Exposure routes of environmental microcontaminants with different modes of actions in the zebrafish

Abstract

This study investigates the importance of water and food as exposure sources of three model contaminants with different physico-chemical properties and modes of action to zebrafish. 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 approaches. 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 developmental stage, 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 defense.

Background

Many aquatic ecosystems are under continuous pressure related to different types of human activities that have a strong impact on the structural and functional organisation of these systems. One of the most serious threats is the continued release and accumulation of microcontaminants to the environment that end up in different compartments (e.g. pesticides, pharmaceuticals). Today we are dealing with thousands of substances of which the environmental concentrations may vary orders of magnitude in time and space and which are either as individual compounds or as part of a cocktail potentially very dangerous for aquatic life (1).

These different compounds can be classified on the basis of their chemical structure and resulting physical and chemical properties or effects on man and environment. The toxicity of a substance depends on the mode of action and how this interferes with normal cellular and organismal functions (2). Once introduced in the aquatic environment chemicals distribute among the different abiotic compartments (water column, sediments,…) from which they are taken up by different types of organisms and enter various food webs. The toxic substances may occur as different chemical species (i.e. ionised, complexed, colloid…) or forms (i.e. dissolved in water, bound to food, suspended matter…). Such amalgamate of matrices that can interact with chemicals, can highly influence the overall availability of these compounds. As such the dose that enters the organism and the resulting adverse effects may vary considerably among exposure routes even when the total concentration during exposure is more or less comparable.

To properly evaluate the potential impact of a microcontaminant on an aquatic organism or ecosystem it is important to know the exposure sources and the biological availability of the substances within these sources (3). Studies have shown that the way in which an organism is exposed has a major impact on the accumulation kinetics and toxicity of a pollutant. It has been shown for a number of metals that exposure via the water phase and uptake across the gills was considerable more toxic than exposure via the food and uptake across the gut. The effect was independent of the exposure time and the accumulation kinetics indicating that the observed differences in toxicity are related to differences in internal compartmentalisation and processing (4). This implies that for a relevant environmental impact assessment of a substance the relative importance of the different exposure routes has to be taken into account and determined in what way and to what extend the different routes contribute to the overall toxic effects. Within this context it has to been pointed out that the current water quality regulations are almost entirely based on assays in which the test species are only exposed via the water to the toxic substance.

Adverse effects through food-inflicted exposure are largely neglected at present.
Under realistic circumstances organisms are almost always exposed to a multitude of substances with different modes of action. Several studies have shown that the biological effects of two or more substances together may be different from the effects caused by the individual compounds. In the most simple cases effects are additive, but also potentiation, synergistic and antagonistic effects can occur (5). Various mixture analysis models have been developed which, in general, refer to the mode of action of a chemical. However, for most toxic substances we know relatively little about their mode of action and about the molecular responses of aquatic organisms. A better understanding of these processes may lead to more accurate predictions of mixture toxicity and could lead to the identification of reliable biomarkers of exposure and effects that could provide explanations for the differences in toxicity observed under different exposure scenarios as well as in mixtures.

Research Project - Aims

Recent developments in molecular biology (genomics) and protein analysis (proteomics) make it possible to study the effects of toxic chemicals on biological systems in much more detail than was possible before (6). In this project we will combine the possibilities provided by these new approaches and technologies with more conventional physiological and toxicological approaches and methods to address some of the most important key issues in aquatic toxicology. More specifically we will investigate the relationship between toxicant exposure routes, bioaccumulation kinetics and organismal responses and mechanistic effects. For this study we will use the zebrafish (Danio rerio) as model organism. The zebrafish is highly suitable in experimental studies and toxicity testing because of its fast growth and rapid maturation (within 3 months). The physiology and biochemistry of the species is well known and it is also a model organism in developmental biology. At this moment this is also the only ecotoxicological test organisms for which gene expression micro-arrays are commercially available.

Within the framework of the project we want to address the following questions:
1. What is the relative importance of different exposure routes for uptake, compartmentalisation and toxicity of model priority microcontaminants with different modes of action (e.g. ion and osmo-regulation, neural communication, endocrine metabolism)?
2. What are the molecular effects of exposure to the model compounds via water and/or food at the level of gene expression and protein synthesis? Is it possible to discriminate the mode of action of the substances depending on their exposure route using their “omics” profile? Can we identify microcontaminant-, exposure route- and effect-specific responses for development of reliable biomarkers?
3. How do the mechanisms of toxicity of single compounds compare with the mechanisms of mixtures? What is the overall contribution of the exposure routes to the toxicity of mixtures? How can differences in exposure route and toxic effects be reflected at the level of gene expression and protein synthesis?
Within the framework of this study we will work with three model compounds representative for compounds which differ in physico-chemical properties (e.g. water-lipid solubility) and mode of action (i.e. cadmium, lindane and 4-(para)-nonylphenol). The model compounds are microcontaminants of environmental concern and appear on the recent priority list of 33 compounds developed within the Framework of the European Water Framework Directive.