Raimo O. Salonen

Heterogeneities in Inflammatory and Cytotoxic Responses of RAW 264.7 Macrophage Cell Line to Urban Air Coarse, Fine, and Ultrafine Particles From Six European Sampling Campaigns

We investigated the cytotoxic and inflammatory activities of size-segregated particulate samples (particulate matter, PM) from contrasting air pollution situations in Europe. Coarse (PM10−2.5), fine (PM2.5−0.2), and ultrafine (PM0.2) particulate samples were collected with a modified Harvard high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the samples for 24 h. Selected inflammatory mediators, nitric oxide (NO) and cytokines (tumor necrosis factor alpha [TNFα], interleukin 6 [IL-6], macrophage inflammatory protein-2 [MIP-2]), were measured together with cytotoxicity (MTT test), and analysis of apoptosis and cell cycle (propidium iodide staining). The PM10−2.5 samples had a much higher inflammatory activity than the PM2.5−0.2 and PM0.2 samples, but the PM2.5−0.2 samples showed the largest differences in inflammatory activity, and the PM0.2 samples in cytotoxicity, between the sampling campaigns. The PM2.5−0.2 samples from traffic environments in springtime Barcelona and summertime Athens had the highest inflammatory activities, which may be related to the high photochemical activity in the atmosphere during the sampling campaigns. The PM0.2 sample from wintertime Prague with proven impacts from local coal and biomass combustion had very high cytotoxic and apoptotic activities and caused a distinct cell cycle arrest. Thus, particulate size, sources, and atmospheric transformation processes affect the toxicity profile of urban air particulate matter. These factors may explain some of the heterogeneity observed in particulate exposure-response relationships of human health effects in epidemiological studies.

Effects of solubility of urban air fine and coarse particles on cytotoxic and inflammatory responses in RAW 264.7 macrophage cell line

We investigated the inflammatory and cytotoxic activities of the water-soluble and -insoluble as well as organic-solvent-soluble and -insoluble fractions of urban air fine (PM(2.5-0.2)) and coarse (PM(10-2.5)) particulate samples. The samples were collected with a high volume cascade impactor (HVCI) in 7-week sampling campaigns of selected seasons in six European cities. Mouse macrophage cells (RAW 264.7) were exposed to the samples for 24 h. The production of nitric oxide (NO) and proinflammatory cytokines (TNFalpha, IL-6), and cytotoxicity (MTT-test, apoptosis, cell cycle) were measured. The inflammatory and cytotoxic responses in both size ranges were mostly associated with the insoluble particulate fractions. However, both the water- and organic-solvent-soluble particulate fractions induced TNFalpha production and apoptosis and had some other cytotoxic effects. Soil-derived water-soluble and -insoluble components of the chemical PM(2.5-0.2) mass closure had consistent positive correlations with the responses, while the correlations were negative with the secondary inorganic anions (NO(3)(-), NH(4)(+), non-sea-salt SO(4)(2-)) and particulate organic matter (POM). With the PM(10-2.5) samples, sea salt and soluble soil components correlated positively with the induced toxic responses. In this size range, a possible underestimation of the insoluble, soil-related compounds containing Si and Ca, and biological components of POM, increased uncertainties in the evaluation of associations of the mass closure components with the responses. It is concluded that insoluble components of the complex urban air particulate mixture exert the highest inflammatory and cytotoxic activities in the macrophage cell line but, at the same time, they may operate as carriers for active water- and lipid-soluble components.

Dose and time dependency of inflammatory responses in the mouse lung to urban air coarse, fine, and ultrafine particles from six european cities

We investigated the dose and time dependency of inflammatory and cytotoxic responses to size-segregated urban air particulate samples in the mouse lung. Coarse (PM10−2.5), fine (PM2.5−0.2), and ultrafine (PM0.2) particles were collected in six European cities (Duisburg, Prague, Amsterdam, Helsinki, Barcelona, Athens) in selected seasons using a modified Harvard high-volume cascade impactor. Healthy C57Bl/6J mice were intratracheally exposed to the particulate samples in a 24-h dose-response study (1, 3, and 10 mg/kg) and in 4-, 12-, and 24-h time course studies (10 mg/kg). After the exposures, the lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: total cell number, cell differential, total protein, and lactate dehydrogenase (LDH) and cytokine (tumor necrosis alpha [TNF-α], interleukin-6 [IL-6], and keratinocyte-derived chemokine [KC]) concentrations. In general, PM10−2.5 samples had higher inflammatory activity than PM2.5−0.2 samples. PM0.2 samples showed negligible inflammatory activity. PM10−2.5 and PM2.5−0.2 samples caused large increases in BALF cytokine concentrations at 4 h, but not at 12 or 24 h, after exposure. The BALF total cell number and total protein concentrations increased significantly at 12 h for both the PM10−2.5 and PM2.5−0.2 samples, but only PM10−2.5 samples produced consistent, significant increases at 24 h after exposure. There was more heterogeneity in BALF cytokine and neutrophil cell number responses to PM2.5−0.2 samples than to PM10−2.5 samples between the sampling campaigns. Thus, particle size, sources, and atmospheric transformation processes affect the inflammatory activity and response duration of urban air particulate matter in the mouse lung.

Inflammation and tissue damage in mouse lung by single and repeated dosing of urban air coarse and fine particles collected from six European cities

The authors have previously demonstrated heterogeneities in the inflammatory activities of urban air fine (PM2.5–0.2) and coarse (PM10–2.5) particulate samples collected from six European cities with contrasting air pollution situations. The same samples (10 mg/kg) were intratracheally instilled to healthy C57BL/6J mice either once or repeatedly on days 1, 3, and 6 of the study week. The lungs were lavaged 24 h after the single dose or after the last repeated dosing. In both size ranges, repeated dosing of particles increased the total cell number in bronchoalveolar lavage fluid (BALF) more than the respective single dose, whereas cytokine concentrations were lower after repeated dosing. The lactate dehydrogenase (LDH) responses increased up to 2-fold after repeated dosing of PM2.5–0.2 samples and up to 6-fold after repeated dosing of PM10–2.5 samples. PM10–2.5 samples evoked a more extensive interstitial inflammation in the mouse lungs. The constituents with major contributions to the inflammatory responses were oxidized organic compounds and transition metals in PM2.5–0.2 samples, Cu and soil minerals in PM10–2.5 samples, and Zn in both size ranges. In contrast, poor biomass and coal combustion were associated with elevated levels of polycyclic aromatic hydrocarbons (PAHs) and a consistent inhibitory effect on the inflammatory activity of PM2.5–0.2 samples. In conclusion, repeated intratracheal instillation of both fine and coarse particulate samples evoked enhanced pulmonary inflammation and cytotoxicity compared to single-dose administration. The sources and constituents of urban air particles responsible for these effects appear to be similar to those encountered in the authors’ previous single-dose study.

Chemical Compositions Responsible for Inflammation and Tissue Damage in the Mouse Lung by Coarse and Fine Particulate Samples from Contrasting Air Pollution in Europe

Inflammation is regarded as an important mechanism in mortality and morbidity associated with exposures of cardiorespiratory patients to urban air particulate matter. We investigated the association of the chemical composition and sources of urban air fine (PM2.5−0.2) and coarse (PM10−2.5) particulate samples with the inflammatory activity in the mouse lung. The particulate samples were collected during selected seasons in six European cities using a high-volume cascade impactor. Healthy C57BL/6J mice were intratracheally instilled with a single dose (10 mg/kg) of the particulate samples. At 4, 12, and 24 h after the exposure, the lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: cell number, total protein, and cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6, and KC). Dicarboxylic acids and transition metals, especially Ni and V, in PM2.5−0.2 correlated positively and some secondary inorganic ions (NO3-, NH4+) negatively with the inflammatory activity. Total organic matter and SO42- had no consistent correlations. In addition, the soil-derived constituents (Ca2+, Al, Fe, Si) showed positive correlations with the PM2.5−0.2-induced inflammatory activity, but their role in PM10−2.5 remained obscure, possibly due to largely undefined biogenic material. Markers of poor biomass and coal combustion, i.e., monosaccharide anhydrides and As, were associated with elevated PAH contents in PM2.5−0.2 and a consistent immunosuppressive effect. Overall, our results support epidemiological findings that the local sources of incomplete combustion and resuspended road dust are important in urban air particulate pollution-related health effects.

Effects of Sample Preparation on Chemistry, Cytotoxicity, and Inflammatory Responses Induced by Air Particulate Matter

Abstract Methanol is used for high-efficiency extraction of air particulate (PM) mass from the sampling substrate in the high-volume cascade impactor. Sonication is needed during extraction and when dissolving dried PM samples in liquids used in exposure studies. We investigated the effects of these procedures on the PM chemistry and PM-induced cytotoxic and inflammatory responses in mouse macrophages. Untreated and methanol-treated ambient air reference PM samples (SRM1649a, EHC-93) and diesel PM (SRM1650) were tested after different sonication durations (5–30 min). Furthermore, the time dependency of the responses to SRM1649a, EHC-93, and a fine PM sample from Helsinki was investigated. Methanol pretreatment increased on average by 24% and 21% the recovery of water-soluble metals from SRM1649a and EHC-93, but not SRM1650. It had no systematic effect on the recoveries of inorganic secondary ions (NO3-, SO42-, NH4+) or the sum of genotoxic PAH compounds from the three reference samples. Nitric oxide (NO) response to SRM1650 was strongly enhanced by methanol pretreatment, whereas the cytotoxic or inflammatory responses to the ambient air PM samples (EHC-93, SRM1649a) were only slightly modified. Sonication duration was a modifying factor only in connection to SRM1650. Maximal interleukin (IL)-1 production was observed earlier (8 h) than maximal tumor necrosis factor (TNF) α and IL-6 productions (24 h), which indicates the importance to know the optimal time points for measurement of the selected response parameters. In conclusion, methanol extraction and reasonable sonication duration are not likely to modify the cytotoxic and inflammatory potency of ambient air PM samples, but some responses to air PM, rich in organic compounds, can be modified.

Low-level exposure to ambient particulate matter is associated with systemic inflammation in ischemic heart disease patients

Short-term exposure to ambient air pollution is associated with increased cardiovascular mortality and morbidity. This adverse health effect is suggested to be mediated by inflammatory processes. The purpose of this study was to determine if low levels of particulate matter, typical for smaller cities, are associated with acute systemic inflammation. Fifty-two elderly individuals with ischemic heart disease were followed for six months with biweekly clinical visits in the city of Kotka, Finland. Blood samples were collected for the determination of inflammatory markers interleukin (IL)-1β, IL-6, IL-8, IL-12, interferon (IFN)γ, C-reactive protein (CRP), fibrinogen, myeloperoxidase and white blood cell count. Particle number concentration and fine particle (particles with aerodynamic diameters <2.5 μm (PM(2.5))) as well as thoracic particle (particles with aerodynamic diameters <10 μm (PM(10))) mass concentration were measured daily at a fixed outdoor measurement site. Light-absorbance of PM(2.5) filter samples, an indicator of combustion derived particles, was measured with a smoke-stain reflectometer. In addition, personal exposure to PM(2.5) was measured with portable photometers. During the study period, wildfires in Eastern Europe led to a 12-day air pollution episode, which was excluded from the main analyses. Average ambient PM(2.5) concentration was 8.7 μg/m(3). Of the studied pollutants, PM(2.5) and absorbance were most strongly associated with increased levels of inflammatory markers; most notably with C-reactive protein and IL-12 within a few days of exposure. There was also some evidence of an effect of particulate air pollution on fibrinogen and myeloperoxidase. The concentration of IL-12 was considerably (227%) higher during than before the forest fire episode. These findings show that even low levels of particulate air pollution from urban sources are associated with acute systemic inflammation. Also particles from wildfires may exhibit pro-inflammatory effects.

Toxicological effects of emission particles from fossil- and biodiesel-fueled diesel engine with and without DOC/POC catalytic converter

There is increasing demand for renewable energy and the use of biodiesel in traffic is a major option when implying this increment. We investigated the toxicological activities of particulate emissions from a nonroad diesel engine, operated with conventional diesel fuel (EN590), and two biodiesels: rapeseed methyl ester (RME) and hydrotreated fresh vegetable oil (HVO). The engine was operated with all fuels either with or without catalyst (DOC/POC). The particulate matter (PM1) samples were collected from the dilution tunnel with a high-volume cascade impactor (HVCI). These samples were characterized for ions, elements, and polycyclic aromatic hydrocarbon (PAH) compounds. Mouse RAW264.7 macrophages were exposed to the PM samples for 24 h. Inflammatory mediators, (TNF-α and MIP-2), cytotoxicity, genotoxicity, and oxidative stress (reactive oxygen species [ROS]) were measured. All the samples displayed mostly dose-dependent toxicological activity. EN590 and HVO emission particles had larger inflammatory responses than RME-derived particles. The catalyst somewhat increased the responses per the same mass unit. There were no substantial differences in the cytotoxic responses between the fuels or catalyst use. Genotoxic responses by all the particulate samples were at same level, except weaker for the RME sample with catalyst. Unlike other samples, EN590-derived particles did not significantly increase ROS production. Catalyst increased the oxidative potential of the EN590 and HVO-derived particles, but decreased that with RME. Overall, the use of biodiesel fuels and catalyst decreased the particulate mass emissions compared with the EN590 fuel. Similar studies with different types of diesel engines are needed to assess the potential benefits from biofuel use in engines with modern technologies.

Population exposure to fine particles and estimated excess mortality in Finland from an East European wildfire episode

Long-range transported particulate matter (PM) air pollution episodes associated with wildfires in the Eastern Europe are relatively common in Southern and Southeastern Finland. In severe cases such as in August–September 2002, the reduced visibility and smell of the smoke, and symptoms such as irritation of eyes and airways experienced by the population raise the issue into the headlines. Because PM air pollution, in general, has been identified as a major health risk, and the exposures are of repeating nature, the issue warrants a risk assessment to estimate the magnitude of the problem. The current work uses the available air quality data in Finland to estimate population exposures caused by one of the worst episodes experienced in this decade. This episode originated from wildfires in Russia, Belarus, Ukraine, and the Baltic countries. The populations of 11 Southern Finnish provinces were exposed between 26 August and 8 September 2002, for 2 weeks to an additional population-weighted average PM2.5 level of 15.7 μg/m3. Assuming similar effect on mortality for these particles as observed in epidemiological time series studies on urban particles (0.5%–2% increase in mortality per 10 μg/m3, central estimate 1%), this exposure level would be associated with 9–34 cases (17 cases central estimate) of additional mortality. Epidemiological evidence specific to particles from biomass combustion is scarce, affecting also the reliability of the current risk assessment. Do the wildfire aerosols exhibit the same level of toxicity as the urban particles? To shed light on this question, it is interesting to look at the exposure data in relationship to the observed daily mortality in Finland, even though the limited duration of the episode allows only for a weak statistical power. The percentage increases observed (0.8%–2.1% per 10 μg/m3 of fine PM) are in line with the more general estimates for urban PM and those used in the current risk assessment.

Field and laboratory tests of a high volume cascade impactor

Abstract The performance of three different configurations of a recently developed high volume cascade impactor (HVCI) system was tested in both field and laboratory experiments. In the field, the HVCI was run simultaneously with a virtual impactor and a low pressure impactor using a 3- or 4-day sampling duration. The study was carried out in downtown Helsinki between June and September 2001. As the field test results were not consistent with the previous laboratory calibration results provided by the manufacturer, the collection efficiency of each field-tested HVCI stage and two additional stages was measured in the laboratory as a function of particle size, using monodisperse dioctyl sebacate test aerosol. The collection efficiency curve for stage 1, impacting particles on a greased aluminum plate, was steep. In stages 2–4, polyurethane foam was used as collection substrate, which made the collection efficiency curves less steep than that of stage 1. According to the present laboratory calibration and field experiments, the HVCI configuration that suits best for physicochemical and toxicological characterization of ambient air aerosol particles consists of an Andersen high volume PM 10 -inlet and of three HVCI stages with the 50% collection efficiency at 2.4, 0.9 and 0.2 μm in aerodynamic diameter, respectively.