Tine Vandenbrouck

A biology-based approach for mixture toxicity of multiple endpoints over the life cycle.

Typical approaches for analyzing mixture ecotoxicity data only provide a description of the data; they cannot explain observed interactions, nor explain why mixture effects can change in time and differ between endpoints. To improve our understanding of mixture toxicity we need to explore biology-based models. In this paper, we present an integrated approach to deal with the toxic effects of mixtures on growth, reproduction and survival, over the life cycle. Toxicokinetics is addressed with a one-compartment model, accounting for effects of growth. Each component of the mixture has its own toxicokinetics model, but all compounds share the effect of body size on uptake kinetics. The toxicodynamic component of the method is formed by an implementation of dynamic energy budget theory; a set of simple rules for metabolic organization that ensures conservation of mass and energy. Toxicant effects are treated as a disruption of regular metabolic processes such as an increase in maintenance costs. The various metabolic processes interact, which means that mixtures of compounds with certain mechanisms of action have to produce a response surface that deviates from standard models (such as ‘concentration addition’). Only by separating these physiological interactions from the chemical interactions between mixture components can we hope to achieve generality and a better understanding of mixture effects. For example, a biology-based approach allows for educated extrapolations to other mixtures, other species, and other exposure situations. We illustrate our method with the interpretation of partial life-cycle data for two polycyclic aromatic hydrocarbons in Daphnia magna.

Gene transcription profiles, global DNA methylation and potential transgenerational epigenetic effects related to Zn exposure history in Daphnia magna

A reduced level of DNA methylation has recently been described in both Zn-exposed and non-exposed offspring of Daphnia magna exposed to Zn. The hypothesis examined in this study is that DNA hypomethylation has an effect on gene transcription. A second hypothesis is that accumulative epigenetic effects can affect gene transcription in non-exposed offspring from parents with an exposure history of more than one generation. Transcriptional gene regulation was studied with a cDNA microarray. In the exposed and non-exposed hypomethylated daphnids, a large proportion of common genes were similarly up- or down-regulated, indicating a possible effect of the DNA hypomethylation. Two of these genes can be mechanistically involved in DNA methylation reduction. The similar transcriptional regulation of two and three genes in the F0 and F1 exposed daphnids on one hand and their non-exposed offspring on the other hand, could be the result of a one-generation temporary transgenerational epigenetic effect, which was not accumulative.

Gene transcription and higher-level effects of multigenerational Zn exposure in Daphnia magna.

Zn exposure of Daphnia magna during one generation has been shown to modulate gene transcription differently in Zn exposed organisms compared to their non-exposed offspring. Here we studied the transcriptional gene regulation with a cDNA microarray in D.magna exposed to Zn for three generations (F0-F2). For the first time molecular effects of multigeneration toxicant exposure in D. magna are described. Out of 73 differentially transcribed genes in the F1Zn exposed generation (compared to the F1 control), only seven genes were also differentially transcribed in the same direction in the F0Zn exposed daphnids (up or down, compared to the F0 control). The majority of the differentially transcribed unigenes in F1Zn exposed daphnids (78%) were not differentially transcribed in the F0Zn exposed organisms. This indicates that Zn exposure affected other molecular pathways in the second exposed generation, although a reduced reproduction and a reduction in juvenile growth were observed in both Zn exposed generations, compared to the respective controls. In the third Zn exposed generation (F2), no reduction in growth or reproduction compared to the control was observed. This acclimation was reflected in a significantly lower number of differentially transcribed genes, compared to the Zn exposed F0 and F1 generations.

Sublethal effects of waterborne uranium exposures on the zebrafish brain:transcriptional responses and alterations of the olfactory bulb ultrastructure

The toxic action modes of uranium (U) in fish are still scarcely known. U is known to modify the acetylcholinesterase activity in the fish brain. To gain further insight into U neurotoxicity in fish, we examined transcriptional responses in the brain of the zebrafish, Danio rerio, exposed to 15 microg L(-1) and 100 microg L(-1) of waterborne U for 3 and 10 days. In parallel, an ultrastructure analysis of the neuropil of the olfactory bulb, an area in the brain of fish sensitive to metal contamination, was performed after 10 days of U exposure. This combined transcriptomic and histological study is the first report performed in the brain and specifically the olfactory bulb of fish exposed to U. We found that 56 transcripts responded to the metal exposure, and the anatomical structure of the olfactory bulb was damaged. The greatest gene response occurred at the lower U concentration and the numbers of responding genes common to any two U exposures were much smaller than those unique to each exposure. These data showed that the intensity of gene response may not correlate positively with toxicant concentrations according to our experimental design. Instead, different patterns of gene expression are expected for each exposure. Gene responses were categorized into eight functional classes, and the transcriptional responses of genes involved in the olfactory system were significantly affected. Collectively, the data suggest that genes in the olfactory region may be ecologically relevant and sensitive transcriptional biomarkers of U waterborne exposure.

Can metal stress induce transferable changes in gene transcription in Daphnia magna

DNA methylation has recently been reported in Daphnia magna, which indicates the possible presence of epigenetic mechanisms regulating gene expression in this species. As such, effects of transient chemical exposure could be transferred through epigenetic inheritance to non-exposed generations. In this study, in the Zn-exposed daphnids, a large number of genes were found to be differentially transcribed, amongst which transcription and translation related genes (downregulated), genes associated with oxidative stress (upregulated) and different types of metabolism-related genes (mostly upregulated). In the two subsequent generations of non-exposed daphnids, a considerable number of differentially regulated genes were observed, indicating an effect of Zn-exposure in the non-exposed progeny. However, none of the differentially transcribed genes observed in the Zn-exposed generation were regulated in the same direction in both non-exposed subsequent generations. The exposure of D. magna to a sublethal Zn concentration for one generation did not result in a stable transgenerational epigenetic effect with consequences for reproductive output nor was a stably epigenetically inheritable effect observed on the transcription of any of the studied genes. An important observation was the large number of genes that were differentially transcribed between different control generations with no pre-exposure history. These genes were not considered in the analysis of the effect of Zn exposure on gene transcription. This differential regulation between subsequent control generations was attributed to possible differences in synchronization of the molting and reproductive cycle of the daphnids in the different generations. This finding is of major importance for the interpretation and design of future microarray experiments with adult Daphnia.

Development of a microarray for Enchytraeus Albidus (oligochaeta): preliminary tool with diverse applications

Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress-induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus.

Integration of molecular with higher-level effects of dietary zinc exposure in Daphnia magna.

We exposed Daphnia magna for 21 days to dietary Zn, incorporated in a diet of the green alga Pseudokirchneriella subcapitata at 720 microg Zn/g dry wt and compared its response to D. magna fed with a control diet (116 microg Zn/g dry wt). Exposure to dietary Zn resulted in an increased body burden of D. magna (93.7 microg/g dry wt vs. 61.3 microg/g dry wt in the control) but did not affect survival, growth, or feeding rate. Only reproduction was significantly reduced from the 2nd brood onwards. Gene expression analysis, using microarray analysis and RT-PCR, showed that dietary Zn exposure resulted in the differential expression of several genes involved in molting-associated processes (i.e., chitin binding, cuticle metabolism), especially after 6 days of exposures (but not after 13 or 21 days of exposure). Monitoring of time to molt and intermolt-period confirmed this molting effect at the organism level in the first week of exposure. The data suggest a possible link between Zn-induced effects on molting-related processes and reproductive inhibition, but this link is only obvious for effects on the 2nd brood size and not for later broods. Reproductive inhibition in later broods may also be explained by a disturbed mitochondrial function, but more research is clearly needed to give a more definitive integrated explanation of the observed effects at the molecular and organism level.

Nickel response in function of temperature differences: effects at different levels of biological organization in Daphnia magna.

In this study, gene transcription profiling in combination with the assessment of systemic parameters at individual and population levels were applied to study the (toxic) effects induced through temperature stress in the presence or the absence of an additional chemical stressor (nickel) in Daphnia magna. It was illustrated that lower temperatures were mainly characterized by a reduction of growth and lipid content, while higher temperatures caused an increase of both endpoints. Many of the differentially regulated transcripts could be correlated with processes affected at higher hierarchical levels of biological organization. Gene clusters with probable roles in producing offspring (peak expression at 22°C), enhancing the metabolic rate (temperature related expression) and translational processes (increased expression at 14°C) were identified. However, it was not possible to pinpoint a specific subset of genes, exclusively responding to temperature or nickel and allowing a retrospective identification of the particular stressor. Overall, extreme temperatures caused a higher level of stress in the organisms in comparison to nickel exposure. Moreover, organisms subjected to the natural stressor appeared to be less capable of dealing with the additional chemical stressor and as a result activate or repress more gene pathways.

Physiological and molecular effect assessment versus physico-chemistry based mode of action schemes: Daphnia magna exposed to narcotics and polar narcotics.

Structural analogues are assumed to elicit toxicity via similar predominant modes of action (MOAs). Currently, MOA categorization of chemicals in environmental risk assessment is mainly based on the physicochemical properties of potential toxicants. It is often not known whether such classification schemes are also supported by mechanistic biological data. In this study, the toxic effects of two groups of structural analogues (alcohols and anilines) with predefined MOA (narcotics and polar narcotics) were investigated at different levels of biological organization (gene transcription, energy reserves, and growth). Chemical similarity was not indicative of a comparable degree of toxicity and a similar biological response. Categorization of the test chemicals based on the different biological responses (growth, energy use, and gene transcription) did not result in a classification of the predefined narcotics versus the predefined polar narcotics. Moreover, gene transcription based clustering profiles were indicative of the observed effects at higher level of biological organization. Furthermore, a small set of classifier genes could be identified that was discriminative for the clustering pattern. These classifier genes covaried with the organismal and physiological responses. Compared to the physico-chemistry based MOA classification, integrated biological multilevel effect assessment can provide the necessary MOA information that is crucial in high-quality environmental risk assessment. Our findings support the view that transcriptomics tools hold considerable promise to be used in biological response based mechanistic profiling of potential (eco)toxicants.

Enchytraeus albidus Microarray: Enrichment, Design, Annotation and Database (EnchyBASE)

Enchytraeus albidus (Oligochaeta) is an ecologically relevant species used as standard test organisms for risk assessment. Effects of stressors in this species are commonly determined at the population level using reproduction and survival as endpoints. The assessment of transcriptomic responses can be very useful e.g. to understand underlying mechanisms of toxicity with gene expression fingerprinting. In the present paper the following is being addressed: 1) development of suppressive subtractive hybridization (SSH) libraries enriched for differentially expressed genes after metal and pesticide exposures; 2) sequencing and characterization of all generated cDNA inserts; 3) development of a publicly available genomic database on E. albidus. A total of 2100 Expressed Sequence Tags (ESTs) were isolated, sequenced and assembled into 1124 clusters (947 singletons and 177 contigs). From these sequences, 41% matched known proteins in GenBank (BLASTX, e-value≤10-5) and 37% had at least one Gene Ontology (GO) term assigned. In total, 5.5% of the sequences were assigned to a metabolic pathway, based on KEGG. With this new sequencing information, an Agilent custom oligonucleotide microarray was designed, representing a potential tool for transcriptomic studies. EnchyBASE (http://bioinformatics.ua.pt/enchybase/) was developed as a web freely available database containing genomic information on E. albidus and will be further extended in the near future for other enchytraeid species. The database so far includes all ESTs generated for E. albidus from three cDNA libraries. This information can be downloaded and applied in functional genomics and transcription studies.