A systems biology approach to the importance of the exposure route on stress susceptibility in zebrafish

In this ecotoxicological study, the biological effects of a major class of environmental pollutants are studied using a systems biology approach, assessing these effects across different levels of biological organisation and investigates the importance of water and food as exposure sources of two model contaminants with different physico-chemical properties and modes of action to zebrafish (Danio rerio).

The experiments are conducted in the laboratory, exposing zebrafish to these two model contaminants. It is explored to what extend uptake via water or food results in different responses and toxic effects. The responses and effects are studied at molecular, cellular and organismal level using genomics, proteomics and physiological (energy stores, swimming performance, condition factor, tissue and whole body contaminant concentrations) approaches at different points in time (acute/chronic).

The project aims to provide a in depth understanding of how different substances interact with a model system taking into account key factors such as exposure route and exposure time. The results of the genomics and proteomics analysis should considerable enlarge our understanding of the molecular mechanisms of toxicity and defence.

The first objective of this study was to investigate the overall stress response to cadmium. Cadmium is a metal with limited, or in most species unknown, biological function that is widely distributed in the modern environment as a result of natural processes and anthropogenic activities. Cadmium is a common pollutant in most surface waters and can cause adverse effects on organisms inhabiting these bodies of water. Consequently, the toxic effects of cadmium have been widely studied in several fish species and include, among others, disturbances of ion and osmoregulation, respiration and enzyme function at the physiological and biochemical levels. Furthermore, a wide range of effects has been described at the gene and protein expression level. However, in most of these studies, integration of different levels of biological organisation within the same experimental setup was not achieved.

In this study all the observations were performed on the same exposure group and where possible on the same samples (energy budget, metal concentration, proteomics and transcriptomics). The organismal effects were investigated using the swimming performance in swimming tunnels (critical swimming speed) and the fish condition factor.The critical swimming speed (Ucrit) was determined after 29 days of exposure. The condition factor (CF) was calculated from individual wet weight and fork length. At the biochemical level tissue specific energy reserves were calculated from protein concentration, lipid concentration and glycogen concentration measurement. Tissue specific and whole body metal accumulation was measured using ICP-MS in gill, liver, intestine and muscle. Both transcriptomics (microarrays) and proteomics (2D-DIGE) analyses were performed to gain insight in the effects of metal stress at the molecular level. Two-dimensional differential gel electrophoresis was performed on liver tissue protein extracts. Images were analysed using Decyder and Delta2D.

Although the analysis of proteomics and transcriptomics data is still ongoing, evidence of resulting effects at different levels of biological organisation has already been demonstrated, as shown in the declining physiological fitness, as expressed by the condition factor and the critical swimming speed, the depletion of liver lipid concentrations and the transcriptional impact on several important biochemical pathways.

Future research focuses on the stress response to cadmium exposure via food and an organic pollutant via water and food as exposure routes in zebrafish.