slide. Data were statistically analyzed with the Kastenbaum–Bowman test (Kastenbaum and Bowman, 1970).
3. Results 3.1. PM10 and PAHs Concentrations of PM10 ranged between 10 and 30 mg/m3 and were highest in the urban area (Table 1). The concentrations of 17 PAHs measured by GC/MS, present in the organic extracts, were also highest in the urban area (Table 2). Eight of the measured PAHs are listed as IARC recognized carcinogenic PAHs, namely benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, Chrysene, dibenzo(a,h)anthracene, and indeno(1,2,3, c,d)pyrene (IARC, 1987). The carcinogenic PAHs contribute largely to the total amount of chemically analyzed PAHs: 20% for the samples from the industrial origin, 33% from the rural origin, and 45% for the urban origin. 3.2. Salmonella mutagenicity test Without addition of S9, only the highest concentration of urban and industrial extracts were mutagenic, particles and rural extracts were not mutagenic. In the presence of S9, extracts and particles of urban (Fig. 1), industrial, and rural areas tested mutagenic in all areas (Table 3).
Table 1 Data on sampling: sample period, total volume of filtered air, total amount of PM10 collected and mean sampled concentration (mg/m3) Area
City name
Sample period
Volume (m3)
PM10 collected (mg)
PM10 (mg/m3)
Urban Rural Industrial
Borgerhout Peer Berendrecht
Sept. 9–25, 2000 Sept. 9–25, 2000 Sept. 9–25, 2000
358 356 346
10.4 5.4 4.5
30 15 13
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
113
3.3. Vitotox test
3.4. Comet assay
Table 4 shows the results of the genotoxicity of PM10 and the extracts using S. typhimurium TA 104 recN 2-4 and pr1 tester strains. The organic extracts of PM10 were toxic at the highest doses. This toxicity can be due to traces of solvents used for the extraction procedure. Merely the response curve of PM10 from the urban area reaches the 1.5 ratio, and is consequently considered genotoxic. Fig. 2 graphically illustrates the results of organic extracts from particles collected in the urban area.
A 24-h exposure of both particles and organic extracts from each area seemed to cause significant DNA damage; however, no clear dose–response relationships were established (Table 5). Particles from the urban area appeared toxic at the highest dose, S9 detoxified the sample. Overall, addition of S9 enhances the DNAdamaging effect. Fig. 3 demonstrates the results of organic extracts of particles collected in the urban area. 3.5. Micronucleus test
Table 2 Concentrations of 17 different PAHs (ppm) measured by GC/MS in organic extracts of filter samples of all areas PAHs
Urban (ng/mL)
Rural (ng/mL)
Industrial (ng/mL)
Acenaphtene Acenaphtylene Anthracene Benzo(a)anthracene Benzo(a)pyrene Benzo(b)fluoranthene Benzo(e)pyrene Benzo(g,h,i)perylene Benzo(k)fluoranthene Chrysene Dibenzo(a,h)anthracene Fluoranthene Fluorene Indeno(1,2,3,c,d)pyrene Naphtalene Phenantrene Pyrene
21.5 0 4.6 10.7 6.1 18.4 12.3 15.3 4.6 12.3 0 12.3 7.6 7.7 0 16.9 12.2
19.44 0 2.43 4.05 1.62 6.48 2.43 2.43 1.62 2.43 0 4.05 4.05 1.62 0 0.81 4.86
12.96 10.53 7.29 4.05 0.81 5.67 2.43 2.43 1.62 2.43 0 3.24 4.05 1.62 0 12.15 12.15
Total
162.5 ng/mL 5.5 ng/m3 air
58.3 ng/mL 2 ng/m3 air
83.4 ng/mL 2.8 ng/m3 air
Note: The data were adjusted for the trace amounts of PAHs detected in the blank filter extracts. PAHs marked with an () are carcinogenic PAHs (group 2A or 2B) according to IARC (1987).
Following exposure to particles in suspension, insufficient binuclear cells were generated due to toxicity. Hence, no data are shown for particle exposure conditions. PM10 extracts were analyzed at the highest Table 3 Mutagenicity of PM10 tested with the Salmonella mutagenicity test and the organic extracts from three areas with and without addition of S9 Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 2.5 5 10 20
19.2 17.3 18.3 19.3 25
19.8 19 19.7 23 27.7
19.2 19 20.7 17.3 26
19.8 20.7 20 24.3 24.3
19.2 18 18 22.7 26
19.8 18.3 19 20.7 26.3
0 2.5 5 10 20
23.8 29.3 30.3 40.3 48.7
23 31 35.3 43.3 48.7
23.8 22.3 27 32 42.3
23 25 26.7 29 33.7
23.8 29 33 37 45.7
23 24.3 28 32.3 38.7
The mean number of revertants (mean of three replicas) is shown in relation to the concentration of the organic extract (rev/m3 air eq). Concentration 0 m3 air eq/mL is the suspension or organic extracts of blank filters, uniformly treated as the loaded filters. Data printed in bold are significantly (Po0:05) different from the blank (one-way ANOVA, combined with the Tuckey Post Hoc test).
60
Nr of revertants
50 40
*
*
*
*
30 20 10 0 0m³ air eq / mL
2.5m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL 20m³ air eq / mL
Fig. 1. Mutagenicity of organic extracts of PM10 from the urban area with addition of S9, tested with the Salmonella mutagenicity test. The mean number of revertants (mean of three replicas) is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq/mL is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with a one-way ANOVA, combined with the Turkey post hoc test.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
114
Table 4 Data on genotoxicity in the Vitotox test of PM10 and the organic extracts from three areas with and without addition of S9 Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 2.5 5 10 20
1.0 1.3 1.4 1.4 1.5
1.1 1.1 1.1 1.2 TOX
0.98 1.0 1.2 1.2 1.3
1.1 1.0 1.1 1.3 TOX
0.98 1.2 1.0 1.0 0.9
1.1 1.1 1.2 TOX TOX
0 2.5 5 10 20
1.0 1.1 1.1 1.2 1.5
1.1 1.0 1.0 TOX TOX
1.0 1.0 1.1 1.2 1.3
1.1 1.2 1.2 TOX TOX
1.0 1.0 1.0 0.9 1.0
1.1 1.0 1.1 1.4 TOX
Note: The max S/N (recN2-4)/max S/N (pr1) is shown for each condition. Concentration 0 m3 air eq/mL is the suspension or extract of blank filters. Data printed in bold are considered genotoxic, at this condition the max S/N (recN2-4)/max S/N (pr1) reaches 1.5. TOX means the max S/N (recN2-4)/max S/N (pr1) reaches below 0.8 and the condition is toxic for the bacteria.
RecN 2-4
2
SIGNAL/NOISE
Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 5 10 20
1.6 1.8 3.9 TOX
1.4 1.7 2.5 3.2
1.6 3 3.2 1.8
1.4 1.5 3.4 2.2
1.6 2.1 1.9 2.2
1.4 2 3 2
0 5 10 20
1.9 2.8 2.0 4.6
1.9 1.5 4.1 4.8
1.9 2.6 3.3 2.4
1.9 1.9 2.1 2.0
1.9 2.2 2.6 2.4
1.9 1.8 3.1 3.0
Note: The % tail-DNA number (median of 50 cells) of each exposure condition is presented. Concentration 0 m3 air eq/mL is the suspension or extract of blank filters. Data printed in bold are significantly (Po0.05) different from the blank, assessed with the non-parametric Median test.
dose (20 m3/mL) (Table 6). If the sample exposure showed a significantly increased micronucleated cell frequency, further dilutions were examined. A significantly increased number of micronucleated binuclear cells was found exclusively as a result of exposure to the organic extract of PM10 collected in the urban area, a clear dose–response relationship was found (Fig. 4). The protocol was only performed without addition of S9.
2.5
1.5
1
4. Discussion
0.5
0 0
60
120
180
240
TIME(min)
Pr1
2.5
2
SIGNAL/NOISE
Table 5 Results of DNA damage measured with the Comet assay after 24 h exposure of human whole blood to PM10 and the corresponding organic extracts from three areas with and without addition of S9
1.5
1
0.5
0 0
60
120
180
240
TIME(min) Fig. 2. Genotoxicity in the Vitotox test of the organic extracts of PM10 from the urban area with addition of S9. The signal/noise ratio (light production of exposed/nonexposed bacteria) of the recN2-4 and pr1 strain is shown in relation to the time (min) for all four tested concentrations of the organic extract (m3 air eq/mL). Compounds are considered toxic when S/N decreases below 0.8. Concentration 0 m3 air eq is the extract of blank filters. (J) 2.5 m3 air eq/mL, (&) 5 m3 air eq/mL, (}) 10 m3 air eq/mL, and (n) 20 m3 air eq/mL.
The major objective of this study was to investigate the genotoxic potency of both ambient PM10 and the adsorbed organic compounds on PM10, collected at three monitoring sites (2-week periods): Antwerp, an urban site with high traffic pollution, Berendrecht, a site with industrial pollution, and Peer, a rural site. Furthermore, the predictive value of standard chemical PAH analysis for the toxicological effect of PM10 was evaluated. For these purposes, PM10 samples and the organic extracts of PM10 were screened by four genotoxicity tests: the Salmonella mutagenicity test, qthe Vitotox test, the Comet assay, and the Micronucleus test. Comparison of the PAH concentrations of the three sites shows considerable differences with the urban site having the highest concentration (ng/m3) for PAHs and the carcinogenic PAHs in particular (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo ðg; h; iÞperylene; benzo(k)fluoranthene, chrysene, dibenzo(a,h)anthracene and indeno(1,2,3,c,d)pyrene; IARC, 1987). The Salmonella mutagenicity test was performed using the S. typhimurium TA98 strain, a sensitive strain
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118 5
115
*
% DNA in tail
4
*
3 2 1 0
0m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL
20m³ air eq / mL
Fig. 3. DNA damage measured with the Comet assay as a result of exposure to the organic extract of PM10 from the urban area with addition of S9. The % tail-DNA number (median of 50 cells) is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with the nonparametric Median test.
Table 6 Number of binucleated cells with micronuclei on a total of 1000 binuclear cells Extracts
0 5 10 20
Urban
Rural
Industrial
3 3 9 16
3 — — 2
3 — — 2
Note: Human whole blood was exposed to PM10 and the corresponding organic extracts from three areas. Concentration 0 m3 air eq/ml is the extract of blank filters. Data printed in bold are significantly (Po0:05) different from the blank (Kastenbaum–Bowman test).
for analysis of air pollutants (Bronzetti et al., 1997; Cˇerna´ et al., 1999; Zhao et al., 2002; Binkova´ et al., 2003). Another frequently used strain, TA100, is valuable for screening purposes as well, but former research (unpublished) showed that this strain adds little for the investigation of airborne particulates; results are comparable to those achieved with assessment by the TA98 strain (Claxton et al., 2001). The Salmonella mutagenicity test could assign—essentially equal—mutagenic potency to all three samples. When S9 was added for metabolization, exposure to urban particles produced the most revertants, and exposure to rural particles produced the least revertants. Organic extracts of the corresponding particles yielded the same pattern, but the differences between the sites were more pronounced. Without S9 addition, only the peak doses (20 m3/mL) of the industrial and urban sites were mutagenic. Samples with mutagenic potency in the Salmonella mutagenicity test have to be regarded as potentially carcinogenic for humans, depending on the sample type (Maron and Ames, 1983). Risks are considered highest in urban sites. Results obtained with the Vitotox test were inconsistent and difficult to interpret. This may be due to
toxicity at high doses (Table 4). Toxicity may be due to traces of organic solvents from the extraction procedure. Merely urban PM10 in suspension showed a dose– response curve that reaches a ratio of 1.5 and should be considered genotoxic. This is in agreement with results of the Salmonella mutagenicity test. The Comet assay also points to PM10 from urban origin as the most DNA-damaging sample for both particles in suspension and organic extracts. In addition, particles in suspension were toxic for the exposed leukocytes. Toxicity was neutralized by S9. However, dose-dependent relationships were not established, as has been reported before (Don Porto Carero et al., 2001). No differences were observed between particles from industrial and rural sources. The genotoxicity of PM10 originating from the urban site was obvious from results obtained by the Micronucleus test. A dose–response relationship was observed upon exposure of lymphocytes to the organic extract from urban particles. Exposure to particles from rural or industrial sites yielded no statistical differences with the Micronucleus test. Sufficient binuclear cells were only assessed on slides with cells exposed to organic extracts. Particles in suspension were toxic for lymphocytes; consequently, no micronuclei were counted. Chromosomal loss and chromosomal breakage can be measured reliably in the micronucleus test, which are important events in genotoxicity and are also associated with enhanced carcinogenic risks. Organic compounds adsorbed on particles from the urban source are potentially more carcinogenic than particles from the industrial and rural source. PM10 from the urban site was the most genotoxic, as shown by the four different in vitro assays. Linear dose– response relationships were only established with the Salmonella mutagenicity test and the Micronucleus test. Genotoxicity increased in the presence of S9. This finding is confirmed by other studies (Vinitketkumnuen et al., 2002).
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
116 20
µNed BN cells / 1000 BN cells
18
*
16 14 12
*
10 8 6 4 2 0
0m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL
20m³ air eq / mL
Fig. 4. Chromosomal damage assessed with the micronucleus assay after exposure to the organic extract of PM10 from the urban area. The number of binucleated cells with micronuclei on a total of 1000 binuclear cells is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with the Kastenbaum– Bowman test.
Organic compounds adsorbed on the particles are important for the genotoxic potency of PM10, as shown by parallel analysis of original particles and the corresponding organic extracts. This tendency can be confirmed by chemical PAH analysis, although the low PAH concentrations cannot explain the measured level of genotoxicity based on the knowledge of minimal effective genotoxic concentrations of individual PAHs (Mersch-Sundermann et al., 1994; Yaffe et al., 2001; White, 2002and unpublished data). Conclusively, genotoxicity of air particulate matter correlates with the amount of 16 EPA PAHs, although the chemical analyses are not predictive of the extent of genotoxic activity of the samples. Differences in genotoxic effects among sampling sites may be partly due to differences in PM10 concentration (mg/m3), which were highest in the urban site. Nevertheless, PM10 concentrations at the rural and industrial sites are comparable, although the noxious effect of industrial particles is more pronounced as seen in the Salmonella mutagenicity test. If standardized for equal concentration (mg/mL), urban particles are far more potent than industrial and rural particles in the Micronucleus test. This small-scale study suggests that the source-type determines the biological activity of the airborne particles, and traffic-related particles are most genotoxic. The abundant contribution of traffic exhaust to the cancer risk caused by exposure to ambient air is often discussed in literature (Lewtas et al., 1992). When screening test samples for genotoxic potency, it is recommended not to use a single in vitro test because different substances can utilize various mechanisms for causing a genotoxic effect. According to our achieved results, a combined application of the Salmonella mutagenicity test and the Micronucleus test can be recommended for the evaluation of airborne particu-
lates. The advantage of this test combination is the complete assessment of gene mutations, chromosomal mutations, as well as genome mutations. Furthermore, both a bacterial test and an assay on human cells was used. Despite the use of a battery of tests and the use of human cells, in vitro tests cannot give a definite answer on the carcinogenicity of PM10 for humans. Extrapolation of the results can lead to a hypothesis for risk assessment of exposure to air pollution. As indicated by this research, exposure to inhalable airborne particles, from urban origin, can cause genotoxic and potentially carcinogenic effects. These findings are in line with studies performed and described by other authors (Miguel et al., 1990; Bronzetti et al., 1997; Cˇerna´ et al., 2000; Buschini et al., 2001; Binkova´ et al., 2003; De Martinis et al., 1999; Zhao et al., 2002). Several epidemiological and human biomonitoring studies attempted to calculate the accountability of particle air pollution to cause lung cancer (Dockery et al., 1993; Cohen, 2000; Pope et al., 2002). Although important triggers for lung cancer, such as tobacco and diet, interfere with the measurements of such studies, it is clear that areas with high air pollution load have a significantly higher incidence of lung cancer compared to less-polluted areas. In vitro studies are useful for explaining and supporting hypotheses from epidemiological studies. On the other hand, in vitro studies are valuable when incorporated in routine monitoring of air pollution. These chemical measurements should be complemented with genotoxicity studies of inhalable airborne particles by in vitro test systems such as the bacterial Salmonella mutagenicity test and the Micronucleus test to indicate the potential hazard of airborne particles. Chemical PAH analysis alone is insufficient to predict the hazardous effect of PM10 on human cells.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
Acknowledgments The authors acknowledge the financial and technical support of the VMM (Flemish Environment Agency). We would like to thank Janssen Pharmaceutical for supplying S9, metabolic fraction.
References Binkova´, B., Cˇerna´, M., Pastorkova´, A., Jelı´ nek, R., Benesˇ , I., Nova´k, J., Sˇra´m, R.J., 2003. Biological activities of organic compounds adsorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000–2001. Mutat. Res. 525, 43–59. Bronzetti, G., Cini, M., Paoli, M., Ciacchini, G., Giaconi, V., Morichetti, E., 1997. Mutagenicity and chemical analysis of airborne particulate matter collected in Pisa. J. Environ. Pathol. Toxicol. Oncol. 16 (2&3), 147–156. Brunekreef, B., Holgate, S.T., 2002. Air pollution and health. Lancet 360, 1233–1242. Buschini, A., Cassoni, F., Anceschi, E., Pasini, L., Poli, P., Rossi, C., 2001. Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay. Chemosphere 44 (8), 1723–1736. Cˇerna´, M., Pastorkova´, A., Vrbı´ kova´, V., Sˇmı´ d, J., Ro¨ssner, P., 1999. Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic. Mutat. Res. 444, 373–386. Cˇerna´, M., Pochmanova´, D., Pastorkova´, A., Benesˇ , I., Lenı´ cˇek, I., Topinka, J., Binkova´, B., 2000. Genotoxicity of urban air pollutants in the Czech Republic Part I. Bacterial mutagenicity potencies of organic compounds absorbed on PM10 particulates. Mutat. Res. 469, 71–82. Claxton, L.D., Warren, S., Zweidinger, R., Creason, J., 2001. A comparative assessment of Boise, Idaho, ambient air fine particle samples using the plate and microsuspension Salmonella mutagenicity assays. Sci. Tot. Environ. 275 (1–3), 95–108. Cohen, A.J., 2000. Outdoor air pollution and lung cancer. Environ. Health Perspect. 108 (4), 743–750. De Martinis, B.S., Kado, N.Y., de Carvalho, L.R.F., Okamoto, R.A., Gundel, L.A., 1999. Genotoxicity of fractionated organic material in airborne particles from Sa˜o Paulo, Brazil. Mutat. Res. 446, 83–94. Dockery, D.W., Pope III, C.A., Xu, X., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris Jr., B.G., Speizer, F.E., 1993. An association between air pollution and mortality in six US cities. N. Engl. J. Med. 329, 1753–1759. Don Porto Carero, A., Hoet, P.H.M., Verschaeve, L., Schoeters, G., Nemery, B., 2001. Genotoxic effects of carbon black particles, diesel exhaust particles and urban air particulates and their extracts on a human alveolar epithelial cell line (A549) and a human monocytic cell line (THP-1). Environ. Mol. Mutagen. 37, 155–163. Fenech, M., 2000. The in vitro micronucleus test. Mutat. Res. 455, 81–95. Fenech, M., Morley, A.A., 1985. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Goldberg, M.S., Burnett, R.T., Bailar III, J.C., Brook, J., Bonvalot, Y., Tamblyn, R., Singh, R., Valois, M.F., Vincent, R., 2001. The association between daily mortality and ambient air particle pollution in Montreal, Quebec. 1. Nonaccidental mortality and 2. Cause-specific mortality. Environ. Res. 86 (1), 12–25 and 26–36. Goldsmith, C.A., Kobzik, L., 1999. Particulate air pollution and asthma: a review of epidemiological and biological studies. Rev. Environ. Health 14, 121–134.
117
Health Effects Institute, 2002. Understanding the Health Effects of Components of the Particulate Matter Mix: Progress and Next Steps. HEI Perspectives. Health Effects Institute, Boston, MA. Hornberg, C., Maciuleviciute, L., Seemayer, N.H., Kainka, E., 1998. Induction of sister chromatide exchanges in human tracheal epithelial cells by the fractions PM10 and PM2.5 of airborne particulates. Toxicol. Lett. 96/97, 215–220. Hsiao, W.L.W., Mo, Z.-Y., Fang, M., Shi, X.-M., Wang, F., 2000. Cytotoxicity of PM2.5 and PM2.5–10 ambient air pollutants assessed by the MTT and the Comet assays. Mutat. Res. 471, 45–55. IARC, 1987. Monographs on the evaluation of carcinogenic risk to humans, overall evaluations of carcinogenicity: an updating of IARC Monographs. International Agency for Research on Cancer, Lyon, 1–42 (7). Kastenbaum, M.A., Bowman, K.O., 1970. Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9, 549–552. Katsouyanni, K., Pershagen, G., 1997. Ambient air pollution exposure and cancer. Cancer Cause Control 8 (3), 284–291. Knaapen, A.M., Shi, T., Borm, P.J.A., Schins, R.P.F., 2002. Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter. Mol. Cell. Biochem. 234/235, 317–326. Lewtas, J., Lewis, C., Zweidinger, R., Stevens, R., Cupitt, L., 1992. Sources of genotoxicity and cancer risk in ambient air. Pharmacogenetics 2 (6), 288–296. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Mersch-Sundermann, V., Schneider, U., Klopman, G., Rosenkranz, H.S., 1994. SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds. Mutagenesis 9 (2), 205–224. Miguel, A.G., Daisey, J.M., Sousa, J.A., 1990. Comparative study of the mutagenic and genotoxic activity associated with inhalable particulate matter in Rio de Janeiro air. Environ. Mol. Mutagen. 15 (1), 36–43. Milieumaatschappij, V., 2002. Luchtkwaliteit in het Vlaamse gewest, 2001. VMM, Erembodegem. Monarca, S., Crebelli, R., Feretti, D., Zanardini, A., Fuselli, S., Filini, L., Resola, S., Bonardelli, P.G., Nardi, G., 1997. Mutagens and carcinogens in size-classified air particulates of a northern Italian town. Sci. Tot. Environ. 205 (2–3), 137–144. Nyberg, F., Gustavsson, P., Jarup, L., Bellander, T., Berglind, N., Jakobsson, R., Pershagen, G., 2000. Urban air pollution and lung cancer in Stockholm. Epidemiology 11 (5), 487–495. Pope III, C.A., Burnett, R.T., Thun, M.J., Calle, E.E., Krewski, D., Ito, K., Thurston, G.D., 2002. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. J. Am. Med. Assoc. 289 (9), 1132–1141. Salvi, S., Holgate, S.T., 1999. Mechanisms of particulate matter toxicity. Clin. Exp. Allergy 29, 1187–1194. Samet, J.M., Dominici, F., Curriero, F.C., Coursac, I., Zeger, S.L., 2000. Fine particulate air pollution and mortality in 20 US cities. N. Engl. J. Med. 343, 1742–1749. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184–191. Takafuji, S., Nakagawa, T., 2000. Air pollution and allergy. J. Invest. Allergol. Clin. Immunol. 10, 5–10. Verschaeve, L., Van Gompel, J., Thilemans, L., Regniers, L., Vanparys, P., van der Lelie, D., 1999. Vitotoxs bacterial genotoxicity and toxicity test for the rapid screening of chemicals. Environ. Mol. Mutagen. 33, 240–248. Vinitketkumnuen, U., Kalayanamitra, K., Chewonarin, T., Kamens, R., 2002. Particulate matter, PM 10 & PM 2.5 levels, and airborne
ARTICLE IN PRESS 118
E. Brits et al. / Environmental Research 96 (2004) 109–118
mutagenicity in Chiang Mai, Thailand. Mutat. Res. 519 (1–2), 121–131. White, P., 2002. The genotoxicity of priority polycyclic aromatic hydrocarbons in complex mixtures. Mutat. Res. 515, 85–98. Wieringa, M.H., Weyler, J., Nele, V., Van Hoof, K., Van Bastelaer, F.J., Van Sprundel, M., Vermeire, P., 1998. Prevalence of respiratory symptoms: marked differences within a small geographic area. Int. J. Epidemiol. 27, 630–635.
Yaffe, D., Cohen, Y., Arey, J., Grosovsky, A.J., 2001. Multimedia analysis of PAHs and nitro-PAH daughter products in the Los Angeles Basin. Risk Anal. 21, 275–294. Zhao, X., Wan, Z., Chen, G., Zhu, H., Jiang, S., Yao, J., 2002. Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China. Mutat. Res. 514, 177–192.
Environmental Research 96 (2004) 109–118
Genotoxicity of PM10 and extracted organics collected in an industrial, urban and rural area in Flanders, Belgium$ Ethel Brits,* Greet Schoeters, and Luc Verschaeve Vito (Flemish Institute for Technological Research), Department of Environmental Toxicology, Boeretang 200, 2400 Mol, Belgium Received 9 July 2003; received in revised form 11 March 2004; accepted 22 March 2004
Abstract The variation in the genotoxic potency of PM10 in vitro in relation to the particle source type was investigated. Particles were collected at one urban, one rural, and one industrial site in Flanders. Genotoxicity was assessed using four different in vitro test systems exposed to PM10 in suspension and to the organic extracts of PM10. Two of these systems were bacterial assays: the Salmonella mutagenicity test and the Vitotox test. In addition, the Comet assay and Micronucleus test were performed using human blood cells. Results show that exposure to PM10 and the organic extracts from both urban and industrial areas causes significant genetic damage. The Salmonella mutagenicity test was most suitable for the screening of PM10 and the organic extracts; the Micronucleus test was most suitable only for the screening of organic extracts, and original particles were toxic for the exposed lymphocytes. Clear dose–response curves were not established in the Comet and Vitotox assay, and organic extracts were apparently toxic in the latter. The total polycyclic aromatic hydrocarbon content of the organic extracts, as measured with GC/MS, ranged between 1 and 6 ng/m3. Results obtained in this study suggest that PM10 causes DNA damage and mutations. The use of biological tests for the screening of air samples is useful to complement air quality control by chemical measurements. r 2004 Elsevier Inc. All rights reserved. Keywords: PM10; PAH; Genotoxicity; Mutagenicity; In vitro
1. Introduction Airborne particulate matter, currently considered a major air pollutant, causes adverse health effects (Brunekreef and Holgate, 2002). Epidemiological studies in US and European cities have demonstrated an association between PM10 pollution episodes and increased acute and chronic respiratory morbidity (Goldsmith and Kobzik, 1999; Takafuji and Nakagawa, 2000), increased mortality (Dockery et al., 1993; Samet et al., 2000; Goldberg et al., 2001), and increased lung cancer risk (Katsouyanni and Pershagen, 1997; Cohen, 2000; Nyberg et al., 2000; Pope et al., 2002). Adverse health effects are mainly ascribed to inhalable particles with an aerodynamic diameter of less than 10 mM (PM10) (Hornberg et al., 1998; Salvi and Holgate, 1999; Hsiao et al., 2000). Differences in response have $ This study was performed in order of and supported by the VMM, Flemish Environment Agency. *Corresponding author. Fax: +32-14-58-26-57. E-mail address: [email protected] (E. Brits).
0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.03.006
been observed among cities; however, epidemiological studies are not sensitive enough to differentiate in composition of the particles. To explore the variation in toxic potency of the particles in relation to their composition and origin, additional toxicological studies are needed. A better understanding of the underlying toxic mechanisms of PM10 will allow more effective measurement to reduce adverse health effects associated with particulate matter (Health Effects Institute, 2002). PM10 proved to be genotoxic in diverse test systems (De Martinis et al., 1999; Cˇerna´ et al., 2000; Buschini et al., 2001; Binkova´ et al., 2003). This may be partly explained by organic substances adsorbed on the particle surface, such as polycyclic aromatic hydrocarbons (PAHs), nitro-polycyclic aromatic compounds, dioxins, and metals. The relative contribution of these pollutants to PM10 may depend on the particle sources and meteorological conditions. Therefore, the spatial and temporal monitoring of PM10 as a genotoxic pollutant is considered important for the evaluation of health risks for populations in polluted areas. This hazard may depend on local factors such as industrial
ARTICLE IN PRESS 110
E. Brits et al. / Environmental Research 96 (2004) 109–118
emissions, traffic exhaust, and domestic and agricultural emissions. Until now, PM10 monitoring in Flanders was restricted to the measurement of particle concentrations and sporadically PM10 is chemically characterized (Milieumaatschappij, 2002). This approach is insufficient for hazard identification, considering the complexity of PM10. In this study, PM10 air samples and organic extracts of PM10 were characterized for their genotoxic potency. The particles were collected at three monitoring sites: Antwerp, an urban site characterized by high traffic pollution and relatively moderate industrial pollution; Berendrecht, a site with high industrial pollution and moderate traffic pollution; and Peer, a rural site with moderate overall pollution. PM10 and the organic extracts were evaluated for genotoxicity using a battery of four different bioassays, including two bacterial tests and two assays using human leukocytes as target cells. The bioassays used for measuring the genotoxic activity of PM10 and the derived organic extracts were selected to evaluate different, but complementary genotoxic endpoints: SOS repair in response to DNA damage (Vitotox test); DNA damage (Alkaline single-cell gel electrophoresis or Comet assay); gene mutations (Salmonella mutagenicity test); chromosome/genome mutations (Micronucleus test). The Ames Salmonella typhimurium assay is a widely accepted bacterial assay, appropriate to detect mutagens in airborne particulates (Monarca et al., 1997; Bronzetti et al., 1997; De Martinis et al., 1999; Cˇerna´ et al., 2000). Recently, new bacterial tests have been developed for the detection of DNA-damaging agents, including the Vitotox test, a short-term assay used in this study. The Vitotox test measures DNA damage as a result of SOS induction and has shown to be very sensitive at detecting genotoxic activity of pure compounds, but not yet at detecting complex environmental mixtures (Verschaeve et al., 1999). The alkaline Comet assay, applied on human leukocytes, measures DNA strand breaks in alkali-labile sites of individual cells and is often used for environmental monitoring (Don Porto Carero et al., 2001; Buschini et al., 2001; Knaapen et al., 2002). The Micronucleus test on human lymphocytes detects structural and numerical chromosomal aberrations; it has been included in this study as an easier and more rapid alternative to the conventional chromosomal aberration test (Fenech, 2000). The organic extracts of PM10 samples were chemically analyzed for their PAH content using gas chromatography and mass spectrometry (GC/MS). The aim of the study was to compare the genotoxic potency of PM10 from different sites. Additionally, the organic extracts derived from PM10 were tested in comparison with the original particles to evaluate whether the genotoxicity is attributed to the particles
and/or the adsorbed organic compounds. The predictive value of chemical PAH analysis for genotoxic characterization of airborne particles was examined. Based on the whole evaluation, the suitability of the selected bioassays for the screening of PM10 was assessed.
2. Materials and methods 2.1. Air sampling and sample preparation PM10 samples were collected at three sample sites in Flanders. The selected sites are included in the PM10 monitoring network of the Flemish Environmental Agency (VMM), and epidemiological data concerning the prevalence of asthma were available (Wieringa et al., 1998). At the urban site (Borgerhout, the city center of Antwerp), the sampling point was located at a distance of 15 m from a street with high traffic density. The industrial site (Berendrecht) is located at the North of Antwerp in close proximity to a chemical industry and the harbor. The sampling site was located near the motorway in a green environment, yet within a 2-km radius of the industry. The rural site (Peer) is a small, green village in the Province of Limburg. The sampling site was located in a sports park, 2 km outside the center of the village. Particles were collected on preweighed Teflon-coated glass fiber filters (Emfab, Pallflex, Gelman) using a Pourbaix low-volume sampler provided by the Flemish Environment Agency (VMM), during 2 weeks in September 2000. The sampling flow was 0.9 m3/h. The volume of filtered air was 358 m3. Gravimetric analysis of the particulates was standardized by conditioning the filters for 48 h at 25.5 C and 50% humidity before and after PM10 collection. PM10-containing filters were cut into four equal parts, and pooled per fourth and per site. The particle suspension was made by shaking two parts (2/4) of the filters overnight at 37 C in sterile, bidistilled water supplemented with 0.1% Triton X-100 (Sigma). Subsequently, filters were removed from the suspension and sterile bidistilled water supplemented with 0.1% Triton X-100 was added to obtain a suspension of particles per milliliter from 20 m3-filtered air. Another one-fourth was extracted with Dionex ASE 200 Accelerated Solvent Extraction in tetrahydrofurane/n-hexane (20/80) at a pressure of 140 bar and a temperature of 100 C. The solvent was evaporated under N2. The residual matter was redissolved in 10% dimethyl sulfoxide (DMSO) in water to obtain 20 m3 of air volume equivalents per 1 mL of extract volume. Another one-fourth was identically extracted with ASE, but the samples were spiked with 120 mL internal standard. These samples were chemically analyzed with GC/MS as described below.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
2.2. Chemical analysis The PAH concentrations were determined by GC/MS in the extracts. The analysis was carried out using a Carlo Erba MFC 500 capillary GC, Carlo Erba A200S autosampler, and Fisons QMD 1000 low-resolution mass spectrometer. The GC/MS system was equipped with a 30 m 0.25 mm 0.25 mm DB-5MS fused silica GC column. Helium carrier gas was used at a flow rate of 1 mL/min. The temperature program consisted of an isothermal hold at 125 C for 1 min, an increase of 20 C/ min to 205 C and a second increase of 10 C/min to 305 C followed by a 15-min isothermal hold. 2.3. Genotoxicity assays 2.3.1. Salmonella mutagenicity test The Salmonella plate incorporation test was performed according to Maron and Ames (1983). S. typhimurium strain TA98 was kept at 80 C until use. One-hundred microliters of the TA98 strain was added to a 50-mL Erlenmeyer, containing 20 mL of Nutrient Broth growth medium (25 g/L Nutrient Broth No. 2 (Oxoid) in sterile distilled water). Two mg/mL 4-NQO and 20 mg/mL benzo(a)pyrene in the presence of a metabolic activation system were used as positive control. As a metabolic system, S9 was used, and it was stored at 80 C until use. Prior to use, the S9 was combined with an Ames mutagenicity test tablet (Boehringer) dissolved in 18 mL sterile distilled water containing the necessary cofactors such as NADP and G-6-P. The S9 fraction was 10% of the S9-mix volume. The test was performed in plates containing two layers of agar, the bottom agar (minimal glucose plate, Oxoid) for providing a suitable support media and the top agar (0.6% agar, Difco and 0.5% NaCl) for applying the test chemical, metabolic activation reagent if required, and the tester strain. Two milliliters of the top agar with histidine/biotin (Sigma) (0.05 mM), S9 mix, or phosphate buffer (0.5 mL), test chemicals (100 mL), and the desired tester strain (100 mL) was poured over a minimal glucose agar plate. Per dose, three replicate plates were made, five replicates for the negative control. Plates were incubated at 37 C for 48 h. Colonies on each plate were counted. Results were expressed as the number of revertants in function of the mass of PM10 and volume of air sampled. The results were evaluated using one-way ANOVA combined with the Tukey post hoc test (Statistica, Statsoft). The level of significance was set at Po0:05: 2.3.2. Vitotox test The Vitotox test was performed according to Verschaeve et al. (1999). The test is based on S. typhimurium TA104 recN2-4 bacteria that contain
111
the lux operon of Vibrio fischeri under transcriptional control of the recN gene, which is part of the SOS system. After incubation of the bacteria with a genotoxic compound, the recN promoter was derepressed, resulting in expression of the lux operon, and consequently, the expression of light production in function of genotoxicity. As an internal control system, the S. typhimurium strain TA104pr1 strain was used. This is a constitutive light-producing strain with a lux operon under control of the strong promoter pr1. S. typhimurium strains TA104recN2-4 and TA104pr1 were kept at 80 C until use. Twenty microliters of each strain (TA104recN2-4 and TA104pr1) was added to 5 mL of a normal bacterial growth medium. Bacterial cultures were then incubated overnight in an Innova 4000 (New Brunswick Scientific) rotative incubator shaker at 250 rpm and at 37 C. Cultures were then diluted 10-fold. Fifty microliters of each strain (TA104recN2-4 and TA104pr1) of the overnight culture was added to 2.5-mL growth medium. Bacterial cultures were then incubated on an Innova 4000 environmental shaker at 250 rpm and at 37 C (1 h) to obtain log phase growth. 4-NQO was used as positive control for the test without metabolic activation in a final concentration of 4 pg/mL and benzo(a)pyrene in the presence of a metabolic activation system in a final concentration of 8 mg/mL. The S9 was stored at 80 C until use. Prior to use, the S9 was combined with NADP and G-6-P (Sigma). The S9 fraction was 10% of the S9-mix volume. In the final measurement plate, this was again 10-fold diluted to a 1% final solution. The samples without S9mix were provided with phosphate buffer to keep the number of bacteria the same. In each well of the microtiter plate, 90 mL of the 1 h culture was mixed with 10 mL of the test compound. Luminescence was measured with a Microlumat LB96B luminometer (EG&G Berthold) or with a Luminoskan Ascent (Labsystems) including the following parameters: 1 s/well; cycle time=5 min; 60 cycles; incubation temperature=30 C. The signal-to-noise ratio (S/N), being the light production of exposed cells divided by the light production of nonexposed cells, was calculated for each measurement. A test compound was considered genotoxic when the max S/N (recN2-4)/max S/N (pr1)41.5, a clear dose–response curve was generated, and the signal was not generated in the first 20 min.
2.3.3. Comet assay The Comet assay or single-cell gel electrophoresis assay using human leukocytes was performed as described by Singh et al. (1988), with slight modifications. Human whole blood was exposed to three dilutions of the organic extract for 24 h at 37 C in a rotative shaker, with and without S9.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
112
Microscopic slides were coated with 80 mL of 1% normal melting point agarose (Sigma). When this layer had solidified, a second layer containing 5-mL whole blood sample mixed with 300 mL of 0.8% low melting point agarose (Sigma) was placed on the slides. After 5 min of solidification on ice, slides were immersed for minimum 1 h in ice-cold freshly prepared lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 1.2% Tris) with 1% Triton X-100 and 10% DMSO added fresh to lyse cells and to allow DNA unfolding. The slides were then placed on a horizontal gel electrophoresis tank. The unit was filled with electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, 0.1% 8-hydroxyquinoline, 2% DMSO, 17 C) and the slides were placed in this alkaline buffer for 40 min to allow DNA denaturation. Electrophoresis was carried out for 20 min at 1 V/cm (300 mA). After electrophoresis, the slides were rinsed gently three times with neutralization buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 mg/mL) and covered with a coverslip. Slides were stored at 4 C in sealed boxes until analysis. A total of 50 randomly captured comets from each slide were examined at 400 magnification using an epifluorescence microscope (Zeiss). Measurements were performed with a computerized image analysis system (Komet 3.1, Kinetic Imaging Ltd., Liverpool, UK). To quantify DNA damage, the percentage of DNA content in the comet tail was measured. The results were evaluated using the nonparametric Median test (Statistica, Statsoft). The level of significance was set at Po0:05: 2.3.4. Micronucleus test The protocol was used as described in Fenech and Morley (1985) with slight modifications. Supplemented medium was prepared by adding 15% fetal calf serum (Greier), 2% phytohemagglutinin (Sigma), 1% glutamine (Sigma), and 1% penicillin–streptomycin (Gibco) to culture medium RPMI 1640 (Gibco). The supplemented medium was filtered through a 0.22-mm filter. Whole human blood (0.5 mL) was added to 5 mL of the medium and 50 mL of the organic extract (in three dilutions) and cultivated at 37 C for 44 h. To stop the cell cycle in the binuclear stage, 100 mL of cytochalasine B (Sigma) (0.6 mg/mL) was added. The lymphocytes were further cultivated at 37 C for 28 h. After centrifugation (1200 rpm for 10 min), the pellet was resuspended
in 5 mL hypotonic solution 0.075 M KCl. The cell suspension was centrifuged again at 1200 rpm for 10 min, the cells in the pellet were fixed in 5 mL methanol and acetic acid 3:1, and three drops of 37% formaldehyde were added. Following centrifugation, the pellet was resuspended in 5 mL and fixed in three steps. Finally, the pellet was resuspended in 4 mL of fixator, this cell suspension was stored at 20 C for at least 16 h. Then, the cells were sedimented at 1200 rpm for 10 min, supernatant was discarded, and the pellet was resuspended in 2 mL fixator, and sedimented again. Microscopic slides were prepared in duplicate from this final cell suspension, air-dried, and stained with 50% May–Grunwald (Sigma), following a 10% Giemsa (Sigma) solution. One thousand binucleated cells were counted on each slide. Data were statistically analyzed with the Kastenbaum–Bowman test (Kastenbaum and Bowman, 1970).
3. Results 3.1. PM10 and PAHs Concentrations of PM10 ranged between 10 and 30 mg/m3 and were highest in the urban area (Table 1). The concentrations of 17 PAHs measured by GC/MS, present in the organic extracts, were also highest in the urban area (Table 2). Eight of the measured PAHs are listed as IARC recognized carcinogenic PAHs, namely benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, Chrysene, dibenzo(a,h)anthracene, and indeno(1,2,3, c,d)pyrene (IARC, 1987). The carcinogenic PAHs contribute largely to the total amount of chemically analyzed PAHs: 20% for the samples from the industrial origin, 33% from the rural origin, and 45% for the urban origin. 3.2. Salmonella mutagenicity test Without addition of S9, only the highest concentration of urban and industrial extracts were mutagenic, particles and rural extracts were not mutagenic. In the presence of S9, extracts and particles of urban (Fig. 1), industrial, and rural areas tested mutagenic in all areas (Table 3).
Table 1 Data on sampling: sample period, total volume of filtered air, total amount of PM10 collected and mean sampled concentration (mg/m3) Area
City name
Sample period
Volume (m3)
PM10 collected (mg)
PM10 (mg/m3)
Urban Rural Industrial
Borgerhout Peer Berendrecht
Sept. 9–25, 2000 Sept. 9–25, 2000 Sept. 9–25, 2000
358 356 346
10.4 5.4 4.5
30 15 13
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
113
3.3. Vitotox test
3.4. Comet assay
Table 4 shows the results of the genotoxicity of PM10 and the extracts using S. typhimurium TA 104 recN 2-4 and pr1 tester strains. The organic extracts of PM10 were toxic at the highest doses. This toxicity can be due to traces of solvents used for the extraction procedure. Merely the response curve of PM10 from the urban area reaches the 1.5 ratio, and is consequently considered genotoxic. Fig. 2 graphically illustrates the results of organic extracts from particles collected in the urban area.
A 24-h exposure of both particles and organic extracts from each area seemed to cause significant DNA damage; however, no clear dose–response relationships were established (Table 5). Particles from the urban area appeared toxic at the highest dose, S9 detoxified the sample. Overall, addition of S9 enhances the DNAdamaging effect. Fig. 3 demonstrates the results of organic extracts of particles collected in the urban area. 3.5. Micronucleus test
Table 2 Concentrations of 17 different PAHs (ppm) measured by GC/MS in organic extracts of filter samples of all areas PAHs
Urban (ng/mL)
Rural (ng/mL)
Industrial (ng/mL)
Acenaphtene Acenaphtylene Anthracene Benzo(a)anthracene Benzo(a)pyrene Benzo(b)fluoranthene Benzo(e)pyrene Benzo(g,h,i)perylene Benzo(k)fluoranthene Chrysene Dibenzo(a,h)anthracene Fluoranthene Fluorene Indeno(1,2,3,c,d)pyrene Naphtalene Phenantrene Pyrene
21.5 0 4.6 10.7 6.1 18.4 12.3 15.3 4.6 12.3 0 12.3 7.6 7.7 0 16.9 12.2
19.44 0 2.43 4.05 1.62 6.48 2.43 2.43 1.62 2.43 0 4.05 4.05 1.62 0 0.81 4.86
12.96 10.53 7.29 4.05 0.81 5.67 2.43 2.43 1.62 2.43 0 3.24 4.05 1.62 0 12.15 12.15
Total
162.5 ng/mL 5.5 ng/m3 air
58.3 ng/mL 2 ng/m3 air
83.4 ng/mL 2.8 ng/m3 air
Note: The data were adjusted for the trace amounts of PAHs detected in the blank filter extracts. PAHs marked with an () are carcinogenic PAHs (group 2A or 2B) according to IARC (1987).
Following exposure to particles in suspension, insufficient binuclear cells were generated due to toxicity. Hence, no data are shown for particle exposure conditions. PM10 extracts were analyzed at the highest Table 3 Mutagenicity of PM10 tested with the Salmonella mutagenicity test and the organic extracts from three areas with and without addition of S9 Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 2.5 5 10 20
19.2 17.3 18.3 19.3 25
19.8 19 19.7 23 27.7
19.2 19 20.7 17.3 26
19.8 20.7 20 24.3 24.3
19.2 18 18 22.7 26
19.8 18.3 19 20.7 26.3
0 2.5 5 10 20
23.8 29.3 30.3 40.3 48.7
23 31 35.3 43.3 48.7
23.8 22.3 27 32 42.3
23 25 26.7 29 33.7
23.8 29 33 37 45.7
23 24.3 28 32.3 38.7
The mean number of revertants (mean of three replicas) is shown in relation to the concentration of the organic extract (rev/m3 air eq). Concentration 0 m3 air eq/mL is the suspension or organic extracts of blank filters, uniformly treated as the loaded filters. Data printed in bold are significantly (Po0:05) different from the blank (one-way ANOVA, combined with the Tuckey Post Hoc test).
60
Nr of revertants
50 40
*
*
*
*
30 20 10 0 0m³ air eq / mL
2.5m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL 20m³ air eq / mL
Fig. 1. Mutagenicity of organic extracts of PM10 from the urban area with addition of S9, tested with the Salmonella mutagenicity test. The mean number of revertants (mean of three replicas) is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq/mL is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with a one-way ANOVA, combined with the Turkey post hoc test.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
114
Table 4 Data on genotoxicity in the Vitotox test of PM10 and the organic extracts from three areas with and without addition of S9 Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 2.5 5 10 20
1.0 1.3 1.4 1.4 1.5
1.1 1.1 1.1 1.2 TOX
0.98 1.0 1.2 1.2 1.3
1.1 1.0 1.1 1.3 TOX
0.98 1.2 1.0 1.0 0.9
1.1 1.1 1.2 TOX TOX
0 2.5 5 10 20
1.0 1.1 1.1 1.2 1.5
1.1 1.0 1.0 TOX TOX
1.0 1.0 1.1 1.2 1.3
1.1 1.2 1.2 TOX TOX
1.0 1.0 1.0 0.9 1.0
1.1 1.0 1.1 1.4 TOX
Note: The max S/N (recN2-4)/max S/N (pr1) is shown for each condition. Concentration 0 m3 air eq/mL is the suspension or extract of blank filters. Data printed in bold are considered genotoxic, at this condition the max S/N (recN2-4)/max S/N (pr1) reaches 1.5. TOX means the max S/N (recN2-4)/max S/N (pr1) reaches below 0.8 and the condition is toxic for the bacteria.
RecN 2-4
2
SIGNAL/NOISE
Urban
Rural
Industrial
Particles
Extracts
Particles
Extracts
Particles
Extracts
0 5 10 20
1.6 1.8 3.9 TOX
1.4 1.7 2.5 3.2
1.6 3 3.2 1.8
1.4 1.5 3.4 2.2
1.6 2.1 1.9 2.2
1.4 2 3 2
0 5 10 20
1.9 2.8 2.0 4.6
1.9 1.5 4.1 4.8
1.9 2.6 3.3 2.4
1.9 1.9 2.1 2.0
1.9 2.2 2.6 2.4
1.9 1.8 3.1 3.0
Note: The % tail-DNA number (median of 50 cells) of each exposure condition is presented. Concentration 0 m3 air eq/mL is the suspension or extract of blank filters. Data printed in bold are significantly (Po0.05) different from the blank, assessed with the non-parametric Median test.
dose (20 m3/mL) (Table 6). If the sample exposure showed a significantly increased micronucleated cell frequency, further dilutions were examined. A significantly increased number of micronucleated binuclear cells was found exclusively as a result of exposure to the organic extract of PM10 collected in the urban area, a clear dose–response relationship was found (Fig. 4). The protocol was only performed without addition of S9.
2.5
1.5
1
4. Discussion
0.5
0 0
60
120
180
240
TIME(min)
Pr1
2.5
2
SIGNAL/NOISE
Table 5 Results of DNA damage measured with the Comet assay after 24 h exposure of human whole blood to PM10 and the corresponding organic extracts from three areas with and without addition of S9
1.5
1
0.5
0 0
60
120
180
240
TIME(min) Fig. 2. Genotoxicity in the Vitotox test of the organic extracts of PM10 from the urban area with addition of S9. The signal/noise ratio (light production of exposed/nonexposed bacteria) of the recN2-4 and pr1 strain is shown in relation to the time (min) for all four tested concentrations of the organic extract (m3 air eq/mL). Compounds are considered toxic when S/N decreases below 0.8. Concentration 0 m3 air eq is the extract of blank filters. (J) 2.5 m3 air eq/mL, (&) 5 m3 air eq/mL, (}) 10 m3 air eq/mL, and (n) 20 m3 air eq/mL.
The major objective of this study was to investigate the genotoxic potency of both ambient PM10 and the adsorbed organic compounds on PM10, collected at three monitoring sites (2-week periods): Antwerp, an urban site with high traffic pollution, Berendrecht, a site with industrial pollution, and Peer, a rural site. Furthermore, the predictive value of standard chemical PAH analysis for the toxicological effect of PM10 was evaluated. For these purposes, PM10 samples and the organic extracts of PM10 were screened by four genotoxicity tests: the Salmonella mutagenicity test, qthe Vitotox test, the Comet assay, and the Micronucleus test. Comparison of the PAH concentrations of the three sites shows considerable differences with the urban site having the highest concentration (ng/m3) for PAHs and the carcinogenic PAHs in particular (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo ðg; h; iÞperylene; benzo(k)fluoranthene, chrysene, dibenzo(a,h)anthracene and indeno(1,2,3,c,d)pyrene; IARC, 1987). The Salmonella mutagenicity test was performed using the S. typhimurium TA98 strain, a sensitive strain
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118 5
115
*
% DNA in tail
4
*
3 2 1 0
0m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL
20m³ air eq / mL
Fig. 3. DNA damage measured with the Comet assay as a result of exposure to the organic extract of PM10 from the urban area with addition of S9. The % tail-DNA number (median of 50 cells) is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with the nonparametric Median test.
Table 6 Number of binucleated cells with micronuclei on a total of 1000 binuclear cells Extracts
0 5 10 20
Urban
Rural
Industrial
3 3 9 16
3 — — 2
3 — — 2
Note: Human whole blood was exposed to PM10 and the corresponding organic extracts from three areas. Concentration 0 m3 air eq/ml is the extract of blank filters. Data printed in bold are significantly (Po0:05) different from the blank (Kastenbaum–Bowman test).
for analysis of air pollutants (Bronzetti et al., 1997; Cˇerna´ et al., 1999; Zhao et al., 2002; Binkova´ et al., 2003). Another frequently used strain, TA100, is valuable for screening purposes as well, but former research (unpublished) showed that this strain adds little for the investigation of airborne particulates; results are comparable to those achieved with assessment by the TA98 strain (Claxton et al., 2001). The Salmonella mutagenicity test could assign—essentially equal—mutagenic potency to all three samples. When S9 was added for metabolization, exposure to urban particles produced the most revertants, and exposure to rural particles produced the least revertants. Organic extracts of the corresponding particles yielded the same pattern, but the differences between the sites were more pronounced. Without S9 addition, only the peak doses (20 m3/mL) of the industrial and urban sites were mutagenic. Samples with mutagenic potency in the Salmonella mutagenicity test have to be regarded as potentially carcinogenic for humans, depending on the sample type (Maron and Ames, 1983). Risks are considered highest in urban sites. Results obtained with the Vitotox test were inconsistent and difficult to interpret. This may be due to
toxicity at high doses (Table 4). Toxicity may be due to traces of organic solvents from the extraction procedure. Merely urban PM10 in suspension showed a dose– response curve that reaches a ratio of 1.5 and should be considered genotoxic. This is in agreement with results of the Salmonella mutagenicity test. The Comet assay also points to PM10 from urban origin as the most DNA-damaging sample for both particles in suspension and organic extracts. In addition, particles in suspension were toxic for the exposed leukocytes. Toxicity was neutralized by S9. However, dose-dependent relationships were not established, as has been reported before (Don Porto Carero et al., 2001). No differences were observed between particles from industrial and rural sources. The genotoxicity of PM10 originating from the urban site was obvious from results obtained by the Micronucleus test. A dose–response relationship was observed upon exposure of lymphocytes to the organic extract from urban particles. Exposure to particles from rural or industrial sites yielded no statistical differences with the Micronucleus test. Sufficient binuclear cells were only assessed on slides with cells exposed to organic extracts. Particles in suspension were toxic for lymphocytes; consequently, no micronuclei were counted. Chromosomal loss and chromosomal breakage can be measured reliably in the micronucleus test, which are important events in genotoxicity and are also associated with enhanced carcinogenic risks. Organic compounds adsorbed on particles from the urban source are potentially more carcinogenic than particles from the industrial and rural source. PM10 from the urban site was the most genotoxic, as shown by the four different in vitro assays. Linear dose– response relationships were only established with the Salmonella mutagenicity test and the Micronucleus test. Genotoxicity increased in the presence of S9. This finding is confirmed by other studies (Vinitketkumnuen et al., 2002).
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
116 20
µNed BN cells / 1000 BN cells
18
*
16 14 12
*
10 8 6 4 2 0
0m³ air eq / mL
5m³ air eq / mL
10m³ air eq / mL
20m³ air eq / mL
Fig. 4. Chromosomal damage assessed with the micronucleus assay after exposure to the organic extract of PM10 from the urban area. The number of binucleated cells with micronuclei on a total of 1000 binuclear cells is shown in relation to the concentration of the organic extract (m3 air eq/mL). Concentration 0 m3 air eq is the extract of blank filters. () Means significantly (Po0:05) different from the blank, assessed with the Kastenbaum– Bowman test.
Organic compounds adsorbed on the particles are important for the genotoxic potency of PM10, as shown by parallel analysis of original particles and the corresponding organic extracts. This tendency can be confirmed by chemical PAH analysis, although the low PAH concentrations cannot explain the measured level of genotoxicity based on the knowledge of minimal effective genotoxic concentrations of individual PAHs (Mersch-Sundermann et al., 1994; Yaffe et al., 2001; White, 2002and unpublished data). Conclusively, genotoxicity of air particulate matter correlates with the amount of 16 EPA PAHs, although the chemical analyses are not predictive of the extent of genotoxic activity of the samples. Differences in genotoxic effects among sampling sites may be partly due to differences in PM10 concentration (mg/m3), which were highest in the urban site. Nevertheless, PM10 concentrations at the rural and industrial sites are comparable, although the noxious effect of industrial particles is more pronounced as seen in the Salmonella mutagenicity test. If standardized for equal concentration (mg/mL), urban particles are far more potent than industrial and rural particles in the Micronucleus test. This small-scale study suggests that the source-type determines the biological activity of the airborne particles, and traffic-related particles are most genotoxic. The abundant contribution of traffic exhaust to the cancer risk caused by exposure to ambient air is often discussed in literature (Lewtas et al., 1992). When screening test samples for genotoxic potency, it is recommended not to use a single in vitro test because different substances can utilize various mechanisms for causing a genotoxic effect. According to our achieved results, a combined application of the Salmonella mutagenicity test and the Micronucleus test can be recommended for the evaluation of airborne particu-
lates. The advantage of this test combination is the complete assessment of gene mutations, chromosomal mutations, as well as genome mutations. Furthermore, both a bacterial test and an assay on human cells was used. Despite the use of a battery of tests and the use of human cells, in vitro tests cannot give a definite answer on the carcinogenicity of PM10 for humans. Extrapolation of the results can lead to a hypothesis for risk assessment of exposure to air pollution. As indicated by this research, exposure to inhalable airborne particles, from urban origin, can cause genotoxic and potentially carcinogenic effects. These findings are in line with studies performed and described by other authors (Miguel et al., 1990; Bronzetti et al., 1997; Cˇerna´ et al., 2000; Buschini et al., 2001; Binkova´ et al., 2003; De Martinis et al., 1999; Zhao et al., 2002). Several epidemiological and human biomonitoring studies attempted to calculate the accountability of particle air pollution to cause lung cancer (Dockery et al., 1993; Cohen, 2000; Pope et al., 2002). Although important triggers for lung cancer, such as tobacco and diet, interfere with the measurements of such studies, it is clear that areas with high air pollution load have a significantly higher incidence of lung cancer compared to less-polluted areas. In vitro studies are useful for explaining and supporting hypotheses from epidemiological studies. On the other hand, in vitro studies are valuable when incorporated in routine monitoring of air pollution. These chemical measurements should be complemented with genotoxicity studies of inhalable airborne particles by in vitro test systems such as the bacterial Salmonella mutagenicity test and the Micronucleus test to indicate the potential hazard of airborne particles. Chemical PAH analysis alone is insufficient to predict the hazardous effect of PM10 on human cells.
ARTICLE IN PRESS E. Brits et al. / Environmental Research 96 (2004) 109–118
Acknowledgments The authors acknowledge the financial and technical support of the VMM (Flemish Environment Agency). We would like to thank Janssen Pharmaceutical for supplying S9, metabolic fraction.
References Binkova´, B., Cˇerna´, M., Pastorkova´, A., Jelı´ nek, R., Benesˇ , I., Nova´k, J., Sˇra´m, R.J., 2003. Biological activities of organic compounds adsorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000–2001. Mutat. Res. 525, 43–59. Bronzetti, G., Cini, M., Paoli, M., Ciacchini, G., Giaconi, V., Morichetti, E., 1997. Mutagenicity and chemical analysis of airborne particulate matter collected in Pisa. J. Environ. Pathol. Toxicol. Oncol. 16 (2&3), 147–156. Brunekreef, B., Holgate, S.T., 2002. Air pollution and health. Lancet 360, 1233–1242. Buschini, A., Cassoni, F., Anceschi, E., Pasini, L., Poli, P., Rossi, C., 2001. Urban airborne particulate: genotoxicity evaluation of different size fractions by mutagenesis tests on microorganisms and comet assay. Chemosphere 44 (8), 1723–1736. Cˇerna´, M., Pastorkova´, A., Vrbı´ kova´, V., Sˇmı´ d, J., Ro¨ssner, P., 1999. Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic. Mutat. Res. 444, 373–386. Cˇerna´, M., Pochmanova´, D., Pastorkova´, A., Benesˇ , I., Lenı´ cˇek, I., Topinka, J., Binkova´, B., 2000. Genotoxicity of urban air pollutants in the Czech Republic Part I. Bacterial mutagenicity potencies of organic compounds absorbed on PM10 particulates. Mutat. Res. 469, 71–82. Claxton, L.D., Warren, S., Zweidinger, R., Creason, J., 2001. A comparative assessment of Boise, Idaho, ambient air fine particle samples using the plate and microsuspension Salmonella mutagenicity assays. Sci. Tot. Environ. 275 (1–3), 95–108. Cohen, A.J., 2000. Outdoor air pollution and lung cancer. Environ. Health Perspect. 108 (4), 743–750. De Martinis, B.S., Kado, N.Y., de Carvalho, L.R.F., Okamoto, R.A., Gundel, L.A., 1999. Genotoxicity of fractionated organic material in airborne particles from Sa˜o Paulo, Brazil. Mutat. Res. 446, 83–94. Dockery, D.W., Pope III, C.A., Xu, X., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris Jr., B.G., Speizer, F.E., 1993. An association between air pollution and mortality in six US cities. N. Engl. J. Med. 329, 1753–1759. Don Porto Carero, A., Hoet, P.H.M., Verschaeve, L., Schoeters, G., Nemery, B., 2001. Genotoxic effects of carbon black particles, diesel exhaust particles and urban air particulates and their extracts on a human alveolar epithelial cell line (A549) and a human monocytic cell line (THP-1). Environ. Mol. Mutagen. 37, 155–163. Fenech, M., 2000. The in vitro micronucleus test. Mutat. Res. 455, 81–95. Fenech, M., Morley, A.A., 1985. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Goldberg, M.S., Burnett, R.T., Bailar III, J.C., Brook, J., Bonvalot, Y., Tamblyn, R., Singh, R., Valois, M.F., Vincent, R., 2001. The association between daily mortality and ambient air particle pollution in Montreal, Quebec. 1. Nonaccidental mortality and 2. Cause-specific mortality. Environ. Res. 86 (1), 12–25 and 26–36. Goldsmith, C.A., Kobzik, L., 1999. Particulate air pollution and asthma: a review of epidemiological and biological studies. Rev. Environ. Health 14, 121–134.
117
Health Effects Institute, 2002. Understanding the Health Effects of Components of the Particulate Matter Mix: Progress and Next Steps. HEI Perspectives. Health Effects Institute, Boston, MA. Hornberg, C., Maciuleviciute, L., Seemayer, N.H., Kainka, E., 1998. Induction of sister chromatide exchanges in human tracheal epithelial cells by the fractions PM10 and PM2.5 of airborne particulates. Toxicol. Lett. 96/97, 215–220. Hsiao, W.L.W., Mo, Z.-Y., Fang, M., Shi, X.-M., Wang, F., 2000. Cytotoxicity of PM2.5 and PM2.5–10 ambient air pollutants assessed by the MTT and the Comet assays. Mutat. Res. 471, 45–55. IARC, 1987. Monographs on the evaluation of carcinogenic risk to humans, overall evaluations of carcinogenicity: an updating of IARC Monographs. International Agency for Research on Cancer, Lyon, 1–42 (7). Kastenbaum, M.A., Bowman, K.O., 1970. Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9, 549–552. Katsouyanni, K., Pershagen, G., 1997. Ambient air pollution exposure and cancer. Cancer Cause Control 8 (3), 284–291. Knaapen, A.M., Shi, T., Borm, P.J.A., Schins, R.P.F., 2002. Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter. Mol. Cell. Biochem. 234/235, 317–326. Lewtas, J., Lewis, C., Zweidinger, R., Stevens, R., Cupitt, L., 1992. Sources of genotoxicity and cancer risk in ambient air. Pharmacogenetics 2 (6), 288–296. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Mersch-Sundermann, V., Schneider, U., Klopman, G., Rosenkranz, H.S., 1994. SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds. Mutagenesis 9 (2), 205–224. Miguel, A.G., Daisey, J.M., Sousa, J.A., 1990. Comparative study of the mutagenic and genotoxic activity associated with inhalable particulate matter in Rio de Janeiro air. Environ. Mol. Mutagen. 15 (1), 36–43. Milieumaatschappij, V., 2002. Luchtkwaliteit in het Vlaamse gewest, 2001. VMM, Erembodegem. Monarca, S., Crebelli, R., Feretti, D., Zanardini, A., Fuselli, S., Filini, L., Resola, S., Bonardelli, P.G., Nardi, G., 1997. Mutagens and carcinogens in size-classified air particulates of a northern Italian town. Sci. Tot. Environ. 205 (2–3), 137–144. Nyberg, F., Gustavsson, P., Jarup, L., Bellander, T., Berglind, N., Jakobsson, R., Pershagen, G., 2000. Urban air pollution and lung cancer in Stockholm. Epidemiology 11 (5), 487–495. Pope III, C.A., Burnett, R.T., Thun, M.J., Calle, E.E., Krewski, D., Ito, K., Thurston, G.D., 2002. Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. J. Am. Med. Assoc. 289 (9), 1132–1141. Salvi, S., Holgate, S.T., 1999. Mechanisms of particulate matter toxicity. Clin. Exp. Allergy 29, 1187–1194. Samet, J.M., Dominici, F., Curriero, F.C., Coursac, I., Zeger, S.L., 2000. Fine particulate air pollution and mortality in 20 US cities. N. Engl. J. Med. 343, 1742–1749. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184–191. Takafuji, S., Nakagawa, T., 2000. Air pollution and allergy. J. Invest. Allergol. Clin. Immunol. 10, 5–10. Verschaeve, L., Van Gompel, J., Thilemans, L., Regniers, L., Vanparys, P., van der Lelie, D., 1999. Vitotoxs bacterial genotoxicity and toxicity test for the rapid screening of chemicals. Environ. Mol. Mutagen. 33, 240–248. Vinitketkumnuen, U., Kalayanamitra, K., Chewonarin, T., Kamens, R., 2002. Particulate matter, PM 10 & PM 2.5 levels, and airborne
ARTICLE IN PRESS 118
E. Brits et al. / Environmental Research 96 (2004) 109–118
mutagenicity in Chiang Mai, Thailand. Mutat. Res. 519 (1–2), 121–131. White, P., 2002. The genotoxicity of priority polycyclic aromatic hydrocarbons in complex mixtures. Mutat. Res. 515, 85–98. Wieringa, M.H., Weyler, J., Nele, V., Van Hoof, K., Van Bastelaer, F.J., Van Sprundel, M., Vermeire, P., 1998. Prevalence of respiratory symptoms: marked differences within a small geographic area. Int. J. Epidemiol. 27, 630–635.
Yaffe, D., Cohen, Y., Arey, J., Grosovsky, A.J., 2001. Multimedia analysis of PAHs and nitro-PAH daughter products in the Los Angeles Basin. Risk Anal. 21, 275–294. Zhao, X., Wan, Z., Chen, G., Zhu, H., Jiang, S., Yao, J., 2002. Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China. Mutat. Res. 514, 177–192.