Inflammation by Mixtures - Exploring the fundamentals of proinflammatory responses by complex combustion-derived pollutants

Diesel exhaust particles (DEP) and other combustion particles constitute an important fraction of ambient air pollutants in urban environments. Such particles may cause or exacerbate inflammatory responses in the body. Chronic, low-grade inflammation is considered a central mechanism for development of a variety of diseases, such as cardiovascular disease, asthma, allergies, cancer and diabetes. DEP constitute a complex mixture of thousands of different organic chemicals bound to the surface of nano-sized carbon particles. The organic chemicals are considered to be the primary cause of the proinflammatory reactions in the cells. However, it still remains unclear what types of compounds or compounds groups contribute the most to these effects. Clarifying this is a main goal of the present project. Organic chemicals from two different DEPs and one wood smoke particle (WSP) have been extracted/fractionated by two different techniques. From each of the three particles, 9 fractions of organic chemicals have been extracted and separated according to polarity (from very water soluble to very lipophilic fractions). The chemical composition of these 27 fractions is now characterized with respect to organic carbon content, distribution of polycyclic aromatic hydrocarbons (PAHs) and their oxidation products as well as other major classes of compounds including alkanes, carboxylic acids, aldehydes and high molecular weight species. The 27 fractions have been tested for effects on general cytotoxicity and inflammatory effects human lung cells and vascular endothelial cells, as well as in a 3D-model of the alveolar/endothelial-barrier. Our results suggest that lipophilic (fat soluble) constituents are rapidly released form particles deposited in the alveoli, and translocates across the epithelial layer reaching the vascular endothelium. Lipophilic compounds also appeared to be most important for cytotoxicity (cell death), oxidative stress, and activation of proinflammatory genes. Oxidative stress, which has been considered central in the toxicity of particulate matter, seems, however, to be a high-dose phenomenon that may be less relevant for real-life exposure concentrations. Some hydrophilic (water soluble) constituents may also seem to contribute to proinflammatory responses. Moreover, inflammatory reactions was only induced by extracts from DEP and not WSP. However, extracts of both DEP and WSP exacerbated existing proinflammatory processes. This is of particular interest as lower concentrations of air pollution primarily is considered to affect people with existing lung- and pulmonary diseases, conditions often involving chronic inflammation. A sub-selection of fractions from the DEP with the highest organic content have been examined for effects on calcium signaling and regulation of proinflammatory genes in human endothelial HMEC-1 cells at the University of Rennes, France. The results indicate that only some of the fractions of organic chemicals from DEP may stimulate calcium signaling. These effects are now being linked to activation of specific receptors and calcium channels. The ability to induce strong activation of proinflammatory genes appear to be associated with the ability to stimulate calcium signaling. Activation of calcium signaling appears to be a particularly sensitive response activated at low concentrations. The mechanisms of calcium signaling was therefore examined in detail. The role of the aryl hydrocarbon receptor (AhR), which is activated by PAhs and other lipophilic compounds, appeared to play a key role in the regulation of these calcium responses. AhR also appeared to be involved in the proinflammatory effects in endothelial cells, indicating that calcium responses and inflammation at least partly could be triggered through comparable mechanisms. The results suggest that lipophilic chemicals are central for the biological effects. The role of AhR further suggest that PAHs may be among the key drivers of the observed effects, but that the traditional carcinogenic PAHs not necessarily are the most important. Experiments with pyrene, a non-carcinogenic PAH found to occur in relatively high levels in combustion particles, appeared to confirm this. This sheds new light on the potential toxicities of non-carcinogenic PAHs, and may have implications for future risk assessment of PAHs in outdoor air. However, the results suggest that the effects to a considerable degree are due to interaction between different constituents in DEP and wood smoke particles. Clarifying how chemicals in DEP interact to produce inflammation in cells may provide information on more general mechanisms for how environmental contaminants affect our immune system. The results of the project also suggest that more complete combustion processes, reducing the formation of organic chemicals, may have the potential to reduce the health risk from combustion-derived particulate matter.

Environmental chemicals from multiple sources have been suggested to contribute to development of a variety of multifactorial diseases including cardiovascular diseases, asthma and allergies, cancer and metabolic syndromes, as well as obesity, by inducing or exacerbating chronic, low-grade inflammation. Real-life exposure of most individual chemicals tends to be below inflammogenic levels. However, different pollutants of complex mixtures may co-operate to produce a significant inflammatory outcome throu gh so-called coctail effects. Yet, our understanding of chemical-induced inflammation is still scarce, in particular in relation to combinatory effects of various chemicals in mixtures. Moreover, preexisting inflammation appears to be a risk factor for di sease development by environmental pollutants. We have recently observed that inflamed cells may be more sensitive and respond differently to chemical exposure, than healthy cells. Clarifying what types of environmental chemicals are the most potent drive rs of inflammation, the mechanisms of combinatory effects of chemical mixtures, and synergies between chemical exposure and preexisting inflammation may give fundamental contribution to risk assessment and expand our understanding of susceptibility factor s for adverse health effects. Such knowledge is also needed to establish causality to reported epidemiological associations. The applied project will explore fundamental mechanisms of proinflammatory effects from complex chemical mixtures by using diesel exhaust particles (DEP) as model exposure. DEP is an important contributor to urban air pollution, with effects mostly attributed to complex mixtures of soluble chemicals adhered to the particle surface. We will mainly focus on effects in lung epithelial cells based on our recent findings. In addition, limited studies will be initiated on adipocytes, to explore a novel field of potentially high importance in chronic effects of environmental chemicals in general.