Particulate matter from car exhaust alters function of human iPSC-derived microglia.

BACKGROUND: Air pollution is recognized as an emerging environmental risk factor for neurological diseases. Large-scale epidemiological studies associate traffic-related particulate matter (PM) with impaired cognitive functions and increased incidence of neurodegenerative diseases such as Alzheimer's disease. Inhaled components of PM may directly invade the brain via the olfactory route, or act through peripheral system responses resulting in inflammation and oxidative stress in the brain. Microglia are the immune cells of the brain implicated in the progression of neurodegenerative diseases. However, it remains unknown how PM affects live human microglia. RESULTS: Here we show that two different PMs derived from exhausts of cars running on EN590 diesel or compressed natural gas (CNG) alter the function of human microglia-like cells in vitro. We exposed human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGLs) to traffic related PMs and explored their functional responses. Lower concentrations of PMs ranging between 10 and 100 µg ml-1 increased microglial survival whereas higher concentrations became toxic over time. Both tested pollutants impaired microglial phagocytosis and increased secretion of a few proinflammatory cytokines with distinct patterns, compared to lipopolysaccharide induced responses. iMGLs showed pollutant dependent responses to production of reactive oxygen species (ROS) with CNG inducing and EN590 reducing ROS production. CONCLUSIONS: Our study indicates that traffic-related air pollutants alter the function of human microglia and warrant further studies to determine whether these changes contribute to adverse effects in the brain and on cognition over time. This study demonstrates human iPSC-microglia as a valuable tool to study functional microglial responses to environmental agents.

Toxicological evaluation and concentration of airborne PM0.1 in high air pollution period in Guangzhou, China.

The emissions and exposure limits for airborne PM0.1 are lacking, with limited scientific data for toxicity. Therefore, we continuously monitored and calculated the number and mass concentrations of airborne PM0.1 in December 2017, January 2018 and March 2018 during the high pollution period in Guangzhou. We collected PM0.1 from the same period and analyzed their chemical components. A549, THP-1 and A549/THP-1 co-cultured cells were selected for exposure to PM0.1, and evaluated for toxicological responses. Our aims are to 1) measure and analyze the number and mass concentrations, and chemical components of PM0.1; 2) evaluate and compare PM0.1 toxicity to different airway cells models at different time points. Guangzhou had the highest mass concentration of PM0.1 in December 2017, while the number concentration was the lowest. Chemical components in PM0.1 vary significantly at different time periods, and the correlation between the chemical composition or source of PM0.1 and the mass and number concentration of PM0.1 was dissimilar. Exposure to PM0.1 disrupted cell membranes, impaired mitochondrial function, promoted the expression of inflammatory mediators, and interfered with DNA replication in the cell cycle. The damage caused by exposure to PM0.1 at different times exhibited variations across different types of cells. PM0.1 in March 2018 stimulated co-cultured cells to secrete more inflammatory mediators, and CMA was significantly related to the expression of them. Our study indicates that it is essential to monitor both the mass and number concentrations of PM0.1 throughout all seasons annually, as conventional toxicological experiments and the internal components of PM0.1 may not effectively reveal the health damages caused by elevated number levels of PM0.1.

Transcriptomic alterations in the olfactory bulb induced by exposure to air pollution: Identification of potential biomarkers and insights into olfactory system function.

This study evaluated how exposure to the ubiquitous air pollution component, ultrafine particles (UFPs), alters the olfactory bulb (OB) transcriptome. The study utilised a whole-body inhalation chamber to simulate real-life conditions and focused on UFPs due to their high translocation and deposition ability in OBs as well as their prevalence in ambient air. Female C57BL/6J mice were exposed to clean air or to freshly generated combustion derived UFPs for two weeks, after which OBs were dissected and mRNA transcripts were investigated using RNA sequencing analysis. For the first time, transcriptomics was applied to determine changes in mRNA expression levels occurring after subacute exposure to UFPs in the OBs. We found forty-five newly described mRNAs to be involved in air pollution-induced responses, including genes involved in odorant binding, synaptic regulation, and myelination signalling pathway, providing new gene candidates for future research. This study provides new insights for the environmental science and neuroscience fields and nominates future research directions.

Air pollution exposure increases ABCB1 and ASCT1 transporter levels in mouse cortex.

Membrane transporters are important for maintaining brain homeostasis by regulating the passage of solutes into, out of, and within the brain. Growing evidence suggests neurotoxic effects of air pollution exposure and its contribution to neurodegenerative disorders, including Alzheimer's disease (AD), yet limited knowledge is available on the exact cellular impacts of exposure. This study investigates how exposure to ubiquitous solid components of air pollution, ultrafine particles (UFPs), influence brain homeostasis by affecting protein levels of membrane transporters. Membrane transporters were quantified and compared in brain cortical samples of wild-type and the 5xFAD mouse model of AD in response to subacute exposure to inhaled UFPs. The cortical ASCT1 and ABCB1 transporter levels were elevated in wild-type and 5xFAD mice subjected to a 2-week UFP exposure paradigm, suggesting impairment of brain homeostatic mechanisms. This study provides new insight on the molecular mechanisms underlying adverse effects of air pollution on the brain.

Black carbon toxicity dependence on particle coating: Measurements with a novel cell exposure method.

Black carbon (BC) is a component of ambient particulate matter which originates from incomplete combustion emissions. BC is regarded as an important short-lived climate forcer, and a significant public health hazard. These two concerns have made BC a focus in aerosol science. Even though, the toxicity of BC particles is well recognized, the mechanism of toxicity for BC as a part of the total gas and particle emission mixture from combustion is still largely unknown and studies concerning it are scarce. In the present study, using a novel thermophoresis-based air-liquid interface (ALI) in vitro exposure system, we studied the toxicity of combustion-generated aerosols containing high levels of BC, diluted to atmospheric levels (1 to 10 µg/m3). Applying multiple different aerosol treatments, we simulated different sources and atmospheric aging processes, and utilizing several toxicological endpoints, we thoroughly examined emission toxicity. Our results revealed that an organic coating on the BC particles increased the toxicity, which was seen as larger genotoxicity and immunosuppression. Furthermore, aging of the aerosol also increased its toxicity. A deeper statistical analysis of the results supported our initial conclusions and additionally revealed that toxicity increased with decreasing particle size. These findings regarding BC toxicity can be applied to support policies and technologies to reduce the most hazardous compositions of BC emissions. Additionally, our study showed that the thermophoretic ALI system is both a suitable and useful tool for toxicological studies of emission aerosols.

Subacute inhalation of ultrafine particulate matter triggers inflammation without altering amyloid beta load in 5xFAD mice.

Epidemiological studies reveal that air pollution exposure may exacerbate neurodegeneration. Ultrafine particles (UFPs) are pollutants that remain unregulated in ambient air by environmental agencies. Due to their small size (<100 nm), UFPs have the most potential to cross the bodily barriers and thus impact the brain. However, little information exists about how UFPs affect brain function. Alzheimer's disease (AD) is the most common form of dementia, which has been linked to air pollutant exposure, yet limited information is available on the mechanistic connection between them. This study aims to decipher the effects of UFPs in the brain and periphery using the 5xFAD mouse model of AD. In our study design, AD mice and their wildtype littermates were subjected to 2-weeks inhalation exposure of UFPs in a whole-body chamber. That subacute exposure did not affect the amyloid-beta accumulation. However, when multiple cytokines were analyzed, we found increased levels of proinflammatory cytokines in the brain and periphery, with a predominant alteration of interferon-gamma in response to UFP exposure in both genotypes. Following exposure, mitochondrial superoxide dismutase was significantly upregulated only in the 5xFAD hippocampi, depicting oxidative stress induction in the exposed AD mouse group. These data demonstrate that short-term exposure to inhaled UFPs induces inflammation without affecting amyloid-beta load. This study provides a better understanding of adverse effects caused by short-term UFP exposure in the brain and periphery, also in the context of AD.

Genotoxic and inflammatory effects of spruce and brown coal briquettes combustion aerosols on lung cells at the air-liquid interface.

Solid fuel usage in residential heating and cooking is one of the largest sources of ambient and indoor air particulate matter, which causes adverse effects on the health of millions of peoples worldwide. Emissions from solid fuel combustion, such as biomass or coal, are detrimental to health, but toxicological responses are largely unknown. In the present study, we compared the toxicological responses regarding cytotoxicity, inflammation and genotoxicity of spruce (SPR) and brown coal briquette (BCB) combustion aerosols on human alveolar epithelial cells (A549) as well as a coculture of A549 and differentiated human monocytic cells (THP-1) into macrophages exposed at the air-liquid interface (ALI). We included both the high emissions from the first hour and moderate emissions from the third hour of the batch combustion experiment in one ALI system, whereas, in the second ALI system, we exposed the cells during the whole 4-hour combustion experiment, including all combustion phases. Physico-chemical properties of the combustion aerosol were analysed both online and offline. Both SPR and BCB combustion aerosols caused mild cytotoxic but notable genotoxic effects in co-cultured A549 cells after one-hour exposure. Inflammatory response analysis revealed BCB combustion aerosols to cause a mild increase in CXCL1 and CXCL8 levels, but in the case of SPR combustion aerosol, a decrease compared to control was observed.

TUBE Project: Transport-Derived Ultrafines and the Brain Effects.

The adverse effects of air pollutants on the respiratory and cardiovascular systems are unquestionable. However, in recent years, indications of effects beyond these organ systems have become more evident. Traffic-related air pollution has been linked with neurological diseases, exacerbated cognitive dysfunction, and Alzheimer's disease. However, the exact air pollutant compositions and exposure scenarios leading to these adverse health effects are not known. Although several components of air pollution may be at play, recent experimental studies point to a key role of ultrafine particles (UFPs). While the importance of UFPs has been recognized, almost nothing is known about the smallest fraction of UFPs, and only >23 nm emissions are regulated in the EU. Moreover, the role of the semivolatile fraction of the emissions has been neglected. The Transport-Derived Ultrafines and the Brain Effects (TUBE) project will increase knowledge on harmful ultrafine air pollutants, as well as semivolatile compounds related to adverse health effects. By including all the major current combustion and emission control technologies, the TUBE project aims to provide new information on the adverse health effects of current traffic, as well as information for decision makers to develop more effective emission legislation. Most importantly, the TUBE project will include adverse health effects beyond the respiratory system; TUBE will assess how air pollution affects the brain and how air pollution particles might be removed from the brain. The purpose of this report is to describe the TUBE project, its background, and its goals.

Inflammatory responses of urban air PM modulated by chemical composition and different air quality situations in Nanjing, China.

The health risks of air pollutants and ambient particulate matter (PM) are widely known. PM composition and toxicity have shown substantial spatiotemporal variability. Yet, the connections between PM composition and toxicological and health effects are vaguely understood. This is a crucial gap in knowledge that needs to be addressed in order to establish air quality guidelines and limit values that consider the chemical composition of PM instead of the current assumption of equal toxicity per inhaled dose. Here, we demonstrate further evidence for varying toxicological effects of urban PM at equal mass concentrations, and estimate how PM composition and emission source characteristics influenced this variation. We exposed a co-culture model mimicking alveolar epithelial cells and macrophages with size-segregated urban ambient PM collected before, during, and after the Nanjing Youth Olympic Games 2014. We measured the release of a set of cytokines, cell cycle alterations, and genotoxicity, and assessed the spatiotemporal variations in these responses by factorial multiple regression analysis. Additionally, we investigated how a previously identified set of emission sources and chemical components affected these variations by mixed model analysis. PM-exposure induced cytokine signaling, most notably by inducing dose-dependent increases of macrophage-regulating GM-CSF and proinflammatory TNFα, IL-6, and IL-1ß concentrations, modest dose-dependent increase for cytoprotective VEGF-A, but very low to no responses for anti-inflammatory IL-10 and immunoregulatory IFNγ, respectively. We observed substantial differences in proinflammatory cytokine production depending on PM sampling period, location, and time of day. The proinflammatory response correlated positively with cell cycle arrest in G1/G0 phase and loss of cellular metabolic activity. Furthermore, PM0.2 caused dose-dependent increases in sub-G1/G0 cells, suggesting increased DNA degradation and apoptosis. Variations in traffic and oil/fuel combustion emissions contributed substantially to the observed spatiotemporal variations of toxicological responses.

Urban air PM modifies differently immune defense responses against bacterial and viral infections in vitro.

Epidemiological evidence has shown the association between exposure to ambient fine particulate matter (PM) and increased susceptibility to bacterial and viral respiratory infections. However, to date, the underlying mechanisms of immunomodulatory effects of PM remain unclear. Our objective was to explore how exposure to relatively low doses of urban air PM alters innate responses to bacterial and viral stimuli in vitro. We used secondary alveolar epithelial cell line along with monocyte-derived macrophages to replicate innate lung barrier in vitro. Co-cultured cells were first exposed for 24 h to PM2.5-1 (particle aerodynamic diameter between 1 and 2.5 µm) and subsequently for an additional 24 h to lipopolysaccharide (TLR4), polyinosinic-polycytidylic acid (TLR3), and synthetic single-stranded RNA oligoribonucleotides (TLR7/8) to mimic bacterial or viral stimulation. Toxicological endpoints included pro-inflammatory cytokines (IL-8, IL-6, and TNF-α), cellular metabolic activity, and cell cycle phase distribution. We show that cells exposed to PM2.5-1 produced higher levels of pro-inflammatory cytokines following stimulation with bacterial TLR4 ligand than cells exposed to PM2.5-1 or bacterial ligand alone. On the contrary, PM2.5-1 exposure reduced pro-inflammatory responses to viral ligands TLR3 and TLR7/8. Cell cycle analysis indicated that viral ligands induced cell cycle arrest at the G2-M phase. In PM-primed co-cultures, however, they failed to induce the G2-M phase arrest. Contrarily, bacterial stimulation caused a slight increase in cells in the sub-G1 phase but in PM2.5-1 primed co-cultures the effect of bacterial stimulation was masked by PM2.5-1. These findings indicate that PM2.5-1 may alter responses of immune defense differently against bacterial and viral infections. Further studies are required to explain the mechanism of immune modulation caused by PM in altering the susceptibility to respiratory infections.

Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke.

BACKGROUND: Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. METHODS: We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. RESULTS: We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m- 3, 41 mg MJ- 1) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m- 3, 26 mg MJ- 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. CONCLUSIONS: Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects.

Toxicological evaluation of exhaust emissions from light-duty vehicles using different fuel alternatives in sub-freezing conditions.

BACKGROUND: Emissions from road traffic are under constant discussion since they pose a major threat to human health despite the increasingly strict emission targets and regulations. Although the new passenger car regulations have been very effective in reducing the particulate matter (PM) emissions, the aged car fleet in some EU countries remains a substantial source of PM emissions. Moreover, toxicity of PM emissions from multiple new types of bio-based fuels remain uncertain and different driving conditions such as the sub-zero running temperature has been shown to affect the emissions. Overall, the current literature and experimental knowledge on the toxicology of these PM emissions and conditions is scarce. METHODS: In the present study, we show that exhaust gas PM from newly regulated passenger cars fueled by different fuels at sub-zero temperatures, induce toxicological responses in vitro. We used exhaust gas volume-based PM doses to give us better insight on the real-life exposure and included one older diesel car to estimate the effect of the new emissions regulations. RESULTS: In cars compliant with the new regulations, gasoline (E10) displayed the highest PM concentrations and toxicological responses, while the higher ethanol blend (E85) resulted in slightly lower exhaust gas PM concentrations and notably lower toxicological responses in comparison. Engines powered by modern diesel and compressed natural gas (CNG) yielded the lowest PM concentrations and toxicological responses. CONCLUSIONS: The present study shows that toxicity of the exhaust gas PM varies depending on the fuels used. Additionally, concentration and toxicity of PM from an older diesel car were vastly higher, compared to contemporary vehicles, indicating the beneficial effects of the new emissions regulations.

Air quality intervention during the Nanjing youth olympic games altered PM sources, chemical composition, and toxicological responses.

Ambient particulate matter (PM) is a leading global environmental health risk. Current air quality regulations are based on airborne mass concentration. However, PM from different sources have distinct chemical compositions and varied toxicity. Connections between emission control measures, air quality, PM composition, and toxicity remain insufficiently elucidated. The current study assessed the composition and toxicity of PM collected in Nanjing, China before, during, and after an air quality intervention for the 2014 Youth Olympic Games. A co-culture model that mimics the alveolar epithelium with the associated macrophages was created using A549 and THP-1 cells. These cells were exposed to size-segregated inhalable PM samples. The composition and toxicity of the PM samples were influenced by several factors including seasonal variation, emission sources, and the air quality intervention. For example, we observed a size-dependent shift in particle mass concentrations during the air quality intervention with an emphasized proportion of smaller particles (PM2.5) present in the air. The roles of industrial and fuel combustion and traffic emissions were magnified during the emission control period. Our analyses revealed that the PM samples demonstrated differential cytotoxic potencies at equal mass concentrations between sampling periods, locations, and time of day, influenced by variations in the predominant emission sources. Coal combustion and industrial emissions were the most important sources affecting the toxicological responses and displayed the least variation in emission contributions between the sampling periods. In conclusion, emission control mitigated cytotoxicity and oxidative stress for particles larger than 0.2 µm, but there was inadequate evidence to determine if it was the key factor reducing the harmful effects of PM0.2.

Glial smog: Interplay between air pollution and astrocyte-microglia interactions.

Every second we inhale a danger in the air; many particles in the atmosphere can influence our lives. Outdoor air pollution, especially particulate matter is the largest environmental risk factor and has been associated with many cardiovascular and lung diseases. Importantly, air pollution has recently been discovered to also impact the brain. Here, we review the effects of air pollution on glial cells of the brain, astrocytes and microglia, and the tightly controlled interplay between these cell types. We focus on how traffic related air pollutants which include both gaseous and particulate emissions and their secondary products influence the intercellular communication of microglia and astrocytes. Finally, we place these air pollution and glial interactions in a larger context by discussing their impact on neurodegeneration.

Human airway construct model is suitable for studying transcriptome changes associated with indoor air particulate matter toxicity.

In vitro models mimicking the human respiratory system are essential when investigating the toxicological effects of inhaled indoor air particulate matter (PM). We present a pulmonary cell culture model for studying indoor air PM toxicity. We exposed normal human bronchial epithelial cells, grown on semi-permeable cell culture membranes, to four doses of indoor air PM in the air-liquid interface. We analyzed the chemokine interleukin-8 concentration from the cell culture medium, protein concentration from the apical wash, measured tissue electrical resistance, and imaged airway constructs using light and transmission electron microscopy. We sequenced RNA using a targeted RNA toxicology panel for 386 genes associated with toxicological responses. PM was collected from a non-complaint residential environment over 1 week. Sample collection was concomitant with monitoring size-segregated PM counts and determination of microbial levels and diversity. PM exposure was not acutely toxic for the cells, and we observed up-regulation of 34 genes and down-regulation of 17 genes when compared to blank sampler control exposure. The five most up-regulated genes were related to immunotoxicity. Despite indications of incomplete cell differentiation, this model enabled the comparison of a toxicological transcriptome associated with indoor air PM exposure.

Integrating farm and air pollution studies in search for immunoregulatory mechanisms operating in protective and high-risk environments.

BACKGROUND: Studies conducted in farm environments suggest that diverse microbial exposure promotes children's lung health. The underlying mechanisms are unclear, and the development of asthma-preventive strategies has been delayed. More comprehensive investigation of the environment-induced immunoregulation is required for better understanding of asthma pathogenesis and prevention. Exposure to air pollution, including particulate matter (PM), is a risk factor for asthma, thus providing an excellent counterpoint for the farm-effect research. Lack of comparable data, however, complicates interpretation of the existing information. We aimed to explore the immunoregulatory effects of cattle farm dust (protective, Finland) and urban air PM (high-risk, China) for the first time using identical research methods. METHODS: We stimulated PBMCs of 4-year-old children (N = 18) with farm dust and size-segregated PM and assessed the expression of immune receptors CD80 and ILT4 on dendritic cells and monocytes as well as cytokine production of PBMCs. Environmental samples were analysed for their composition. RESULTS: Farm dust increased the percentage of cells expressing CD80 and the cytokine production of children's immune cells, whereas PM inhibited the expression of important receptors and the production of soluble mediators. Although PM samples induced parallel immune reactions, the size-fraction determined the strength of the effects. CONCLUSIONS: Our study demonstrates the significance of using the same research framework when disentangling shared and distinctive immune pathways operating in different environments. Observed stimulatory effects of farm dust and inhibitory effects of PM could shape responses towards respiratory pathogens and allergens, and partly explain differences in asthma prevalence between studied environments.

Emissions and atmospheric processes influence the chemical composition and toxicological properties of urban air particulate matter in Nanjing, China.

Ambient inhalable particulate matter (PM) is a serious health concern worldwide, but especially so in China where high PM concentrations affect huge populations. Atmospheric processes and emission sources cause spatial and temporal variations in PM concentration and chemical composition, but their influence on the toxicological characteristics of PM are still inadequately understood. In this study, we report an extensive chemical and toxicological characterization of size-segregated urban air inhalable PM collected in August and October 2013 from Nanjing, and assess the effects of atmospheric processes and likely emission sources. A549 human alveolar epithelial cells were exposed to day- and nighttime PM samples (25, 75, 150, 200, 300 µg/ml) followed by analyses of cytotoxicity, genotoxicity, cell cycle, and inflammatory response. PM10-2.5 and PM0.2 caused the greatest toxicological responses for different endpoints, illustrating that particles with differing size and chemical composition activate distinct toxicological pathways in A549 cells. PM10-2.5 displayed the greatest oxidative stress and genotoxic responses; both were higher for the August samples compared with October. In contrast, PM0.2 and PM2.5-1.0 samples displayed high cytotoxicity and substantially disrupted cell cycle; August samples were more cytotoxic whereas October samples displayed higher cell cycle disruption. Several components associated with combustion, traffic, and industrial emissions displayed strong correlations with these toxicological responses. The lower responses for PM1.0-0.2 compared to PM0.2 and PM2.5-1.0 indicate diminished toxicological effects likely due to aerosol aging and lower proportion of fresh emission particles rich in highly reactive chemical components in the PM1.0-0.2 fraction. Different emission sources and atmospheric processes caused variations in the chemical composition and toxicological responses between PM fractions, sampling campaigns, and day and night. The results indicate different toxicological pathways for coarse-mode particles compared to the smaller particle fractions with typically higher content of combustion-derived components. The variable responses inside PM fractions demonstrate that differences in chemical composition influence the induced toxicological responses.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

BACKGROUND: In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. METHODS: We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. RESULTS: We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. CONCLUSIONS: All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Particulate emissions from modern and old technology wood combustion induce distinct time-dependent patterns of toxicological responses in vitro.

Toxicological characterisation of combustion emissions in vitro are often conducted with macrophage cell lines, and the majority of these experiments are based on responses measured at 24h after the exposure. The aim of this study was to investigate how significant role time course plays on toxicological endpoints that are commonly measured in vitro. The RAW264.7 macrophage cell line was exposed to PM1 samples (150µg/ml) from biomass combustion devices representing old and modern combustion technologies for 2, 4, 8, 12, 24 and 32h. After the exposure, cellular metabolic activity, cell membrane integrity, cellular DNA content, DNA damage and production of inflammatory markers were assessed. The present study revealed major differences in the time courses of the responses, statistical differences between the studied samples mostly limiting to differences between modern and old technology samples. Early stage responses consisted of disturbances in metabolic activity and cell membrane integrity. Middle time points revealed increases in chemokine production, whereas late-phase responses exhibited mostly increased DNA-damage, decreased membrane integrity and apoptotic activity. Altogether, these results implicate that the time point of measurement has to be considered carefully, when the toxicity of emission particles is characterised in in vitro study set-ups.

Particulate emissions from the combustion of birch, beech, and spruce logs cause different cytotoxic responses in A549 cells.

According to the World Health Organization particulate emissions from the combustion of solid fuels caused more than 110,000 premature deaths worldwide in 2010. Log wood combustion is the most prevalent form of residential biomass heating in developed countries, but it is unknown how the type of wood logs used in furnaces influences the chemical composition of the particulate emissions and their toxicological potential. We burned logs of birch, beech and spruce, which are used commonly as firewood in Central and Northern Europe in a modern masonry heater, and compared them to the particulate emissions from an automated pellet boiler fired with softwood pellets. We determined the chemical composition (elements, ions, and carbonaceous compounds) of the particulate emissions with a diameter of less than 1 µm and tested their cytotoxicity, genotoxicity, inflammatory potential, and ability to induce oxidative stress in a human lung epithelial cell line. The chemical composition of the samples differed significantly, especially with regard to the carbonaceous and metal contents. Also the toxic effects in our tested endpoints varied considerably between each of the three log wood combustion samples, as well as between the log wood combustion samples and the pellet combustion sample. The difference in the toxicological potential of the samples in the various endpoints indicates the involvement of different pathways of toxicity depending on the chemical composition. All three emission samples from the log wood combustions were considerably more toxic in all endpoints than the emissions from the pellet combustion. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1487-1499, 2017.