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

This research addresses some key consequences, effects and differences of the short-term and long-term uptake and accumulation of an essential (Zn) and a non-essential (Cd) metal in the fresh water fish zebrafish (Danio rerio) using a systems biology approach, assessing these effects across different levels of biological organisation and investigating the importance of water and food as exposure routes.

The experiments were conducted in the laboratory, exposing zebrafish to these two metals. Additionally, the effect of exposure route on uptake, response and toxic effects were investigated. The responses and effects were studied at molecular, cellular and organismal level using transcriptomics, proteomics and phenomenological (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 transcriptomics and proteomics analysis should considerable enlarge our understanding of the molecular mechanisms of toxicity and defence.

In the different experiments all the observations were performed on the same exposure group and on the same samples. The condition factor and/or the critical swimming speed were measured as organismal parameters. At the organ level tissue specific energy reserves were calculated from protein concentration, lipid concentration and glycogen concentration measurement. Tissue specific (gill, liver, intestine, brain, gonads and muscle) and whole body metal accumulation was measured using ICP-MS. Both transcriptomics (microarrays) and/or proteomics analyses were performed to gain insight in the effects of metal stress at the molecular level. Direct effects of exposure at these different levels of biological organization were observed. Additionally, the effects of exposure on the metabolism were described separately at the different levels of biological organization. Using co-expression networks these effects were ascertained at different levels of biological organization.

This research not only describes reported effects of metal exposure over different time points and at different concentrations within the same samples in an integrated manner, but also provides a workflow for using co-expression networks of proteomics and transcriptomics data as a means of integrating, condensing and linking data over different levels of biological organization. This integration over biological levels and over time can not only give a broader overview of the toxic effects of cadmium, but can provide a mechanistic sense of direction and causality of the toxic effects. To align the specific strengths of the phenomenological and the high-troughput workflows in this study, higher level biological parameters were not only evaluated in themselves but were also used as a denominator in a biologically motivated data reduction scheme of the proteomics and transcriptomics data.

This integrative approach of simultaneously investigating and linking effects at different levels of biological organization is not often used in (eco)toxicological studies. However, the integration of data gathered at these distinct biological levels over several time points can not only give a broader overview of the toxic effects, but it can also assist in the unravelling of the general toxic mechanisms of cadmium exposure. To align the integrated, ecologically relevant information of the phenomenological workflow and the detailed information of the high-troughput workflows, effects at the organismal and the organ level were not only evaluated separately, but they were also used as a denominator in a biologically motivated data reduction scheme of the proteomics and the transcriptomics data. To supplement the descriptive analysis of proteomics and transcriptomics data, a coexpression network approach, using biological parameters as a screening mechanism, was applied to provide a means to bridge the gap between effects at individual gene or protein level and the complex phenomenological effects.

The analysis of both the waterborne exposure to zinc and the dietary exposure to zinc and cadmium is still ongoing.