The number of chemical products produced by men is enormous. At present the number of chemicals registered by the Chemical Abstract Service exceeds 20 million. Estimations are that over 100.000 compounds are released in the environment in such volumes that they generate a potential threat for men and environment. Only little is known on the toxicological properties of this large group of chemicals. The scarce information available until now consists mainly of acute toxicity data. Long term adverse effects on populations, communities and ecosystems are poorly documented. The lack of chronic and long term data is illustrated by the phenomenon of endocrine disruption. Various chemicals are capable of interfering with the endocrine metabolism of several animal species leading to negative effects on their reproduction. There is a great deal of uncertainty about the possible adverse (endocrine disrupting) character of chemicals for men and environment due to the scarce availability of chronic toxicity data and restricted knowledge on the toxicological working mechanisms. Especially for the ecologically important group of invertebrates no clear assays for mechanistic evaluation of endocrinological pathways disruption are developed yet. It is clear that there is an urgent need for this kind of assays to provide both chronic relevant as well as mechanistic detailed data.
In this research project a cDNA (complementary DNA) array for the freshwater flea Daphnia magna will be developed in order to:
(a) quantify the relationship between the short term biomarker effects and the accompanying changes in population dynamics.
(b) reveal the mechanisms of toxicant-induced effects on the different metabolic pathways.
(c) gain insight in the disruption of endocrine mediated effects by toxic exposure.
Freshwater fleas are considered as one of the most important test organisms for the ecotoxicological evaluation of chemicals. They are used intensively for the impact-evaluation of environmental pollution on freshwater ecosystems because of their little size, their relative short lifecycle, their high sensitivity to a wide range of environmental chemicals and their central role in diverse aquatic food chains.
At present, little genome information on daphnids is available making molecular toxicological studies a difficult task. In our group, we have chosen to use the Suppression Subtractive Hybridization (SSH) PCR method to isolate differentially expressed genes from organisms with clear differences in physiological status. This technique is based on suppression PCR that permits exponential amplification of target cDNAs, whereas amplification of undesired sequences is repressed. The isolation of differentially expressed genes allows the constructing of specific cDNA arrays for ecotoxicological standard test organisms like Daphnia magna. Our working hypothesis was based on the general “metabolic cost†hypothesis. Since chemical stress reduces the amount of energy that is allocated towards growth and reproduction we wanted to collect the most relevant genes related to energy metabolism, growth (molting) and reproduction (life stage-specific) for toxicant characterization. In order to assess the applicability of the custom microarray, the effect of different chemicals on gene expression will be evaluated and adverse effects will be studied at higher levels of biological organization.
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