New methods and instruments for accessing the composition, sources and health-relevance of aerosols: From lung cell exposure based biological effects analysis to single particle laser mass spectrometry
About the Talk:
Air pollution contributes to climate change and represents the largest environmental health-risk worldwide. Inhalation of air toxic organic compounds such as the Polycyclic Aromatic Hydrocarbons (PAHs, known carcinogenic air toxicants) or inorganic compounds such as transition metals (e.g. iron, nickel, zinc, known to induce oxidative stress and inflation processes in the lung tissue) is a well-known cause of morbidity and mortality. In particular combustion aerosol emissions are responsible for acute and chronic health effects such as asthma exacerbation, heart-arrhythmia and -failure, lung cancer or COPD in humans. However, emissions from different combustion sources induce different health outcome and those effects also depend on the type of combustion compliance and fuel. However, up to now only few links between aerosol chemical composition and biological effects have been established and the relative toxicity of different combustion emissions still needs to be investigated. In the framework of the Virtual Helmholtz Institute-HICE (www.hice-vi.eu), physical and chemical properties of combustion emissions and their biological effects on lung cells (human epithelial cells: A549, BEAS2B and RAW/THP1 murine/human macrophages) are comprehensively analysed. For addressing the biological activity and toxicity of the aerosols, the lung cell-cultures were realistically exposed at the air-liquid interface (ALI) using novel mobile automated ALI exposure-systems with 24 or 18 places for inserts with ca. 600.000 lung cells each (Vitrocell GmbH, Germany). The ALI exposure systems are placed a mobile S2-bio safety laboratory (HICE-MobiLab) and cells are exposed with different moisturized combustion aerosols or clean air (reference). After 4h exposure biological effects are analysed by a multi-omics characterisation (transcriptomic & proteomic level) or assays for specific toxicological endpoints (viability, genotoxicity, cell integrity etc.). During all biological tests, the chemical composition and physical parameters of the emissions were thoroughly characterized (gases and particles) using high level instrumental analysis. The acquired biological effects were comprehensively characterized and are put in context with the chemical and physical aerosol data (see Oeder et al.). For validation partly also animals (BL6 mice) are exposed in parallel to the ALI-exposures (RAW cells) and the macrophages from the broncheoalveolar lavage fluid (BALF) are subjected to molecular biological effects analysis as well. The comparison of the effects from mice BALF (in-vivo) and RAW (in-vitro) for exposures with diluted exhaust from a Kubota diesel engine showed that immune response pathways are activated in both cases. Emissions from wood combustion (masonry heater, pellet burner and log wood stove, ship engines (heavy and light fuel oil), car engines (diesel, gasoline and ethanol fuels) were investigated by the ALI-approach. Very recently lignite combustion and aging of wood and lignite aerosol emissions were investigated. The UV-light induced aging of the emission was performed in specially developed, high-throughput flow tube. In summary/conclusion observed biological response-strengths differ considerably for different combustion aerosol sources and are not well correlated to the deposited PM2.5-mass (i.e. partly also strong gas phase effects). This is suggesting large differences in the relative toxicity of the aerosol emissions from different combustion sources and fuel types. The aging experiments induce increased cytotoxicity, an effect which is currently deeper analysed on the molecular biological level. In addition to adverse reactions also supposedly protective effects are observed. For example, the emission from a log-wood stove, exhibiting very high emission of soot, organics, PM2.5 are inducing only relatively mild acute effects in the ALI exposed cells, if compared to diesel or pellet burner emission. The high abundance of antioxidant compounds such polyphenols in the logwood stove-emissions may explain this counter-intuitive observation (see Kanashova et al.). The latter findings are supported by detailed analyses of activated cellular response pathways (GO-term analysis), depicting regulation of pathways such as pro-inflammatory signalling, xenobiotic metabolism, phagocytosis or oxidative stress and findings from the selected animal exposure experiments. The biological analysis is accompanied by comprehensive analyses of the aerosol composition and properties. In recent experiments one of the automated ALI exposure stations was equipped for long-term exposures and successfully applied for up to 48 h exposure times in order to address diluted aerosols (ambient air). Furthermore, also innovative physicochemical measurement techniques are developed which are directed for an on-line analysis of the PM components, which have been detected or verified to be very relevant with respect to their biological/toxicological activity. This includes e.g. the elemental carbon/soot content, the content of PAH and PAH-derivatives (CYP1A1 induction, genotoxicity) and redox-active or toxic metals like Fe or Zn (oxidative stress). One important aspect determining the chemical toxicity of inhaled particles is the local pollutant dose generated at the particle deposition location in the lung. Thus the mixing state of these toxic components in the particle ensemble matters (i.e. the internal or external distribution of components in the particle ensemble). This would favour a single particle-based chemical analysis method. In this context e.g. the so called “tar balls” from combustion or pyrolysis processes are relevant. They consist mainly of organic molecules and exhibit extremely high PAH-contents, suggesting the local override of cellular defence mechanisms at the deposition site in the lung. Our approach for on-line single particle profiling is based on bipolar Time-of-Flight Mass Spectrometry (TOF-MS). Aerosol particles are introduced into the system through an aerodynamic lens and detected and size-classified by laser velocimetry. Subsequently, the organic coating is desorbed by an IR-laser pulse a few microseconds before the combined ionization step of Resonance-Enhanced Multi-Photon Ionization (REMPI) and Laser Desorption/Ionization (LDI) with one single UV-laser pulse (248 nm, KrF Excimer laser) takes place. Here we present a novel route to obtain the full LDI-information of both cations and anions needed for particle source apportionment in combination with health-relevant PAHs by REMPI. Because all ions are formed within one combined ionization step, no further laser beyond the two sources for conventional two-step approaches is required (see Passig 2017 and Schade 2019). Furthermore, experiments with tuneable lasers and different fixed-frequency lasers point out that resonance enhanced LDI (or RELDI) of elements can be achieved if the ionisation laser wavelengths is in resonance with an atomic line of an element. In fact, iron, which is particular important for oxidative stress related health impacts, can be detected by RELDI with greatly enhanced sensitivity using the 248,3 main atomics transition (Fe: 3d64s2→3d64s4p). Energy transfer processes from the ubiquitous iron also support detection of other relevant transition metals. This allows to achieve a “double resonance” enhancement (RELDI and REMPI) of two very health relevant species using spatially shaped 248 nm KrF laser pulses. The new method has been applied for several combustion emission-experiments as well as ambient monitoring and allows a deepened insight into the mixing state of PAHs and metals as well as other compounds (e.g. nitrate, sulphate, organic and elemental carbon etc.) and a single particle resolved detection of relevant air toxicants. The latter information is crucial to determine the source- and aging-state of the particles. Finally, it is concluded that for detection and understanding of the health relevant ambient aerosol parameters of the it is necessary to develop and apply new biological and physicochemical aerosol characterisation methods.
References: Kanashova 2018: Kanashova et al., J. Mol. Clin. Med. 1, 2018, 23-35; Oeder 2015: Öder et al., PLoSOne 2015, https://doi.org/10.1371/journal.pone.0126536; Passig 2017: Passig et al., Anal. Chem. 89 ,2017, 6341-6345; Schade 2019: Schade et al., Anal. Chem. 91, 2019, 10282-10288
Ralf Zimmermann studied Chemistry and Physics at the Technical University Munich (TUM) and the Ludwigs-Maximilian University, Munich. In 1991 he graduated from the TUM (Physical Chemistry, Prof. Boesl). He received his Ph.D. degree in 1995 from the same University with Prof. Kettrup (Ecological Chemistry and Instrumental Analytical Chemistry) and Prof. Schlag (Physical Chemistry). He moved as post-doc to the GSF Research Centre for Environment and Health in Oberschleißheim, Germany and the University of Antwerp (Prof. Adams). Based on research on laser mass spectrometry, dioxin analysis and combustion aerosol monitoring he received in 2001 the hablitation-degree from the Technical University Munich-Weihenstephan.
From 2001 to 2008 Zimmermann was appointed as Associated Professor of Analytical Chemistry at the University of Augsburg as well as head of the Chemistry Department of the bifa-Environmental Institute in Augsburg. Since 2008 he is Full Professor of Analytical Chemistry at the University of Rostock (UR) and in personal union head of the Department “Comprehensive Molecular Analytics” (CMA) at the Helmholtz Zentrum München (HMGU). The Rostock and Munich parts of his research group are united as “Joint Mass Spectrometry Centre”, supported by both of his affiliations.
His research interests are in the field of the physical properties, organic composition as well as biological- and health-effects of anthropogenic aerosol emissions and ambient aerosols. The instrumental analysis (on-line and off-line) of the chemical signature of complex molecular mixtures using high-resolution Mass Spectrometric (MS) approaches, comprehensive chromatographic and thermal analytical techniques as well as novel photo ionisation based MS tools are further focal points of his research interest. This includes the development and application of advanced single particle aerosol mass spectrometric technologies and photo ionisation MS techniques for on-line analysis of gases and vapors. Since 2012 Zimmermann is leading the Virtual Helmholtz Institute “HICE-Aerosol and Health”, which is addressing the composition, toxicology, biological effects and health implications of combustion aerosol emissions in the framework of an international cooperation network (www.vice-vi.eu) and from 2019 on, he is spokesperson of the Helmholtz International Lab aeroHEALTH. Within aeroHEALTH, the HMGU, the Forschungszentrum Jülich (FZJ) and the Weizmann Institute of Science in Israel are investigating within a joint long-term initiative the impact of photochemical ageing and atmospheric processing of aerosol emissions and ambient air on human health.