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.

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.

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.

Molecular responses during cadmium-induced stress in Daphnia magna: integration of differential gene expression with higher-level effects.

DNA microarrays offer great potential in revealing insight into mechanistic toxicity of contaminants. The aim of the present study was (i) to gain insight in concentration- and time-dependent cadmium-induced molecular responses by using a customized Daphnia magna microarray, and (ii) to compare the gene expression profiles with effects at higher levels of biological organization (e.g. total energy budget and growth). Daphnids were exposed to three cadmium concentrations (nominal value of 10, 50, 100microg/l) for two time intervals (48 and 96h). In general, dynamic expression patterns were obtained with a clear increase of gene expression changes at higher concentrations and longer exposure duration. Microarray analysis revealed cadmium affected molecular pathways associated with processes such as digestion, oxygen transport, cuticula metabolism and embryo development. These effects were compared with higher-level effects (energy budgets and growth). For instance, next to reduced energy budgets due to a decline in lipid, carbohydrate and protein content, we found an up-regulated expression of genes related to digestive processes (e.g. alpha-esterase, cellulase, alpha-amylase). Furthermore, cadmium affected the expression of genes coding for proteins involved in molecular pathways associated with immune response, stress response, cell adhesion, visual perception and signal transduction in the present study.

Effluent impact assessment using microarray-based analysis in common carp: a systems toxicology approach.

Effluents are a main source of direct and continuous input of pollutants to the aquatic environment, and can cause ecotoxicological effects at different levels of biological organization. Since gene expression responses represent the primary interaction site between environmental contaminants and biota, they provide essential clues to understand how chemical exposure can affect organismal health. The aim of the present study was to investigate the applicability of a microarray approach for unraveling modes of action of whole effluent toxicity and impact assessment. A chronic toxicity test with common carp (Cyprinus carpio) was conducted where fish were exposed to a control and 100% effluent for 21 days under flow-through conditions. Microarray analysis revealed that effluent treatment mainly affected molecular pathways associated with the energy balance of the fish, including changes in carbohydrate and lipid metabolism, as well as digestive enzyme activity. These gene expression responses were in clear agreement with, and provided additional mechanistic information on various cellular and higher level effects observed for the same effluent. Our results demonstrate the benefit of toxicogenomic tools in a "systems toxicology" approach, involving the integration of adverse effects of chemicals and stressors across multiple levels of biological complexity.

Daphnia magna and ecotoxicogenomics: gene expression profiles of the anti-ecdysteroidal fungicide fenarimol using energy-, molting- and life stage-related cDNA libraries.

In the present study, the existing life stage-specific cDNA library was extended with energy- and molting-related genes using Suppression Subtractive Hybridization PCR and a microarray for the aquatic test organism Daphnia magna was created. A gene set of 2455 fragments was produced belonging to different pathways such as carbohydrate and lipid metabolism, O2 transport and heme metabolism, immune response, embryo development, cuticula metabolism and visual perception pathways. Using this custom microarray, gene expression profiles were generated from neonates exposed to three concentrations of the anti-ecdysteroidal fungicide fenarimol (0.5, 0.75, 1 microg/ml) during 48 h and 96 h. In total, 59 non-redundant genes were differentially expressed, of which more genes were down- than up-regulated. The gene expression data indicated a main effect on molting specific pathways. At the highest concentration, a set of proteolytic enzymes - including different serine proteases and carboxypeptidases - were induced whereas different cuticula proteins were down-regulated (48 h). Moreover, effects on embryo development were demonstrated at the gene expression as well as at the organismal level. The embryo development related gene vitellogenin was differentially expressed after 96 h of exposure together with a significant increase in embryo abnormalities in the offspring. This study suggests that this Daphnia magna microarray is of great further value for the elucidation of molecular mechanisms of toxicity and for the future development of specific biomarkers for hazard characterization.

Use of suppression subtractive hybridization PCR for the development of cDNA arrays for the detection of endocrine disruption in carp (Cyprinus carpio).

The potential of a variety of xenobiotic compounds to modulate or disrupt the endocrine system of humans and wildlife is now widely recognized. In the present study, we developed a molecular tool for the evaluation of endocrine disruption in common carp (Cyprinus carpio). Suppression Subtractive Hybridization PCR was applied for the isolation of a relevant gene set, consisting of gender- and hormone-responsive gene fragments. This resulted in 398 different gene fragments that were most related to endocrine functioning. To investigate the applicability of this gene collection for studying endocrine disruption in fish, the gender-related genes were spotted on a cDNA macroarray, and expression profiles were generated for 17beta-estradiol (E2) and cortisol. Therefore, fish were injected with these hormones, and after 24 h and 96 h RNA was extracted and used for macroarray hybridizations. E2 exposure resulted in a total of 35 differentially expressed genes, whereas cortisol only affected 3 genes spotted on the macroarray. These results indicate the discriminating power of the developed array, and its usefulness to describe the toxicological mode of action of endocrine disruptive chemicals.

Patterns of gene expression in carp liver after exposure to a mixture of waterborne and dietary cadmium using a custom-made microarray.

Gene expression changes in carp liver tissue were studied after acute (3 and 24h) and subchronic (7 and 28 days) exposure to a mixture of waterborne (9, 105 and 480 microg/l) and dietary (9.5, 122 and 144 microg/g) cadmium, using a custom-made microarray. Suppression subtractive hybridization-PCR (SSH-PCR) was applied to isolate a set of 643 liver genes, involved in multiple biological pathways, such as energy metabolism (e.g. glucokinase), immune response (e.g. complement C3) and stress and detoxification (e.g. cytochrome P450 2F2, glutathione-S-transferase pi). These genes were subsequently spotted on glass-slides for the construction of a custom-made microarray. Resulting microarray hybridizations indicated a highly dynamic response to cadmium exposure. At low exposure concentrations (9 microg/l through water and 9.5 microg/g dry weight through food) mostly energy-related genes (e.g. glucokinase, elastase) were influenced, while a general stress response was obvious through induction of several stress-related genes, including hemopexin and cytochrome P450 2F2, at high cadmium concentrations. In addition, fish exposed to the highest cadmium concentrations showed liver damage after 7 days of exposure, as measured by elevated alanine transaminase activity in plasma and increased liver water content (wet-to-dry weight ratio). Moreover, decreased hematocrit and growth were found at the end of the experiment. Altogether this study clearly demonstrated the importance of varying exposure conditions for the characterization of the molecular impact of cadmium and showed that microarray results can provide important information, required to unravel the molecular events and responses related to cadmium exposure.

Neuropharmaceuticals in the environment: mianserin-induced neuroendocrine disruption in zebrafish (Danio rerio) using cDNA microarrays.

Because of their environmental occurrence and high biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. A major bottleneck in their risk assessment is the lack of relevant and specific effect data. We developed an approach using gene expression analysis in quantifying adverse effects of neuroendocrine pharmaceuticals in the environment. We studied effects of mianserin on zebrafish (Danio rerio) gene expression using a brain-specific, custom microarray, with real-time polymerase chain reaction as confirmation. After exposure (0, 25, and 250 microg/L) for 2, 4, and 14 d, RNA was extracted from brain tissue and used for microarray hybridization. In parallel, we investigated the impact of exposure on egg production, fertilization, and hatching. After 2 d of exposure, microarray analysis showed a clear effect of mianserin on important neuroendocrine-related genes (e.g., aromatase and estrogen receptor), indicating that antidepressants can modulate neuroendocrine processes. This initial neuroendocrine effect was followed by a "late gene expression effect" on neuronal plasticity, supporting the current concept regarding the mode of action for antidepressants in mammals. Clear adverse effects on egg viability were seen after 14 d of exposure at the highest concentration tested. Based on the specific molecular impact and the effects on reproduction, we conclude that further investigation of the adverse effects on the brain-liver-gonad axis is needed for a correct ecological risk assessment of antidepressants.

Expression profiling of endocrine-disrupting compounds using a customized Cyprinus carpio cDNA microarray.

Exposure to a variety of anthropogenic compounds has been shown to interfere with normal development, physiology, and reproduction in a wide range of organisms, both in laboratory studies and wildlife. We have developed a Cyprinus carpio cDNA microarray consisting of endocrine-related genes. In the current study, we investigated the applicability of this microarray (1) to study the molecular effects induced by exposure to a variety of endocrine-disrupting compounds (EDCs) in fish and (2) to discriminate the specific transcriptional profiles associated with these compounds. To that purpose, gene expression profiles were generated in livers of juvenile carp exposed to 14 Organization of Economical Cooperation Development (OECD)-recommended reference EDCs (17beta-estradiol, 17alpha-ethinylestradiol, 4-nonylphenol, bisphenol A, tamoxifen, 17alpha-methyltestosterone, 11-ketotestosterone, dibutyl phthalate, flutamide, vinclozolin, hydrocortisone, CuCl(2), propylthiouracil, and a mixture of L-triiodothyronine and L-thyroxine). Our results show that, in addition to some expression similarities between analogous acting substances, each individual compound produced its own unique expression pattern on the array, distinct from the profiles generated by the other compounds. In addition, we were able to identify a minimal subset of genes, which also allowed to discriminate between the different compounds. Overall, our findings suggest that the developed cDNA array has great promise to screen new and existing chemicals on their endocrine-disruptive potential and to identify distinct classes of EDCs.

Dynamics of cadmium accumulation and effects in common carp (Cyprinus carpio) during simultaneous exposure to water and food (Tubifex tubifex).

In this study, the influence of exposure time and concentration on the accumulation of cadmium and the occurrence of adverse effects was investigated when carp were simultaneously exposed through contaminated water and food. As exposure concentrations increased (9, 105, and 480 microg/L through water and 9.5, 122, and 141 microg/g dry weight through food) the accumulation pattern of cadmium changed, with progressively more cadmium being accumulated in the liver and especially the kidney and less in the gills and the intestine. A strong concentration- and time-dependent effect of cadmium exposure on plasma calcium concentrations was observed, leading to a decrease, with approximately 16% in the mid group and up to 50% in the group exposed to the highest cadmium concentration. Also at the highest exposure concentrations, sodium and chloride levels as well as plasma osmolality were significantly reduced, indicating a disturbed ion homeostasis. Furthermore, an increase in plasma alanine transaminase activity indicated liver damage caused by the cadmium accumulation in this organ. At the end of the experiment, general stress responses such as decreased hematocrit, growth, and mortality (17%) also were observed. All together, these results suggest that plasma hypocalcemia as an ion-disruptive phenomenon is the most sensitive effect during cadmium exposure, although the occurrence and magnitude both depend on exposure time and concentration.

Molecular impact of propiconazole on Daphnia magna using a reproduction-related cDNA array.

We have developed a first version cDNA microarray of the cladoceran Daphnia magna. Through Suppression Subtractive Hybridisation PCR (SSH-PCR) 855 life stage-specific cDNAs were collected and used to document the toxicological mode of action of the pesticide propiconazole. DNA sequencing analysis revealed gene fragments related to important functional classes such as embryo development, energy metabolism, molting and cell cycle. Major changes in transcription were observed in organisms exposed for 4 and 8 days to 1 microg/mL. After 4 days a 3-fold down-regulation of the gene encoding the yolk protein, vitellogenin, was observed indicating impaired oocyte maturation. Moreover, genes such as a larval-specific gene and chaperonin were repressed, whereas the heat shock 90 protein and ATP synthase were induced. Organismal effects clearly confirmed the major molecular findings: at the highest concentration (1 microg/mL) adult growth was significantly (p < 0.05) impaired and increased developmental effects in the offspring could be noted. We have demonstrated the potential of microarray analysis in toxicity screening with D. magna. The use of vitellogenin mRNA as a rapid biomarker of reproductive effects in chronic toxicity studies with cladocerans is suggested.

Determination of diazepam in aquatic samples by capillary liquid chromatography-electrospray tandem mass spectrometry.

In recent years growing attention has been paid toward the discharge, presence and potential adverse effects of pharmaceuticals in the environment. Using different existing analytical methods several studies have already identified a variety of drugs in waste-, surface- and drinking water. The monitoring of surface waters for drugs is of great importance because drugs are designed to be biological very active substances. A capillary LC/ES-MS-MS method has been developed that enables the sensitive and specific detection of diazepam in water samples up to 0.1 ng/ml (LOD). It requires neither multiple extraction steps, nor the use of large volumes of organic solvent. Applying this assay we have detected diazepam in 'in/effluent samples' collected in Belgium and demonstrated the applicability for water analysis without off-line pre-concentration of the analyte.

Exposure patterns of perfluorooctane sulfonate in aquatic invertebrates from the Western Scheldt estuary and the southern North Sea.

Over the past decades little research has been conducted on the environmental behavior and effects of fluorinated organochemicals (FOCs). Recently it has been reported that perfluorooctane sulfonic acid (PFOS) is occurring worldwide. Little is known about the PFOS levels in organisms originating from the southern North Sea and the Western Scheldt estuary. In this study, we determined, for the first time, the PFOS-exposure levels in Crangon crangon, Carcinus maenas, and Asterias rubens from these ecosystems. Concentrations on a wet-weight basis in soft tissues of shrimp, crab, and starfish ranged from 19 to 520 ng/g, from 24 to 877 ng/g, and from 9 to 176 ng/g, respectively. These results show the existence of a PFOS pollution gradient in organisms along the Western Scheldt estuary, with the highest concentrations near Antwerp. The range of PFOS levels in shrimp and crab are slightly higher in coastal regions compared with sampling sites in open water. This study shows widespread distribution of PFOS in the Belgian and Dutch marine and estuarine environment at rather high concentrations.

A multivariate biomarker-based model predicting population-level responses of Daphnia magna.

A multivariate model is proposed relating short-term biomarker measurements in Daphnia magna to chronic effects (21-d exposure) occurring at the population level (time to death, mean brood size, mean total young per female, intrinsic rate of natural increase, net reproductive rate, and growth). The results of the short-term exposure (48 h-96 h) to eight model toxicants (cadmium, chromium, mercury, tributyl tin, linear alkylsulfonic acid, sodium pentachlorophenolate, lindane, and 2,4-dichlorophenoxyacetic acid) on the following biomarkers were used for the multivariate model: digestive enzymes (amylase, cellulase, beta-galactosidase, trypsin, and esterase), enzymes of the intermediary metabolism (glycogen phosphorylase, glucose-6-phosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase, and isocitrate dehydrogenase), cellular energy allocation (CEA) (protein, carbohydrate, and lipid content and electron transport activity), and DNA damage and antioxidative stress activity. Using partial least squares to latent structures (PLS), a two-component model was obtained with R2 of 0.68 and a Q2 value of 0.60 based on the combined analysis of a limited number of the 48- and 96-h biomarker responses. For the individual population-level responses, the R2 values varied from 0.66 to 0.77 and the Q2 values from 0.52 to 0.69. Energy-related biomarkers (cellular energy allocation, lipid contents, anaerobic metabolic activity--pyruvate kinase, and lactate dehydrogenase), combined with parameters related to oxidative stress (catalase) and DNA damage measured after 48 and 96 h of exposure, were able to predict long-term effects at higher levels of biological organization.

The missing biomarker link: relationships between effects on the cellular energy allocation biomarker of toxicant-stressed Daphnia magna and corresponding population characteristics.

The cellular energy allocation (CEA) methodology was used to assess the adverse effects of toxic stress on the energy budget of test organisms. This biochemical assay is quantified by determining changes in the available energy reserves, Ea (total carbohydrate, protein, and lipid content) and the energy consumption, Ec (electron transport activity). The CEA methodology was fully explored by using neonates of Daphnia magna exposed for 96 h to six model toxicants (CdCl2, K2Cr2O7, tributyltin chloride, linear alkylbenzene sulfonic acid, sodium pentachlorophenolate, and 2,4-dichlorophenoxyacetic acid). To evaluate the ecological relevance of the CEA parameter, we compared the suborganismal responses with population-level parameters (obtained from 21-d life-table experiments) such as the intrinsic rate of natural increase (rm) and the mean total offspring per female. The observed reductions in CEA values were both the result of a decrease in Ea and an increase in Ec. From all individual CEA components analyzed, the lipid reserve criterion was the most sensitive endpoint studied. Both the CEA-based lowest-observed-adverse-effect concentration (LOAEC) values and the effective concentration of 10% (EC10) values were significantly (p < 0.05) and linearly correlated with the chronic (21-d) LOAEC and EC10 values based on growth, survival, and reproduction. This relationship demonstrates the usefulness of the methodology to predict long-term effects. Furthermore, significant (p < 0.0001) sigmoid relationships between the 96-h CEA value (expressed as percentage relative to the control) and population-level effects were observed.

Perfluorooctane sulfonic acid in bib (Trisopterus luscus) and plaice (Pleuronectes platessa) from the Western Scheldt and the Belgian North Sea: distribution and biochemical effects.

A biomonitoring campaign was conducted in the Belgian North Sea and in the Western Scheldt (The Netherlands) with the primary goal to assess perfluorooctane sulfonic acid (PFOS) contamination and distribution in different biota. This study covers the results obtained for bib (Trisopterus luscus) and plaice (Pleuronectes platessa) and includes the assessment of some stress-related biochemical endpoints. Analysis of liver and muscle PFOS concentrations of both species provided evidence for the existence of a PFOS pollution gradient along the Western Scheldt with higher levels at the upstream locations and a lower degree of PFOS pollution at the marine locations. Cellular necrosis was studied by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the serum. Serum ALT but not serum AST was shown to correlate positively with the PFOS liver concentration in bib (r = 0.44, p < 0.05), indicating that PFOS might contribute to the induction of hepatic damage in bib in the area of study. Analysis of total carbohydrate, lipid, and protein content of bib liver tissue revealed a positive correlation between the protein content and the PFOS liver concentration (r = 0.55, p < 0.01). Whether this is due to induction of compensatory mechanisms, detoxification, or repair processes remains unclear.