Green fuels and human health - toxicity of engine
emissions from 1st and 2nd generation biodiesel fuels
Norway Grants
METHODS & APPROACH
The project involves three different approaches: physical and chemical characterisation of particulate and gaseous diesel emissions (WP1), in vivo inhalation studies in rats (WP2) and in vitro cell culture studies (WP3 and 4). Given the aims of the study, a range of different laboratory techniques will be used:

Emission characterisation and particle sampling: Diesel engine emissions from biodiesel fuels produced in presence and absence of DPF treatment will be physico-chemically characterized by GC-MS method (Gas hromatography–mass spectrometry ), HP-LC method (High-performance liquid chromatography ), LC-MS method (Liquid chromatography–mass spectrometry), DLS method (Dynamic light scattering), BET method (Brunauer–Emmett–Teller ), NanoSight (Nanoparticle Tracking Analysis ).

 

In vivo studies: Ratss will be exposed by inhalation (whole body exposure) to diesel engine emissions from biodiesel fuels produced in presence and absence of DPF treatment. The animals will be terminated on day 7 or 28 after first day of treatment. Bronchoalveolar lavage fluid will be sampled for analysis of infiltrating immunecells (macrophages, neutrophils, eosinophils, basophils, etc.). Tissue samples will be collected for clinical and histomorhological analyses, RT-PCR and ELISA analyses (expression of proinflammatory genes/mediators), colorimetrical methods (oxidative damage to proteins), In vivo Comet assay (DNA damage), Pig-a assay (gene mutations), DNA microarrays, Flow cytometry methods (cytotoxicity).

In vitro studies: Comparative studies of cellular and molecular effects of the different nanoparticles from engine emissions from biodiesel fuels will be explored in primary human bronchial epithelial cells, primary human monocytes differentiated to macrophage-like cells, A549 (human alveolar type-2 like epithelial cell line) and Calu-3 (human bronchial epithelial cell line). Cellular effects will be determined by RT-PCR and PCR arrays (expression of proinflammatory genes/mediators) in cell lines, colorimetrical methods (oxidative damage to proteins), Micronucleus assay + FISH (DNA damage), In vitro Comet assay (DNA damage), Histone γ-2HX foci formation (DNA damage and repair),  Flow cytometry methods (cytotoxicity), Cell cycle analysis.
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Projekt obejmuje trzy różne zadania: fizyko-chemiczną analizę emisji silnika Diesla podczas spalania badanych bio-paliw (WP1), badania in vivo na szczurach (WP2) oraz badania in vitro (WP3 i 4). W celu realizacji powyższych zadań zostanie zastosowany szereg metod badawczych.

Charakterystyka emisji ze spalania biopaliw: Emisje będą charakteryzowane za pomocą metod fizyko-chemicznych:  metoda GC-MS (Gas hromatography–mass spectrometry ), HP-LC  (High-performance liquid chromatography ), LC-MS  (Liquid chromatography–mass spectrometry), DLS  (Dynamic light scattering), BET  (Brunauer–Emmett–Teller ), NanoSight (Nanoparticle Tracking Analysis ).

 

Badania In vivo: Szczury będa inhalowane przez 7 i 28 dni emisjami ze palania biopaliw w obecności filtra cząstek czastył oraz bez filtra. Płyn z ukłądu oddechowego szczurów (Bronchoalveolar lavage) będzie analizowany pod kątem składu komórek układu immunologicznego(makrofagi, neutrofile, eozynofile, bazofile itd.). Tkanki zwierząt zostaną pobrane w celu analiz biochemicznych i histomorfologicznych oraz analizy RT-PCR i ELISA (ekspresja genów pro-zapalnych), badań kolorymetrycznych, testu kometowego, testu Pig-a, mikromacierzy DNA oraz metod cytometrycznych.

In vitro studies: eksperymenty zostana przeprowadzone na ludzkich liniach komórkowych: A549 (human alveolar type-2 like epithelial cell line), BEAS, Calu-3 (human bronchial epithelial cell line). Komórkowe efekty zostaną zbadanie za pomocą analizy RT-PCR i ELISA (ekspresja genów pro-zapalnych), badań kolorymetrycznych, testu kometowego, metod cytometrycznych, testu gamma-H2AX, analizy cyklu komórkowego.
Narodowe Centrum Badań i Rozwoju Polish-Norwegian Research Programme
Project Coordinator: Professor Anna Lankoff
Project Reference: Pol-Nor/201040/72/2013
Duration: 01.11.2013 - 31.10.2016
Budget: 3 077 458,00 PLN