Effect of combustion condition on cytotoxic and inflammatory activity of residential wood combustion particles

Atmospheric Environment 44 (2010) 1691e1698

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Effect of combustion condition on cytotoxic and inflammatory activity of residential wood combustion particles Pasi I. Jalava a, b, *, Raimo O. Salonen a, Kati Nuutinen b, Arto S. Pennanen a, Mikko S. Happo a, b, Jarkko Tissari b, Anna Frey c, Risto Hillamo c, Jorma Jokiniemi b, d, Maija-Riitta Hirvonen a, b a

National Institute for Health and Welfare (THL), Department of Environmental Health, Kuopio, Finland University of Kuopio, Department of Environmental Science, Kuopio, Finland Finnish Meteorological Institute, Air Quality Research, Helsinki, Finland d VTT Technical Research Centre of Finland, Fine Particles, Finland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 August 2009 Received in revised form 20 November 2009 Accepted 31 December 2009

Residential heating is an important local source of fine particles and may cause significant exposure and health effects in populations. We investigated the cytotoxic and inflammatory activity of particulate emissions from normal (NC) and smouldering (SC) combustion in one masonry heater. The PM1e0.2 and PM0.2 samples were collected from the dilution tunnel with a high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the PM-samples for 24 h. Inflammatory mediators, (IL-6, TNFa and MIP-2), and cytotoxicity (MTT-test), were measured. Furthermore, apoptosis and cell cycle of macrophages were analyzed. The HVCI particulate samples were characterized for ions, elements and PAH compounds. Assays of elemental and organic carbon were conducted from parallel low volume samples. All the samples displayed mostly dose-dependent inflammatory and cytotoxic activity. SC samples were more potent than NC samples at inducing cytotoxicity and MIP-2 production, while the order of potency was reversed in TNFa production. SC-PM1e0.2 sample was a significantly more potent inducer of apoptosis than the respective NC sample. After adjustment for the relative toxicity with emission factor (mg MJ
1), the SC-PM emissions had clearly higher inflammatory and cytotoxic potential than the NC-PM emissions. Thus, operational practice in batch burning of wood and the resultant combustion condition clearly affect the toxic potential of particulate emissions. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Fine particles Sources Residential wood combustion Inflammation Apoptosis

1. Introduction Wood combustion in residential heating is one of the most important sources of fine particulate (PM2.5) pollution worldwide (Naeher et al., 2007), which is associated with a substantial disease burden. Residential wood combustion (RWC) accounts for a mere 3.5% of the total heating energy consumption in Finland (situation in 2000/2001) (Sevola et al., 2003), but it has been estimated to be responsible for 25% of the total PM2.5 emissions (Karvosenoja et al., 2008). For comparison, this is a somewhat greater proportion than that for particulate emissions from vehicular traffic (19%). In Denmark, fine particulate (PM2.5) concentrations in residential area with extensive emissions from RWC have risen to levels similar to streets with busy traffic (Glasius et al., 2006). In Helsinki, Finland, * Corresponding author at: National Institute for Health and Welfare, Department of Environmental Health, P.O. Box 95, FI-70701 Kuopio, Finland. Tel.: þ358 20 610 6476; fax: þ358 20 610 6499. E-mail address: Pasi.Jalava@thl.fi (P.I. Jalava). 1352-2310/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2009.12.034

where a substantial number of households are connected to district heating, the contribution of biomass burning to the autumn and winter PM2.5 concentrations has been around 20% (Saarikoski et al., 2008). In cities with more prevalent RWC, that contribution has been estimated as high as 70% of the wintertime PM2.5 in Northern Sweden (Hedberg and Johansson, 2006), 74% in British Columbia, Canada (Jeong et al., 2008), and 90% in Christchurch, New Zealand (McGovan et al., 2002). It is noteworthy that RWC does not affect only the outdoor but also the indoor air quality, even in modern societies (Gustafson et al., 2008). Small-scale wood combustion has been associated with increased cardiac and respiratory hospital admissions in Washington (Norris et al., 1999; Schreuder et al., 2006), California (Lipsett et al., 1997), and Atlanta, GA (Sarnat et al., 2008). These epidemiological data are supported by a chamber study, where exposure to wood combustion emissions has evoked inflammation, increased tendency towards blood coagulation and oxidative stress in healthy subjects (Barregard et al., 2006). It has been observed that wood smoke aerosol may impair immunological and host defence functions, e.g. clearance of

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bacteria from the lungs as well as interfering with other macrophage activities, as reviewed by Zelikoff et al. (2002). In previous toxicological studies, wood combustion particles have also caused inflammation and cytotoxicity (e.g. Karlsson et al., 2006; Kocbach et al., 2008a, b). However, there is little information available on the effect of combustion conditions or the chemical composition of emission particles on the toxicological responses. This is of special interest since the emission factor of biomass combustion is largely dependent on the type of small-scale heating appliance (Purvis et al., 2000; Sternhufvud et al., 2004). Moreover, the way that the appliance is operated, i.e. its air supply, and fuel type and load, affect particulate emissions even from the same heater (Tissari et al., 2007; Jordan and Seen, 2005; Fine et al., 2002). We investigated the toxicological responses to and chemical characteristics of particles emitted from smouldering (SC) and normal (NC) combustion in one masonry heater. The RAW 264.7 macrophage cells were exposed to several doses of particulates to examine their inflammatory and cytotoxic activities. These data were subsequently combined with the emission data and the chemical characteristics of particulate samples. Inflammation is regarded as the main mechanism of particle related adverse effects on cardiorespiratory patients (e.g. Brunekreef and Holgate, 2002; Brook et al., 2004).

glass fiber filters were placed in 50 ml glass tubes filled with methanol. The samples were extracted 2  30 min in an ultrasonic water bath at 20  C. Subsequently, the methanol extracts of the samples or blank filters were concentrated in a rotary evaporator. Thereafter, the methanol suspension containing PM0.2 particles was filtered to remove glass fiber fragments derived from the backup filter. Finally, aliquots of the concentrated suspension, calculated on a mass basis, were dried in glass tubes under nitrogen (99.5%) flow and stored at
20  C. 2.4. Emission calculations The nominal emission values were calculated in relation to energy input to the combustion process (g MJ
1). The mass concentrations (mg m
3) in the flue gas were normalized to an oxygen concentration of 13%. The particle emission and concentration values were corrected with the dilution (Tissari et al., 2007). As shown in Table 1, the mean concentration of carbon monoxide with SC was 3.5-fold, and that of total volatile organics (VOC) 14-fold, PM1 mass 6-fold, particulate organic matter 7-fold, and elemental carbon 2-fold compared to NC. In contrast, the ash and particle number emissions were lower from SC than from NC (Frey et al., 2009; Tissari et al., 2007).

2. Material and methods 2.1. Combustion conditions and characterization of emissions Size-segregated particulate samples were collected from the emissions under two different combustion conditions in a conventional soapstone masonry heater (1.2  0.7  0.5 m; weight 800 kg) as described in detail by Tissari et al. (2007). Shortly, NC at high combustion temperature (flue gas temperature 253  3  C) was achieved by using an appropriate batch size (2.4  0.1 kg) and large logs (mean weight 480 g) of dry birch (moisture 7%). SC was produced by restricting the combustion air supply and slightly overloading (3.5  0.1 kg) the firebox with smaller logs (mean weight 230 g). In SC, a much lower flue gas temperature (159  28  C) was achieved. 2.2. Sampling of wood combustion particles Particulate samples were taken through the isokinetic inlet of the dilution tunnel (ISO 8178-1), with dilution ratios of between 180 and 330 and at an average temperature of 24  C. Each sample consisted of a complete heating cycle (75e80 min) including three SC or four NC batches in the masonry heater. The particles were collected with a modified Harvard high-volume cascade impactor (HVCI) to achieve high efficiency and representative sampling over several particulate size ranges (Sillanpää et al., 2003). The PM10e2.5, PM2.5e1 and PM1e0.2 samples were collected on polyurethane foam (McMaster-Carr, New Brunswick, NJ, USA), while the PM0.2 samples were collected on glass fiber filters (Munktell MGA, Munktell Filter AB, Grycksbo, Sweden). However, the PM10e2.5 and PM2.5e1 sizerange particles were excluded from the final analysis of the toxicological data, since most of the emission particles occur in sizes below 1 mm in diameter. Moreover, it was estimated on the basis of the PAH analysis that the adsorption of potentially harmful gaseous organics, including PAH compounds, onto the PUF sampling material would have caused an artifact in particulate sampling with these two upper stages of the HVCI impactor.

2.5. Chemical characterization of wood combustion particles Two ion chromatographs (Dionex DX500, Dionex Corporation, Sunnyvale, USA) were simultaneously used for analysis of the 2
þ þ þ 2þ anions (Cl
, NO
3 , SO4 , oxalate) and cations (Na , NH4 , K , Mg , 2þ Ca ) (Teinilä et al., 2000). Elemental analysis (Al, As, Cd, Co, Cr, Cu, Ni, V, Fe, Mn, Pb, Zn) was made by using an inductively coupled plasma mass-spectrometer (ICP-MS; Perkin Elmer Sciex Elan 6000, The PerkineElmer Corp., Norwalk, USA) as described earlier (Pakkanen et al., 2001). A total of 34 PAH compounds were analyzed using a gas-chromatograph mass-spectrometer single ion monitoring technique (GCMS-SIM; HP 5890 GC, equipped with a HP 5970B Series Mass Selective Detector, Agilent Technologies, Germany) after extraction of the particulate samples with dichloromethane (Saarnio et al., 2008). Special attention was paid to those PAH compounds that are recommended to be measured from the outdoor air PM10 in Europe (Directive 2004/107/EC). The sum of genotoxic PAH content in particulate samples was calculated on the basis of the WHOeIPCS criteria (WHO, 1998). The chemistry data of the HVCI samples were complemented with the analysis of elemental carbon (EC) and organic carbon (OC) from parallel low volume particulate samples. The OC concentration was multiplied by 1.6 to obtain particulate organic matter (POM) (Frey et al., 2009). 2.6. Cell culture A mouse macrophage cell line RAW 264.7 obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) was cultured

Table 1 Characteristic of particulate and gaseous emissions of the normal combustion (NC) and smouldering combustion (SC) particulate (PM1) samples (Tissari et al., 2008; Frey et al., 2009). Particle number CO OGCa POM EC PM1 (mg MJ
1) (  108 cm
3) (mg MJ
1) (mg MJ
1) (mg MJ
1) (mg MJ
1)

2.3. Sample preparation for chemical analysis and cell studies The size-segregated particulate samples were prepared for the cell experiments using previously validated procedures (Jalava et al., 2005, 2006). In brief, the sampled PUF-strips or quarters of

NC 100 SC 600 a

3.2 1.2

2300 8100

120 1700

50 330

50 120

OGC e Organic gaseous carbon measured with a flame ionization detector.

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in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin streptomycin (Gibco BRL, Paisley, UK). The cells were maintained at 37  C in a 5% CO2 atmosphere. In the experiments, the cell suspension was diluted to 5  105 cells ml
1 and cultured on 6-well plates (Costar, Corning, NY, USA) for 24 h to allow the cells to adhere. One hour before the experiments, 1 ml of fresh medium (37  C) was changed. 2.7. Experimental setup Before the cell exposure, particulate sample tubes were sonicated for 30 min in an ultrasonic water bath (FinnSonic m03) to suspend the size-segregated material into water (Sigma W1503, St. Louis, MO, USA) at a concentration of 5 mg ml
1. The blank samples were treated similarly to the actual particulate samples. DMSO (0.3% v/v at dose 150 mg ml
1) was used to facilitate suspension of the collected particulate mass into water. This concentration was non-toxic to macrophages. The RAW 264.7 macrophages were separately exposed to four doses (15, 50, 150 and 300 mg ml
1) of each particulate sample for 24 h. Three independent experiments were run in duplicate. To rule out possible contamination of particulate samples and methodological artifacts, each plate had also one untreated well as a cell control and a blank sample in a volume corresponding to the dose of 150 mg ml
1. After 24-h exposure, the cells were scraped and resuspended into cell culture medium. The viability of the cells was measured with the MTT-test from the cell suspension (2  100 mL) of each well. The remaining cell suspension was centrifuged and the supernatants were stored at
80  C for subsequent cytokine analysis (Jalava et al., 2005). The cells were washed, suspended in PBS and fixed in 70% (v/v) ethanol for subsequent propidium iodide staining (Penttinen et al., 2005). 2.8. Cytokine analysis The proinflammatory cytokines (IL-6 TNFa) and the chemokine (MIP-2) were immunochemically analyzed from the cell culture medium, using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions as earlier described in detail (Jalava et al., 2005). Absorbances from the 96well plates were spectrophotometrically measured with a multilabel-counter (PerkinElmer Victor3). The results were counted as absorbances and compared to the standard curve of the respective cytokine. Blank samples were run in all experiments in order to rule out methodological artifacts. The time-course of production of the present cytokines in RAW 264.7 macrophages has been previously validated, which motivated the selection of 24 h as a feasible exposure period (Jalava et al., 2005; Penttinen et al., 2005). 2.9. Cell viability analysis Viability of the macrophages was detected with the MTT-test on 96-well plates, and calculated as a percentage by comparing absorbances from cell suspensions exposed to particulate samples with those from corresponding control cells (Jalava et al., 2005). Cell viability assessed by the MTT-test is based on the presence of functioning mitochondria and endoplasmic reticulum in the cell suspension. In the test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide] is transformed to formazan, which is spectrophotometrically detected. The test was also run with particles only and with blank samples to ensure methodological reliability.

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can be identified as apoptotic cells (SubG1) (Darzynkiewicz et al., 1992). This is an indirect method that has been successfully used in some previous studies to assess the proportion of apoptotic cells. The validity of the method has also been confirmed in RAW 264.7 macrophages, as an association between the expression of caspase enzymes and the subG1 proportion of the cells (Penttinen et al., 2005). This method provides also information about the cell cycle of non-apoptotic cells. After PI staining, a total of 10,000 cells per sample were analyzed in a flow cytometer (CyAn ADP, BeckmanCoulter, CO, USA) (Penttinen et al., 2005). Possible interference of the method with particles was checked and found to be negligible. 2.11. Statistical analysis Levene's test for equality of variances was used for all the samples before analyzing the data with the analysis of variance (ANOVA). The results from exposures to actual particulate samples were tested against corresponding blanks as well as with regard to particle dose. Paired comparisons between the different particulate doses within each size range and combustion condition were made by Dunnett's test. ANOVA and Tukey's test were used for comparison of the differences between the combustion conditions. The cell-cycle results were tested with the non-parametric KruskalleWallis test. All the differences were regarded as statistically significant at p < .05. The data were analyzed using the SPSS version 16.0 (SPSS Inc. Chicago, IL, USA). 3. Results 3.1. Particulate emissions from two combustion conditions Under SC condition, the PM1 emission as combined from the two lowest HVCI stages (PM1e0.2 and PM0.2) was on average five times greater than that observed with NC. In the SC experiments, the aerodynamic size of emitted particles was somewhat larger than that with NC. In NC, the dominant size range was PM0.2, whereas with SC it was PM1e0.2. (Table 1). 3.2. Chemical composition of emission particles The samples consisted mostly of POM and EC. These were analyzed from parallel low volume virtual impactor samples (Frey et al., 2009). The percentage contribution of POM to NC-PM1 was 33% whereas with SC-PM1 it was 68%. The corresponding EC values were 32% and 25%. Ions, water-soluble elements and PAH compounds were analyzed from the HVCI samples. The average mass portion of inorganic ions in PM1 was much larger with NC (88 mg mg
1) than with SC (15 mg mg
1). In both cases, potassium, sulfate and chloride were the most abundant ions. The mass portion of trace elements was small, but clearly larger in NC samples compared to SC samples. The principle element under both conditions was zinc (Table 2). The average mass portion of total genotoxic PAH compounds in PM1 was only slightly larger in SC samples compared to NC samples. However, when the total genotoxic PAH emission measured with the HVCI was adjusted for the wood energy content, the release of genotoxic PAHs from SC was 5.5-fold higher (SC 352 vs. NC 64 mg MJ
1).

2.10. Analysis of apoptosis and cell cycle

3.3. Inflammatory and cytotoxic responses to size-segregated particulate samples

Cellular DNA content was analyzed by propidium iodide (PI) staining of permeabilized cells. The cells containing fragmented DNA

The dose-relationships in the responses to size-segregated particulate samples are described in terms of the size range for each

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Table 2 Chemical composition (ng mg
1) of the normal combustion (NC) and smouldering combustion (SC) particulate samples in two size ranges. Constituents

NC

SC

PM0.2

PM1e0.2

PM0.2

PM1e0.2

Inorganic ions

Naþ NHþ 4 Kþ Mg2þ Ca2þ Cl
NO
3 SO2þ 4

18000 22.0 40990 76.3 323 12700 6790 10200

2660 71.7 50700 71.5 249 6600 3500 23400

7580 61.9 5060 119 353 2650 1370 3220

681 84.2 5410 35.6 304 1200 455 1830

Elements

Cd Cr Cu Mn Ni Pb Fe Zn

0.34 1.72 72.2 4.66 1.49 3.78 18.4 468

9.10 0.98 24.2 45.1 0.54 32.6 14.0 3780

0.40 1.28 16.8 1.26 n.a. 1.44 n.a. 242

1.30 0.99 1.71 1.94 n.a. 2.30 n.a. 414

Benzo[a]anthracene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Indeno[1,2,3-cd]pyrene Dibenzo[a,h]anthracene Total genotox PAHa Total PAHb

59.8 54.4 27.1 103 261 16.6 936 1030

78.7 48.5 23.8 91.9 244 13.3 1250 2290

72.7 40.8 21.2 84.1 291 21.3 1090 1450

76.9 42.7 21.1 85.9 301 20.6 1270 2260

PAH compounds

n.a. not applicable. a Genotoxic PAH compounds selected on the basis of the WHOeIPCS (2008) criteria. Above analysis results for the six selected genotoxic PAH criteria compounds listed in the EU Directive (2004/107/EC). b Total PAHs refer to all the 34 compounds analyzed in this study according to the method described in detail by Saarnio et al. (2008).

parameter. Blank samples had negligible activities in all the measured parameters. 3.3.1. Cytokine production Fig. 1A shows the production of the proinflammatory cytokine, TNFa, in macrophages after 24-h exposure to particulate samples collected under either NC or SC conditions. All the samples evoked dose-dependent increases in TNFa production, with the exception

A 1500

B 3500

of the largest dose (300 mg ml
1). Generally, the PM0.2 samples were less potent inducers of TNFa production than the PM1e0.2 samples with both combustion conditions. With the exception of NC-PM0.2, all of the samples evoked statistically significant, dose-dependent inductions of chemokine MIP-2 in the RAW 264.7 macrophages (Fig. 1B). However, both SC particulate samples induced their largest MIP-2 production at the dose of 150 mg ml
1. The SC-PM1e0.2 sample was a significantly more potent inducer of MIP-2 production than the respective NC-PM1e0.2 sample. None of the SC or NC particulate samples caused any significant IL-6 response (data not shown). 3.3.2. Cytotoxicity All the samples from both combustion conditions showed statistically significant, dose-dependent decreases in macrophage viability as assessed by the MTT-test (Fig. 1C). The NC particulate samples decreased cell viability to 68  4% (PM1e0.2) and 89  4% (PM0.2), and the respective SC samples to 47  7% (PM1e0.2) and 54  8% (PM0.2). The SC-PM0.2 sample was a significantly more potent inducer of cytotoxicity than the NC-PM0.2 sample at the dose of 150 mg ml
1. The acute cytotoxicity did not significantly correlate with cytokine production. The SC particulate samples significantly increased apoptosis, when compared to the respective blank samples and the NC particulate samples. This was indicated by the significantly increased proportion of apoptotic cells in the SubG1 phase. Moreover, as shown in Fig. 2, the SC samples tended to induce also other cell-cycle effects: decreased proportion of cells in resting (G1) phase and inhibition of DNA synthesis (S-phase). In contrast, the effects of NC particulate samples did not statistically differ from the blank sample. 3.4. Relative toxicity adjusted with particle concentration and fuel energy content The biochemical responses measured in macrophages were adjusted for the mass emission and the fuel energy content in order to obtain an overall picture of the toxic potential of the particulate emissions under real-life conditions (Table 3). This approach changed the results considerably, when compared to responses to equal mass doses of the SC and NC particulate samples.

* *

500

*

*

*

*

*

2000

* 1500 1000

*

*

Normal Combustion

Smouldering Combustion

* *

*

60

* 40

*

*

* *

0

0

PM1-0.2 PM0.2 PM1-0.2 PM0.2

80

20

500 0

# 120

100

2500

MIP-2 (pg/ml)

TNFα (pg/ml)

3000

1000

C

#

Viability % (of control)

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PM1-0.2 PM0.2 PM1-0.2 PM0.2 Normal Combustion

Smouldering Combustion

PM1-0.2 PM0.2 PM1-0.2 PM0.2 Normal Combustion

Smouldering Combustion

Fig. 1. A) TNFa and B) MIP-2 release, and C) viability of RAW 264.7 macrophages after exposure to four doses (15, 50, 150 and 300 mg ml
1) of PM1e0.2 and PM0.2 samples from normal (NC) and smouldering (SC) combustion. Asterisk indicates statistically significant response compared to control cells *(p < .05; Dunnett's test). Statistical difference between each concentration of the samples in two combustion conditions is indicated with #(p < .05; Tukey's test).

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60 control level

% (of 10 000 cells)

50

Normal combustion Smouldering combustion

-

40

30

-

*

+

* 20

+ +

10

0 PM1-0.2 PM0.2 PM1-0.2 PM0.2 PM1-0.2 PM0.2 PM1-0.2 PM0.2

Sub G1

G1

S

G2/M

Fig. 2. Cell-cycle phases of RAW 264.7 macrophages after exposure to a single dose (150 mg ml
1) of PM1e0.2 and PM0.2 samples from normal (NC) and smouldering (SC) combustion. Asterisk indicates statistically significant difference from control cells * þ and e signs indicate positive or negative differences of the SC samples from the respective NC samples (KruskalleWallis test).

The emission adjustment was performed by calculating the energy contents of the wood logs used in the present combustion experiments. The mass emission per fuel energy for SC was 2.2-fold in PM0.2 and 9.7-fold in PM1e0.2, when compared to NC. The relative toxicological responses adjusted for the mass emissions and fuel energy content are shown in Table 3. The TNFa response to SC-PM0.2 was over 2-fold and that to PM1e0.2 8-fold, when compared to NC. The corresponding MIP-2 responses exhibited 4-fold and over 20-fold differences between the SC and the NC. The adjusted cytotoxicity assessed with the MTT-test for SCPM0.2 was 1.3-fold and for SC-PM1e0.2 7-fold, when compared to the respective NC particulate samples. Moreover, the apoptotic (SubG1) activities of SC-PM0.2 and SC-PM1e0.2 were 10-fold and 30-fold greater than those obtained with the NC samples.

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practice significantly affected both the cytotoxic and inflammatory properties of emission particles from small-scale wood combustion as indicated by the many differences observed between the normal (NC) and smouldering (SC) combustion conditions. However, the overall toxic potential of particulate emissions from the two combustion conditions seemed to be largely determined by the emitted particulate mass per cubic meter of air or per fuel energy. Controlled exposure to wood smoke has been shown to induce inflammation and increased tendency towards coagulation of blood in healthy subjects (Barregard et al., 2006). In previous studies, operational practice, fuel and the heating appliance have affected both the amount and chemical composition of emitted wood combustion particles (e.g. Purvis et al., 2000; Tissari et al., 2007; Jordan and Seen, 2005; Frey et al., 2009). As far as we are aware, this is the first time, when the toxicological properties of particulate emissions have been reported from the same small-scale heater run under different combustion conditions. A poor air supply and overloading of the firebox, as demonstrated in the present study, are common operational deficiencies of wood batch burning in residential heaters and, thus, major potential contributors to poor local air quality. 4.1. Emissions from combustion experiments NC in this study cannot be described as complete or even optimal combustion of wood. This can be seen from the relatively high emission factors of CO, volatile organic compounds and PM1 summarized in Table 1 and reported previously in detail by Tissari et al. (2007). The PM emission from NC (1.8  0.5 g kg
1) was similar to with the emission factors of 0.6e7.2 g kg
1 previously reported for masonry heaters, baking ovens and stoves in other studies (McDonald et al., 2000). The total particulate mass in the emission was clearly larger for SC than for NC, especially in HVCI-PM1e0.2. The particles emitted from SC were also somewhat larger in size and had a much larger content of POM. The larger particle size in SC is likely to be attributable to the condensation of organic vapors onto primary particles and from the formation of agglomerates (Tissari et al., 2008; Frey et al., 2009). The proportion of inorganic ions was clearly larger in NC samples, suggesting that the emission of fine ash particles, composed of alkaline sulfates and chlorides, was higher than in SC. Together with the lower emission of organics, this indicates more complete combustion.

4. Discussion 4.2. Inflammatory responses to particulate samples Little is known about how the operational condition of heating appliances can influence the toxicological properties of emission particles. The present in vitro data revealed that operational

Table 3 Relative responses in cellular parameters to PM0.2 and PM1e0.2 samples. Value 1 refers to the NC-PM0.2 sample. The first four rows are responses to particulate samples calculated on an equal mass dose basis. The last four rows are the same data adjusted for the emission factor (mg MJ
1) for NC and SC.

per mg PM

NC SC

per MJ

NC SC

Apoptosis

MIP-2

TNFa

Cytotoxicity

PM1e0.2 PM0.2 PM1e0.2 PM0.2

4.8 1 6.7 6.7

2.4 1 5.7 2.0

2.0 1 1.6 1.0

3.1 1 5.1 4.4

PM1e0.2 PM0.2 PM1e0.2 PM0.2

2.4 1 33 15

1.2 1 28 4.3

1.0 1 7.9 2.3

1.5 1 25 9.7

Note. Largest values among the samples are given in boldface.

All the particulate samples caused mainly dose-dependent increases in the detected responses. There were some differences in the responses to the particulate samples between SC and NC. On an equal mass dose basis, NC samples tended to induce slightly larger TNFa responses than SC samples, with the exception of the doses 50 mg ml
1 of the PM1e0.2 sample and 150 mg ml
1 of the PM0.2 sample. None of these differences were statistically significant. The reasonably small cytokine responses to both NC and SC particulate samples in our study may be due to the relatively large proportions of potentially suppressive organic compounds in these samples. This view is supported by our recent studies demonstrating immunosuppressive properties of PAH-rich urban air fine particulate samples with sources of poor combustion of biomass and coal for residential heating (Jalava et al., 2007; Happo et al., 2008). In a recent study with human macrophages (Karlsson et al., 2006), particulate samples collected from old-type and modern wood boiler emissions have induced non-significant TNFa responses similar to each other.

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Wood combustion-derived particulate samples have evoked smaller inflammatory responses but greater cytotoxicity than trafficderived particles when tested in a co-culture of alveolar epithelial cells A549 and monocyte cells THP-1 (Kocbach et al., 2008a). Moreover, the non-soluble particulate fraction, but not the extracted organic soluble fraction of wood smoke particles, increased dosedependently both the levels of TNFa and chemokine IL-8 in a human monocyte cell line (THP-1) (Kocbach et al., 2008b). Particle solubility was a major determinant of macrophage responses also in our previous study on urban air fine particles (Jalava et al., 2008). In contrast to TNFa, the chemokine MIP-2 response was larger to SC samples than to NC samples. In our previous study, the MIP-2 response to urban air PM0.2 samples was the greatest with the Prague wintertime sample which in the six European campaigns displayed the largest contribution of PAHs originating from poor biomass and coal combustion (Jalava et al., 2007). In the studies of Kocbach et al. (2008b) and Karlsson et al. (2006), wood smoke particles have been more potent inducers of the chemokine response than of the proinflammatory cytokine response. However, particles derived from modern wood boiler have been more potent inducers of the chemokine IL-8 production than particles from old-fashioned boiler (Karlsson et al., 2006). Overall, traffic-derived particles have been more potent inducers of IL-8 and TNFa responses than wood smoke particles (Karlsson et al., 2006; Kocbach et al., 2008b). The differences between the chemokine and proinflammatory cytokine responses are most likely due to the different capabilities of the chemical constituents of the particulate samples to activate biochemical pathways in macrophages, and also due to different roles of the protein mediators in the cytokine cascade. 4.3. Cytotoxic responses to particulate samples The SC-PM1e0.2 and SC-PM0.2 samples were generally more cytotoxic than the respective NC samples. This finding is also in line with our previous findings with the Prague urban air samples which were collected during the wintertime (Jalava et al., 2007). The most obvious difference between the two combustion conditions was in the particle-induced apoptosis in macrophages, i.e. both SC samples induced larger apoptotic responses than the respective NC samples. Furthermore, all the SC samples decreased the proportion of cells in the G1 phase of cell cycle. The apoptotic response may be associated with the oxidative properties of combustionderived particles (Dellinger et al., 2000), although wood smoke particles may have also protective antioxidant properties (Kjällstrand and Petterson, 2001). In addition to what was presently investigated, wood smoke particles have been reported to evoke DNA damage and oxidative stress (Karlsson et al., 2006; Danielsen et al., 2009). 4.4. Methodological considerations The current results are from macrophage cell line, which represents the major immunological cell type involved in the responses to particles in the lungs. However, there are also other cell types present in the lungs, which may modify the responses induced in inflammatory cells like macrophages. Co-cultures of monocytes and respiratory epithelial cells have been used in some previous studies, which have demonstrated somewhat diverse cytokine patterns between responses to traffic-derived particles and wood smoke particles (e.g. Kocbach et al., 2008a, b). However, it is important to know the basic responses of the key inflammatory cells to the particulate samples before taking more complex mechanistic approaches. The multiple steps of sample collection, extraction and resuspension of the particles may alter their toxicological properties. However, in our approach, the cell exposure is well controlled and the chemical composition of the samples is characterized in detail.

To minimize the aggregation of the samples, we used small, nontoxic amounts of DMSO and effective sonication that provided a more even suspension of the particles into water. Carbonaceous emission particles aggregate also in the diluted and cooled flue gas immediately after their release from combustion process, and their particle size grows rapidly (Tissari et al., 2008). It is possible that extensive cytotoxicity could diminish the cytokine concentrations, produced by the cells. We calculated the correlation between cytokine concentrations and cytotoxicity, and they were negative especially for MIP-2. Whether this is simply due to cytotoxicity or in a more complex manner related to the chemical composition of particulate samples remains unclear in the present dataset. However, in our previous studies on urban air fine particles, high cytotoxicity has taken place concomitantly with both large and minimal cytokine responses, regardless of the number of viable cells. E.g. a high content of soil minerals favoured large cytokine production in macrophages, while a high PAH content was associated with small cytokine production due to immunosuppression (Jalava et al., 2009). 4.5. Relative toxicity adjusted with emission factor The detected relative inflammatory and cytotoxic activities of the NC and SC samples were weighed with the emission factor (mg MJ
1). This was done to estimate the relative toxic potential of the particulate emissions in real-life situations. Emissions from a residential wood combustion appliances cause local air pollution and increase exposure of the people living in their vicinity (Schreuder et al., 2006; Boman et al., 2003). The energy unit-weighed toxicity results revealed that despite the smaller response of some inflammatory parameters to SC particles, the overall toxic potential of particulate emissions can be regarded as much larger with SC than with NC. In PM1e0.2 and PM0.2 size ranges, the adjusted relative toxic potential of SC particles was up to 8-fold greater for TNFa, 23-fold for MIP-2, and 14-fold for apoptosis, when compared to NC particles. 4.6. Potentially causative chemical compositions No correlations between the detected chemical constituents in particulate samples and the respective cellular responses could be calculated, since there were only two data points in both particulate size ranges. However, EC and organic composition, especially that of PAHs, are of special interest in the present particulate samples. EC is non-soluble and adsorbs both organic and inorganic vapors. Therefore, it may act as a carrier of these compounds for uptake into macrophages and respiratory epithelial cells. EC or related particulate metrics has been associated with a variety of health outcomes in epidemiological studies, including mortality (Clancy et al., 2002; Hoek et al., 2002) and exacerbation of ischemic heart disease (Lanki et al., 2006). and increased prevalence of bronchitis in asthmatic children (McConnell et al., 2003). In urban air fine particles, PAH compounds derived from wood and coal combustion have been negatively associated with the particle-induced production of both the proinflammatory cytokines and the chemokines in macrophages (Jalava et al., 2009) and in mouse lung (Happo et al., 2008). Moreover, PAH compounds may cause genotoxicity and evoke oxidative stress (Binkova et al., 2003; Li et al., 2003) and apoptosis (Solhaug et al., 2004). One additional action of PAHs and related combustion-derived organic compounds is adjuvant activity in sensitization to allergens as shown in animal studies (Steerenberg et al., 2006; Schwarze et al., 2007). PAH contents of the present HVCI samples from NC and SC were similar to each other, which may explain the very minor differences in their inflammatory potency. However, the equal enrichment of PAHs in the NC and SC samples cannot explain the greater cytotoxic

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and apoptotic activities of the SC particles. These responses may involve other organics, including reactive derivatives of PAHs, i.e. quinones or nitro PAHs. It is known that quinones formed in combustion processes can produce hydroxyl radicals via redox cycling (Squadrito et al., 2001). There were much higher contents of Cu, Mn, Fe and Zn in NC samples than in SC samples with Zn being clearly the most abundant constituent. The larger metal contents of the NC samples can be explained by higher combustion temperature and the more complete vaporization of these metals. These metals may contribute to the present inflammatory responses in macrophages since previously metals have been shown to induce in vitro production of inflammatory mediators (Merolla and Richards, 2005; Frampton et al., 1999). Thus, operational practice in batch burning of wood and the resultant combustion conditions clearly affect the toxic potential of particulate emission. Ensuring the proper use of residential heating appliances is a major issue when attempting to reduce the emissions of harmful particulate matter. Acknowledgements This research was funded by the National Agency for Technology and Innovation (Tekes) and the Ministry of Social Affairs and Health in Finland. This project belongs to the KANTIVA Bioenergy Research Centre of the University of Kuopio. Laboratory assistance by Ms. Arja Rönkkö is highly appreciated. Dr. Ewen Macdonald is acknowledged for revising the language of the manuscript. References Barregard, L., Sällsten, G., Gustafson, P., Andersson, L., Johansson, L., Basu, S., Stigendal, L., 2006. Experimental exposure to wood smoke particles in healthy humans: effects of markers on inflammation, coagulation, and lipid peroxidation. Inhalation Toxicology 18, 845e853. Binkova, B., Cerna, M., Pastorovka, A., Jelinek, R., Benes, I., Novak, J., Sram, R.J., 2003. Biological activities of organic compounds absorbed onto ambient air particles: comparison between the cities of Teplice and Prague during the summer and winter seasons 2000e2001. Mutation Research 525, 43e59. Boman, B.C., Forsberg, A.B., Jarvholm, B.G., 2003. Adverse health effects from ambient air pollution in relation to residential wood combustion in modern society. Scandinavian Journal of Work, Environment and Health 29, 251e260. Brook, R.D., Franklin, B., Cascio, W., Hong, Y., Howard, G., Lipsett, M., Luepker, R., Mittleman, M., Samet, J., Smith, S.C., Tager, I., 2004. Air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association. Circulation 109, 2655e2671. Brunekreef, B., Holgate, S.T., 2002. Air pollution and health. Lancet 360, 1233e1244. Clancy, L., Goodman, P., Sinclair, H., Dockery, D.W., 2002. Effect of air-pollution control on death rates in Dublin, Ireland: an intervention study. Lancet 360, 1210e1214. Danielsen, H.D., Loft, S., Kocbach, A., Schwarze, P.E., Moller, P., 2009. Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines. Mutation Research 674, 116e122. Darzynkiewicz, Z., Brun, S., Delbino, G., Gorzyca, W., Hotz, M.A., Lassota, P., Traganos, F., 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795e808. Dellinger, B., Pryor, W.A., Cueto, R., Squdrito, G., Deutsch, W.A., 2000. The role of combustion generated radicals in the toxicity of PM2.5. Proceedings of the Combustion Institute 28, 2675e2681. Directive 2004/107/EC of the European Parliament and of the Council of 15 December 2004 relating to arsenic, cadmium, mercury, nickel and polycyclic aromatic hydrocarbons in ambient air 2005. Official Journal L 23. Fine, P.M., Cass, G.R., Simoneit, B.R.T., 2002. Chemical characterization of fine particle emissions from fireplace combustion of woods grown in the Southern United States. Environmental Science and Technology 36, 1442e1451. Frampton, M.W., Ghio, A.J., Samet, J.M., Carson, J.L., Carter, J.D., Devlin, R.B., 1999. Effects of aqueous extracts of PM10 filters from the Utah Valley on human airway epithelial cells. American Journal of Physiology- Lung Cellular and Molecular Physiology 277, L960eL967. Frey, A.K., Tissari, J., Saarnio, K.M., Timonen, H.J., Tolonen-Kivimäki, O., Aurela, M.A., Saarikoski, S.K., Makkonen, U., Hytönen, K., Jokiniemi, J., Salonen, R.O., Hillamo, R.E. J., 2009. Chemical composition and mass size distribution of fine particulate matter emitted by a small masonry heater. Boreal Environment Research 14, 255e271. Glasius, M., Ketzel, M., Wåhlin, P., Jensen, B., Mønster, J., Berkowicz, B., Palmgren, F., 2006. Impact of wood combustion on particle levels in a residential area in Denmark. Atmospheric Environment 40, 7115e7124.

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