Molecular and Cellular Toxicology

Despite recent improvements of water quality in Belgium, there is a historic pollution of heavy metals in the aquatic environment. Different sensitivities of fish towards this metal pollution have been reported. Within this project, we will investigate the differences between rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio) in dealing with the effects of copper exposure. These fish species differ in their tolerance to copper, but the underlying mechanisms have not yet been revealed. Fish will be exposed to two sublethal exposure-conditions; one similar concentration for every fish species and one concentration which is the 10% LC50 96h value for each fish species. After 1h, 12h, 24h, 3days, 1 week and 1 month gill and plasma samples are taken. The intracellular proteome of gill cells as well as the hormonal balance, the role of hormonal influences and oxidative stress conditions during exposure will be evaluated by use of techniques such as iTRAQ, 2DIGE, immunoassays, Real-time PCR and a number of enzymatic assays. When proteome-profiles are constructed and quantitative and qualitative changes in expression of proteins are unravelled, we can search for differentially expressed proteins which can serve as biomarkers.

Over the last decade, increasing concern has been raised towards the potential of a diversity of environmental compounds to interfere with the endocrine system of humans and wildlife. Given the ubiquitous presence of these ‘endocrine disrupting chemicals’ (EDCs) in the aquatic environment – often at high concentrations – fish are inevitably exposed, resulting in a variety of adverse biochemical, physiological and reproductive effects, as demonstrated by researchers worldwide.

Until recently, most studies on endocrine disruption have focused on effects at the physiological level. In this research, we will evaluate the effects of endocrine disruption on a lower (gene expression) and a higher (reproductive behaviour) level of biological organization. Adult zebrafish are exposed to (potential) endocrine disruptors under flow-through conditions. After 4 and 28 days, fish are dissected and gonads are collected for the production of oligonucleotide microarrays. Gonadosomatic index (GSI) and vitellogenin of whole body homogenates are determined after 28 days. During every day of the first and the last week of exposure, fecundity and fertility are evaluated. Hatching is followed up until 4 days post-hatch. On day 4, 14 and 28, reproductive behaviour of 10 zebrafish (5 males and 5 females) in the control and high dose group is recorded at 3 time points, using a 3D-behaviour analysis system. Swimming velocity, swimming height and inter-individual interactions are calculated.

Transgenic cells - be it prokaryotic or eukaryotic - were introduced in environmental toxicology thanks to biotechnology and genetic engineering. Such cellular systems typically combine a promoter::operator, which acts as the sensing element, with a reporter gene coding for an easily detectable protein. In the absence of toxicant the repressor is bound to the operator and expression of the reporter is blocked. Under stress conditions, the repressor is released from the operator and reporter expression is switched on either through direct interaction with the repressor or through a cascade of connected responses (signal transduction). Important aspects in the different transgenic models are a sensitive promoter, which is reliable and relevant for a certain stress, and a reporter gene allowing efficient detection of the gene product thanks to a specific substrate or, ideally detectable as such. Most, if not all, reporter proteins can be detected using optical, immunological or electrochemical methods. Many different sensing strains and cell lines of different genera have already been constructed for the detection of specific analytes such as metals, aromatic compounds and genotoxic compounds.