The temperatures of wood fires play an important role in the extent of particulate pollution; the higher the combustion temperatures, the fewer toxic compounds contained in particulates are emitted through chimneys into the atmosphere. Black soot on the filters of combustion plants is the result of low combustion temperatures and hence ineffective combustion; light dust indicates excellent combustion at high temperatures and thus the generation of low amounts of soot. The physicochemical composition of particulates needs to be determined by investigation at specific laboratories. The same laboratories are also investigating the toxicological properties of particulate matter components. Polycyclic aromatic hydrocarbons constitute a huge proportion of toxicologically active substances and are therefore the object of intensive investigations.

The "Human and Environmental Toxicology" research group at the University of Konstanz under Prof. Dr. Daniel Dietrich is part of a German network of institutes whose work involves assessing the risks of particulate matter resulting from the combustion of wood. The German Biomass Research Centre in Leipzig provides particulates for the project, the TU Hamburg-Harburg and the University of Konstanz are using biotests to investigate the toxicological effect of particulates from wood combustion.

Three doctoral students have been working on developing in vitro assessment methods for the last year. Their work and objectives are defined by three model approaches involving bacteria, worms and cell culture models. The three models are used to simulate the effects of the toxic components contained in environmental particulates on soil-dwelling organisms (worms and bacteria) and on humans (cell culture).

Susanne Gauggel from the University of Konstanz is establishing cell culture models using the A-549 human epithelial lung cell line and the THP-1 human monocyte cell line (white blood cells) amongst others. Gauggel hopes that these human cell lines will provide her with insights into the connection between lung diseases and particulate matter that contains environmental toxins. "On the gene and protein expression level, the reaction of the cell lines to toxins contained in particulate matter such as polycyclic aromatic hydrocarbons, suggests that particulates might have a carcinogenic effect," said Gauggel. The cell models could also prove to be suitable for detecting changes in the expression pattern of specific genes in patients with respiratory diseases, which might be the result of the harmful effects of toxic particulate matter.

Iris Gutiérrez in PD Dr. Wolfgang Ahlf's department at the TU Hamburg-Harburg is using a bacterial model based on the soil bacterium Arthrobacter. "The bacteria contact test reveals the effect of toxic particulates on Arthrobacter globiformis cultures during the bacteria's exponential growth phase, when the bacteria's metabolism is highly active," said Gutiérrez who uses a fluorescent dye to detect the inhibition of a particular enzyme (a dehydrogenase) involved in the cell metabolism. She also uses a test that makes it possible to specifically detect genotoxicity. Iris Gutiérrez explains: "The umu test is a genotoxicity test involving the bacterium Salmonella typhimurium TA 1535/pSK1002. The bacteria are exposed to different concentrations of the substance to be tested. The genotoxins induce the umuC gene, which belongs to the SOS repair system of cells and which counteracts potential damage to the bacterial DNA." By coupling the umuC gene to the lacZ gene of the enzyme ß-galactosidase, Gutiérrez is able to indirectly show the activation of the umuC gene by way of a colour reaction.

In the near future, Gutiérrez is planning to use Saccharomyces cerevisiae yeast cultures to determine the proportion of hormonally active substances in the particulates, especially those that interact with the oestrogen receptor and induce a hormone-like reaction. The oestrogen receptor not only reacts highly specifically with oestradiol, but also with many other substances, although to a much lesser extent. The "Yeast Oestrogen Test" involves genetically modified yeast cells that have been generated for the specific detection of oestrogen-mimicking substances. "The exposure of yeast cells to oestrogen-mimicking substances leads to the activation of a gene that encodes the enzyme ß-galactosidase," said Gutiérrez. The activity of ß-galactosidase, which the yeast cells release into the culture medium, is proportional to the quantity of enzyme released. Enzyme activity is detected by adding a suitable substrate, in this case CPRG (chlorophenol red ß-galactopyranoside; yellow), which is cleaved by ß-galactosidase, resulting in a colour shift from yellow to red. "The intensity of the colour can be measured with a photometer. Therefore, the effect of hormonally active substances contained in particulates can be shown in a very effective way," said Gutiérrez.

Birte Hegemann, another PhD student at the TU Hamburg-Harburg, is studying the reaction of Caenorhabditis elegans to particulate matter. She exposes the transparent worm, which is around one millimetre long, to particulates generated through the combustion of wood in order to investigate potential toxic effects on the worm's reproduction and growth. Amongst other things, Hegemann is looking for genes that have been shown to cause lung disorders, "whose expression in the lung tissue of people suffering from asthma or early degenerative lung diseases differs from that of healthy people. C. elegans is a perfect organism to study these changes," said Hegemann.

The three models use fine silicon dioxide (silica) particles as reference particles; these are free from toxic substances. Using silica allows the researchers to exclude mechanical aspects such as irritation or mechanical injury of the tissue which is typical for silicosis, a form of occupational lung disease caused by inhalation of crystalline silica dust.

Further information:

Susanne Gauggel
Human and Environmental Toxicology
University of Konstanz
Germany
Phone: +49-7531-88-4137
Fax: +49-7531-88-3170
E-mail: susanne.gauggel(at)uni-konstanz.de

Iris Gutiérrez
E-mail: Iris.Gutierrez(at)tu-harburg.de

Birte Hegemann
Hamburg-Harburg University of Technology
Institute of Environmental Technology and Energy Economy
Ecotoxicology
Risk Eißendorfer Straße 40
21073 Hamburg
Tel.: +49 (0) 40 42878 2809
Fax: +49 (0) 40 42878 2315
E-mail: birte.hegemann(at)tu-harburg.de