A combined DNA-microarray and mechanism-specific toxicity approach with zebrafish embryos to investigate the pollution of river sediments

Abstract

The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into the ecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Mechanism-specific biotests indicated a defined hazard potential of the sediment extracts, and microarray analysis revealed several classes of significantly regulated genes which could be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism.

Introduction

Ecotoxicogenomics attempts to link ecotoxicological effects resulting from exposure to toxicants with changes in (specific) gene expression patterns on the assumption that the gene expression in animals is altered in consequence of toxicity, either as a direct or an indirect result of toxicant exposure [1]. The challenge faced by (eco)toxicologists is to define, under a given set of experimental conditions, the characteristic and specific patterns of gene expressions elicited by a toxicant or environmental sample with known or potential toxicity [2], [3]. In the future, such knowledge might be helpful for the development of simulation models to predict the toxic effects by linking molecular biomarkers with population level effects, and, eventually, to even predict the ecologic risk of novel chemicals [2], [3].

Correlations of expression patterns in multiple samples may help to identify differentially expressed genes that play a role in embryogenesis, tissue patterning, organ development, and other physiological processes [4]. The challenge is to discriminate alterations associated with chemical toxicity from changes that are related to other stressors within normal adaptive responses not associated with truly adverse effects in an individual cell, organ, or organism [5]. By careful selecting appropriate sets of genes, one single microarray experiment could possibly combine numerous biologically relevant endpoints, provided the relevance of altered gene expression for the intact organism is truly understood.

For virtually all regulatory applications, endpoints most commonly assessed in toxicity tests with fish are those that can be directly related to effects at the population level: survival, growth (generally during early life-stages), sex ratio, and reproductive success [6]. To some extent, this holds true for bioassays addressing genotoxic effects or measure gene alterations, e.g. the formation of DNA adducts, DNA strand breaks, loss and/or chemical modifications of DNA bases as well as cross-linking of DNA [7], [8]. During the past several decades, a multitude of in vitro techniques have been developed to measure toxicity like toxicant-induced DNA damage or mutagenicity [9], [10], [11], [12], [13], [14], [15], [16], [17]. These assays include the Ames test [9], the Syrian hamster embryo cell transformation assay [18], micronucleus assays [19], measurements of sister chromatid exchange [20], unscheduled DNA synthesis [21] or the single cell gel electrophoresis (comet assay) [16]. Fundamental to all of these methods is the fact that toxicity is often preceded by, and/or results in, alterations in gene expression, with changes in gene expression being potentially far more sensitive, characteristic, and measurable endpoints than the toxicity itself [1]. Therefore, Nuwaysir et al. [1] propose genome-wide gene expression patterns as a tool highly informative of toxicant exposure to complement the established methods described above.

Hamadeh and co-workers reported that chemical-specific patterns of altered gene expression can be identified by high-density microarray analyses of tissues from exposed rats [22]. Likewise, gene expression profile approaches, were successfully used to discriminate between different classes of genotoxins in mice [23] as well as between different endocrine-disrupting compounds in carp [24] and to reliably predict the identity of toxicants in zebrafish [25]. Nevertheless, the specificity, sensitivity, and quantitative capabilities of high throughput transcriptomics/gene expression analyses for environmental applications are still in the early stages of evaluation [26].

Zebrafish eggs and embryos are receiving increasing attention, and in Germany the so-called “fish egg assay” has been validated for use in standardized wastewater assessment [27]. A modified version was submitted as the “Fish embryo test” (FET) to the Organization for Economic Cooperation and Development (OECD) Working Program [28], since the FET could be shown to have an excellent correlation to the acute fish test [29]. Various recent investigations also documented the suitability of zebrafish embryos for microarray investigations [25], [30], [31], [32], [33], [34].

The present study aimed to identify changes in the gene expression of zebrafish embryos as a response to exposure to selected sediment samples from the Rhine River, Germany. Over decades, the Rhine River in Germany had been strongly polluted with industrial and municipal waste waters. In the meantime, however, regulatory measures have been taken, and a good chemical and ecological status has been claimed for the surface waters by the European Water Framework Directive until 2015. In contrast to surface waters, river sediments still contain records of particle bound deposits of toxicants that can be remobilized during flood events [35] and are, e.g. able to cause DNA strand breaks in fish cells after dissociation from these particles by Soxhlet extraction [36]. However, chemical analysis did not correlate well with the genotoxic potential and, thus, the main cause of the effect, if any, remains unclear. Therefore, there is a need for approaches allowing detailed insight into the mechanisms underlying toxic response of model systems at the level of gene expression. DNA microarray techniques might provide such insight by comparing expression patterns elicited by sediment exposure to those produced by known contaminants. The approach is based on a correlation of DNA microarray data with a set of biological assays (e.g., mortality, cytotoxicity, mutagenicity, genotoxicity) as well as with chemical analyses for selected compounds including heavy metals and polycyclic aromatic hydrocarbons.