Cytokine responses induced by diesel exhaust particles are suppressed by PAR-2 silencing and antioxidant treatment, and driven by polar and non-polar soluble constituents.

Adsorbed soluble organics seem to be the main drivers of inflammatory responses induced by diesel exhaust particles (DEP). The specific compounds contributing to this process and the cellular mechanisms behind DEP-induced inflammation are not well known. We have assessed pro-inflammatory effects of DEP and various soluble DEP fractions, in human bronchial epithelial cells (BEAS-2B). DEP increased the expression of interleukin (IL)-6 and CXCL8. Silencing of the aryl hydrocarbon receptor (AhR) by siRNA or pretreatment with AhR-antagonists did not attenuate DEP-induced IL-6 and CXCL8 responses. However, the halogenated aromatic hydrocarbon (HAH)-selective AhR antagonist CH223191 caused a considerable reduction in DEP-induced CYP1A1 expression indicating that this response may be due to dioxin or dioxin-like constituents in DEP. Knock-down of protease activated receptor (PAR)-2 attenuated IL-6 responses without affecting CXCL8. Antioxidants did not affect IL-6 expression after 4h DEP-exposure and only partly reduced CXCL8 expression. However, after 24h exposure antioxidant treatment partly suppressed IL-6 protein release and completely blocked CXCL8 release. Furthermore, a heptane-soluble (non-polar) extract of DEP induced both IL-6 and CXCL8 release, whereas a PBS-soluble (highly polar) extract induced only IL-6. Thus, pro-inflammatory responses in DEP-exposed epithelial cells appear to be the result of both reactive oxygen species and receptor signaling, mediated through combinatorial effects between both non-polar and polar constituents adhered to the particle surface.

Differential proinflammatory responses induced by diesel exhaust particles with contrasting PAH and metal content.

Exposure to diesel engine exhaust particles (DEPs), representing a complex and variable mixture of components, has been linked with cellular production and release of several types of mediators related to pulmonary inflammation. A key challenge is to identify the specific components, which may be responsible for these effects. The aim of this study was to compare the proinflammatory potential of two DEP-samples with contrasting contents of polycyclic aromatic hydrocarbons (PAHs) and metals. The DEP-samples were compared with respect to their ability to induce cytotoxicity, expression and release of proinflammatory mediators (IL-6, IL-8), activation of mitogen-activated protein kinases (MAPKs) and expression of CYP1A1 and heme oxygenase-1 (HO-1) in human bronchial epithelial (BEAS-2B) cells. In addition, dithiothreitol and ascorbic acid assays were performed in order to examine the oxidative potential of the PM samples. The DEP-sample with the highest PAH and lowest metal content was more potent with respect to cytotoxicity and expression and release of proinflammatory mediators, CYP1A1 and HO-1 expression and MAPK activation, than the DEP-sample with lower PAH and higher metal content. The DEP-sample with the highest PAH and lowest metal content also possessed a greater oxidative potential. The present results indicate that the content of organic components may be determinant for the proinflammatory effects of DEP. The findings underscore the importance of considering the chemical composition of particulate matter-emissions, when evaluating the potential health impact and implementation of air pollution regulations.

The occurrence of polycyclic aromatic hydrocarbons and their derivatives and the proinflammatory potential of fractionated extracts of diesel exhaust and wood smoke particles.

Exposure to combustion emissions, including diesel engine exhaust and wood smoke particles (DEPs and WSPs), has been associated with inflammatory responses. To investigate the possible role of polycyclic aromatic hydrocarbons (PAHs) and PAH-derivatives, the DEPs and WSPs methanol extracts were fractionated by solid phase extraction (SPE), and the fractions were analyzed for more than ∼120 compounds. The pro-inflammatory effects of the fractionated extracts were characterized by exposure of bronchial epithelial lung cells (BEAS-2B). Both native DEPs and WSPs caused a concentration-dependent increase in IL-6 and IL-8 release and cytotoxicity. This is consistent with the finding of a rather similar total content of PAHs and PAH-derivatives. Yet, the samples differed in specific components, suggesting that different species contribute to the toxicological response in these two types of particles. The majority of the IL-6 release and cytotoxicity was induced upon exposure to the most polar (methanol) SPE fraction of extracts from both samples. In these fractions hydroxy-PAHs, carboxy-PAHs were observed along with nitro-amino-PAHs in DEP. However, the biological effects induced by the polar fractions could not be attributed only to the occurrence of PAH-derivatives. The present findings indicate a need for further characterization of organic extracts, beyond an extensive analysis of commonly suspected PAH and PAH-derivatives. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Environmental Science and Health, Part A, to view the supplemental file.

The challenge of obtaining correct data for cellular release of inflammatory mediators after in vitro exposure to particulate matter.

Exposure to ambient particulate matter (PM) has been associated with adverse cardiopulmonary effects where inflammation seems to play an important role. Cellular release of inflammatory mediators is therefore commonly measured in in vitro studies of PM-induced effects. However, adsorption of such mediators to PM may interfere with these measurements and thereby possibly also influence the conclusions of such studies. The aim of the present mini review is to provide the reader with an update on what is currently known about adsorption of inflammatory mediators to PM. We also present a step-by-step method for correction of in vitro results, based on mediator adsorption experiments. Moreover, mediator adsorption and its possible consequences are exemplified with a case study demonstrating adsorption of long pentraxin 3 (PTX3) and vascular endothelial growth factor (VEGF) to a selection of PM samples. The highest degree of adsorption was determined to be 65 and 95% for PTX3 and VEGF respectively, and for the various PM samples the degree of adsorption was highly variable. In conclusion, the data and results discussed in this review underscore the importance of assessing and correcting for mediator adsorption, especially in studies involving comparison of effects induced by several different PM samples.

Cell toxicity and oxidative potential of engine exhaust particles: impact of using particulate filter or biodiesel fuel blend.

The link between emissions of vehicular particulate matter (PM) and adverse health effects is well established. However, the influence of new emission control technologies and fuel types on both PM emissions and health effects has been less well investigated. We examined the health impact of PM emissions from two vehicles equipped with or without a diesel particulate filter (DPF). Both vehicles were powered either with diesel (B0) or a 50% v/v biodiesel blend (B50). The DPF effectively decreased PM mass emissions (∼85%), whereas the fuel B50 without DPF lead to less reduction (∼50%). The hazard of PM per unit distance driven was decreased for the DPF-equipped vehicle as indicated by a reduced cytotoxicity, oxidative, and pro-inflammatory potential. This was not evident and even led to an increase when the hazard was expressed on a per unit of mass basis. In general, the PM oxidative potential was similar or reduced for the B50 compared to the B0 powered vehicle. However, the use of B50 resulted in increased cytotoxicity and IL-6 release in BEAS-2B cells irrespective of the expression metric. This study shows that PM mass reduction achieved by the use of B50 will not necessarily decrease the hazard of engine emissions, while the application of a DPF has a beneficial effect on both PM mass emission and PM hazard.

Diesel exhaust particles induce CYP1A1 and pro-inflammatory responses via differential pathways in human bronchial epithelial cells.

BACKGROUND: Exposure to diesel engine exhaust particles (DEPs) has been associated with several adverse health outcomes in which inflammation seems to play a key role. DEPs contain a range of different inorganic and organic compounds, including polycyclic aromatic hydrocarbons (PAHs). During the metabolic activation of PAHs, CYP1A1 enzymes are known to play a critical role. In the present study we investigated the potential of a characterised sample of DEPs to induce cytotoxicity, to influence the expression of CYP1A1 and inflammation-related genes, and to activate intracellular signalling pathways, in human bronchial epithelial cells. We specifically investigated to what extent DEP-induced expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 was regulated differentially from DEP-induced expression of CYP1A1. RESULTS: The cytotoxicity of the DEPs was characterised by a marked time- and concentration-dependent increase in necrotic cells at 4 h and above 200 µg/ml (~ 30 µg/cm2). DEP-induced DNA-damage was only apparent at high concentrations (≥ 200 µg/ml). IL-6, IL-8 and COX-2 were the three most up-regulated genes by the DEPs in a screening of 20 selected inflammation-related genes. DEP-induced expression of CYP1A1 was detected at very low concentrations (0.025 µg/ml), compared to the expression of IL-6, IL-8 and COX-2 (50-100 µg/ml). A CYP1A1 inhibitor (α-naphthoflavone), nearly abolished the DEP-induced expression of IL-8 and COX-2. Of the investigated mitogen-activated protein kinases (MAPKs), the DEPs induced activation of p38. A p38 inhibitor (SB202190) strongly reduced DEP-induced expression of IL-6, IL-8 and COX-2, but only moderately affected the expression of CYP1A1. The DEPs also activated the nuclear factor-κB (NF-κB) pathway, and suppression by siRNA tended to reduce the DEP-induced expression of IL-8 and COX-2, but not CYP1A1. CONCLUSION: The present study indicates that DEPs induce both CYP1A1 and pro-inflammatory responses in vitro, but via differential intracellular pathways. DEP-induced pro-inflammatory responses seem to occur via activation of NF-κB and p38 and are facilitated by CYP1A1. However, the DEP-induced CYP1A1 response does not seem to involve NF-κB and p38 activation. Notably, the present study also indicates that expression of CYP1A1 may represent a particular sensitive biomarker of DEP-exposure.

Pulmonary and cardiovascular effects of traffic-related particulate matter: 4-week exposure of rats to roadside and diesel engine exhaust particles.

Traffic-related particulate matter (PM) may play an important role in the development of adverse health effects, as documented extensively in acute toxicity studies. However, rather little is known about the impacts of prolonged exposure to PM. We hypothesized that long-term exposure to PM from traffic adversely affects the pulmonary and cardiovascular system through exacerbation of an inflammatory response. To examine this hypothesis, Fisher F344 rats, with a mild pulmonary inflammation at the onset of exposure, were exposed for 4 weeks, 5 days/week for 6 h a day to: (a) diluted diesel engine exhaust (PM(DEE)), or: (b) near roadside PM (PM(2.5)). Ultrafine particulates, which are largely present in diesel soot, may enter the systemic circulation and directly or indirectly trigger cardiovascular effects. Hence, we assessed the effects of traffic-related PM on pulmonary inflammation and activity of procoagulants, vascular function in arteries, and cytokine levels in the heart 24 h after termination of the exposures. No major adverse health effects of prolonged exposure to traffic-related PM were detected. However, some systemic effects due to PM(DEE) exposure occurred including decreased numbers of white blood cells and reduced von Willebrand factor protein in the circulation. In addition, lung tissue factor activity is reduced in conjunction with reduced lung tissue thrombin generation. To what extent these alterations contribute to thrombotic effects and vascular diseases remains to be established. In conclusion, prolonged exposure to traffic-related PM in healthy animals may not be detrimental due to various biological adaptive response mechanisms.

Mechanisms involved in ultrafine carbon black-induced release of IL-6 from primary rat epithelial lung cells.

The aims of the present study were to establish to what extent IL-1, and intracellular pathways involving mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB), play a role in ultrafine particle-induced release of IL-6 by primary rat epithelial lung cells. Ultrafine carbon black (Printex 90) induced a concentration- and time-dependent increase in the release of IL-1alpha, IL-1beta and IL-6. The ultrafine carbon black-induced release of IL-6 was completely eliminated by an IL-1 receptor antagonist (IL-1ra). Cellular release of IL-1alpha, IL-1beta and IL-6 was significantly attenuated by curcumin and by inhibitors of the MAPKs ERK1/2 (PD98069), p38 (SB202190) and JNK (SP600125), whereas pyrrolidine dithiocarbamate (PDTC) attenuated the release of IL-6, but not of IL-1alpha and IL-1beta. The effects of curcumin and PDTC may indicate an involvement of NF-kappaB. Furthermore, ultrafine carbon black induced degradation of IkappaBalpha, used as an indicator of NF-kappaB activation, and induced phosphorylation of ERK1/2, p38 and JNK1/2. This degradation and phosphorylation was attenuated by IL-1ra. The present findings provide more insight into the largely unknown mechanisms involved in ultrafine particle-induced release of cytokines from lung cells. The findings suggest that ultrafine carbon black-induced release of IL-6 strongly depends on IL-1 and that activation of MAPKs and NF-kappaB is involved in this response.

Particle-induced cytokine responses in cardiac cell cultures--the effect of particles versus soluble mediators released by particle-exposed lung cells.

Increased levels of particulate matter have been associated with adverse effects in the respiratory as well as the cardiovascular system. The biological mechanisms behind these associations are still unresolved. Among potential mechanisms, particulate matter-associated cardiac effects may be initiated by inhaled small-sized particles, particle components and/or mediators related to inflammation that translocate into the pulmonary circulation. In the present study cytokine responses (interleukin [IL]-6, IL-1beta, and tumor necrosis factor [TNF]-alpha) of primary rat cardiomyocytes and cardiofibroblasts in mono- and cocultures induced by direct exposure to particles, were compared with cytokine responses induced by mediators released by particle-exposed primary rat epithelial lung cells (conditioned media). Cells were exposed to a model ultrafine particle (ultrafine carbon black, Printex 90) and in selected experiments to an urban air particle sample (SRM 1648, St Louis, MO). In lung cell cultures both particle types induced release of IL-6 and IL-1beta, whereas TNF-alpha was only detected upon exposure to St Louis particles. The release of IL-6 by cardiac cells was strongly enhanced upon exposure to conditioned media, and markedly exceeded the response to direct particle exposure. IL-1, but not TNF-alpha, seemed necessary, but not sufficient, for this enhanced IL-6 release. The role of IL-1 was demonstrated by use of an IL-1 receptor antagonist that partially reduced the effect of the conditioned media, and by a stimulating effect on the cardiac cell release of IL-6 by exogenous addition of IL-1alpha and IL-1beta. These in vitro findings lend support to the hypothesis that particle-induced cardiac inflammation and disease may involve lung-derived mediators.

Pro-inflammatory potential of ultrafine particles in mono- and co-cultures of primary cardiac cells.

Inhalation of particulate air pollution has been associated with increased risks for cardiovascular mortality and morbidity, but the underlying mechanisms are still under discussion. One possible pathway may be that inhaled particles cross the air-blood barrier and interact directly with cardiac tissue. The aim of the present study was to examine the pro-inflammatory potential of particles in cardiac cells. Mono- and co-cultures of primary adult male Wistar (Han) rat cardiomyocytes (CMs) and cardiofibroblasts (CFs) were exposed to increasing concentrations of ultrafine (<100nm) carbon black particles (Printex 90). Expression and release of cytokines (IL-6, IL-1beta and TNF-alpha) were measured by using quantitative real-time PCR and ELISA, respectively. Cytotoxicity was estimated by measuring cellular release of lactate dehydrogenase (LDH). A particle concentration-dependent increase in IL-6 release was observed in both CM mono- and co-cultures (EC(50) approximately 57microg/ml). Furthermore, IL-6 levels detected in both control and particle-exposed co-cultures were synergistically increased compared to mono-cultures (10-19-fold, dependent on the exposure). Experiments with contact and non-contact co-cultures indicate that direct cellular contact is of key importance for the enhanced release of IL-6 in co-cultures. An apparent particle-induced release of IL-1beta was only detected in co-cultures. The release of TNF-alpha was low and did not seem notably influenced by particle exposure. Treatment with an IL-1 receptor antagonist apparently eliminated the particle-induced release of IL-6. In conclusion, ultrafine particles have a pro-inflammatory potential in primary cardiac cells. Furthermore, IL-1 seems critical in triggering particle-induced release of IL-6. These pro-inflammatory responses may be elicited when particles are translocated into the pulmonary circulation upon inhalation or administered intravascularly during medical procedures.

From science to policy--when are scientific results certain enough?

The amount of scientific findings linking air pollution with adverse health effects is continuously growing and indicates a need for action to improve air quality. On 21 September 2005, the European Commission published a new draft directive on air quality, as part of the Thematic Strategy on air pollution. This is a long-term plan on how to reduce air pollution in the European Union in the next 15 years. Immediately after its release, the Commission received criticism for not going far enough from various instances, such as research institutions and nongovernmental organizations concerned with health and environmental effects of air pollution. One policy argument for not taking full measures was the argument of scientific uncertainty. In light of this air quality strategy and the corresponding criticism which ensued, the present article discusses how the ambiguity of scientific uncertainty may contribute to impeding the process of translating scientific findings into concrete policy options. As complete certainty is likely to never be achieved, the question arises whether it is possible to determine and agree on clear and applicable definitions of certain levels of scientific certainty. The case referred to in this paper clearly demonstrates a situation with discordant views on the uncertainty of scientific findings. More discussion on how to define scientific uncertainty and how to deal with it would be beneficial for both the scientific and the political communities. Finally, it is important to recognize that scientific evidence is not the only driver influencing policy decisions.