Pasi I. Jalava

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 (10u2009mg/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 24u2009h 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.

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 24u2009h. 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.

Pulmonary inflammation and tissue damage in the mouse lung after exposure to PM samples from biomass heating appliances of old and modern technologies

Current levels of ambient air fine particulate matter (PM(2.5)) are associated with mortality and morbidity in urban populations worldwide. In residential areas wood combustion is one of the main sources of PM(2.5) emissions, especially during wintertime. However, the adverse health effects of particulate emissions from the modern heating appliances and fuels are poorly known. In this study, health related toxicological properties of PM(1) emissions from five modern and two old technology appliances were examined. The PM(1) samples were collected by using a Dekati® Gravimetric Impactor (DGI). The collected samples were weighed and extracted with methanol for chemical and toxicological analyses. Healthy C57BL/6J mice were intratracheally exposed to a single dose of 1, 3, 10 or 15 mg/kg of the particulate samples for 4, 18 or 24h. Thereafter, the lungs were lavaged and bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation, cytotoxicity and genotoxicity. Lungs of 24h exposed mice were collected for inspection of pulmonary tissue damage. There were substantial differences in the combustion qualities of old and modern technology appliances. Modern technology appliances had the lowest PM(1) (mg/MJ) emissions, but they induced the highest inflammatory, cytotoxic and genotoxic activities. In contrast, old technology appliances had clearly the highest PM(1) (mg/MJ) emissions, but their effect in the mouse lungs were the lowest. Increased inflammatory activity was associated with ash related components of the emissions, whereas high PAH concentrations were correlating with the smallest detected responses, possibly due to their immunosuppressive effect.

Seasonal variation in chemical composition of size-segregated urban air particles and the inflammatory activity in the mouse lung

We investigated the seasonal variations in the chemical composition and in vivo inflammatory activity of urban air particulate samples in four size ranges (PM10–2.5, PM2.5–1, PM1–0.2, and PM0.2). The samples were collected in Helsinki using a high-volume cascade impactor (HVCI). Healthy C57BL/6J mice were intratracheally instilled with a single dose (10u2009mg/kg) of the particulate samples. The lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6, and keratinocyte-derived chemokine [KC]) at 4u2009h, and total cell number and total protein concentration at 12u2009h. The PM10–2.5 and PM2.5–1 samples had much higher inflammatory potency than the PM1–0.2 and PM0.2 samples. The relative inflammatory activities of the autumn samples were the highest on an equal mass basis, but when estimated for the particulate mass per cubic meter of air, the springtime samples had the highest inflammatory potential. Resuspended soil material and other non-exhaust particulate material from traffic were associated with a high inflammatory activity of the PM10–2.5 and PM2.5–1 samples. Secondary inorganic ions in the PM1–0.2 and PM0.2 samples had inconsistent negative or positive correlations with the inflammatory activity. There were no systematic seasonal variations in the tracers of incomplete combustion and atmospherically oxidized organics in the PM1–0.2 and PM0.2 samples, which probably explains their low correlations with the inflammatory activity. In conclusion, in a relatively clean Nordic city, the resuspension of road dust and other non-exhaust particulate material from traffic were the major sources of inflammatory activity of urban air inhalable particles.

Toxicological properties of emission particles from heavy duty engines powered by conventional and bio-based diesel fuels and compressed natural gas

BackgroundOne of the major areas for increasing the use of renewable energy is in traffic fuels e.g. bio-based fuels in diesel engines especially in commuter traffic. Exhaust emissions from fossil diesel fuelled engines are known to cause adverse effects on human health, but there is very limited information available on how the new renewable fuels may change the harmfulness of the emissions, especially particles (PM). We evaluated the PM emissions from a heavy-duty EURO IV diesel engine powered by three different fuels; the toxicological properties of the emitted PM were investigated. Conventional diesel fuel (EN590) and two biodiesels were usedu2009−u2009rapeseed methyl ester (RME, EN14214) and hydrotreated vegetable oil (HVO) either as such or as 30% blends with EN590. EN590 and 100% HVO were also operated with or without an oxidative catalyst (DOCu2009+u2009POC). A bus powered by compressed natural gas (CNG) was included for comparison with the liquid fuels. However, the results from CNG powered bus cannot be directly compared to the other situations in this study.ResultsHigh volume PM samples were collected on PTFE filters from a constant volume dilution tunnel. The PM mass emission with HVO was smaller and with RME larger than that with EN590, but both biofuels produced lower PAH contents in emission PM. The DOCu2009+u2009POC catalyst greatly reduced the PM emission and PAH content in PM with both HVO and EN590. Dose-dependent TNFα and MIP-2 responses to all PM samples were mostly at the low or moderate level after 24-hour exposure in a mouse macrophage cell line RAW 264.7. Emission PM from situations with the smallest mass emissions (HVOu2009+u2009cat and CNG) displayed the strongest potency in MIP-2 production. The catalyst slightly decreased the PM-induced TNFα responses and somewhat increased the MIP-2 responses with HVO fuel. Emission PM with EN590 and with 30% HVO blended in EN590 induced the strongest genotoxic responses, which were significantly greater than those with EN590u2009+u2009cat or 100% HVO. The emission PM sample from the CNG bus possessed the weakest genotoxic potency but had the strongest oxidative potency of all the fuel and catalyst combinations. The use of 100% HVO fuel had slightly weaker and 100% RME somewhat stronger emission PM induced ROS production, when compared to EN590.ConclusionsThe harmfulness of the exhaust emissions from vehicle engines cannot be determined merely on basis of the emitted PM mass. The study conditions and the engine type significantly affect the toxicity of the emitted particles. The selected fuels and DOCu2009+u2009POC catalyst affected the PM emission from the heavy EURO IV engine both qualitative and quantitative ways, which influenced their toxicological characteristics. The plain HVO fuel performed very well in emission reduction and in lowering the overall toxicity of emitted PM, but the 30% blend of HVO in EN590 was no better in this respect than the plain EN590. The HVO with a DOCu2009+u2009POC catalyst in the EURO IV engine, performed best with regard to changes in exhaust emissions. However some of the toxicological parameters were significantly increased even with these low emissions.