Identification of proteome markers for drug-induced liver injury in zebrafish embryos.

The zebrafish embryo (ZFE) is a promising alternative non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatic responses related to drug-induced liver injury (DILI). Here, we hypothesize that detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of compounds and to the reduction of rodents used for hepatotoxicity assessment. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of steatosis, cholestasis, and necrosis, and effects compared with negative controls. Protein profiles of the individual compounds were generated using LC-MS/MS. We identified differentially expressed proteins and pathways, but as these showed considerable overlap, phenotype-specific responses could not be distinguished. This led us to identify a set of common hepatotoxicity marker proteins. At the pathway level, these were mainly associated with cellular adaptive stress-responses, whereas single proteins could be linked to common hepatotoxicity-associated processes. Applying several stringency criteria to our proteomics data as well as information from other data sources resulted in a set of potential robust protein markers, notably Igf2bp1, Cox5ba, Ahnak, Itih3b.2, Psma6b, Srsf3a, Ces2b, Ces2a, Tdo2b, and Anxa1c, for the detection of adverse responses.

Time-Integrative Passive sampling combined with TOxicity Profiling (TIPTOP): an effect-based strategy for cost-effective chemical water quality assessment.

This study aimed at demonstrating that effect-based monitoring with passive sampling followed by toxicity profiling is more protective and cost-effective than the current chemical water quality assessment strategy consisting of compound-by-compound chemical analysis of selected substances in grab samples. Passive samplers were deployed in the Dutch river delta and in WWTP effluents. Their extracts were tested in a battery of bioassays and chemically analyzed to obtain toxicity and chemical profiles, respectively. Chemical concentrations in water were retrieved from publicly available databases. Seven different strategies were used to interpret the chemical and toxicity profiles in terms of ecological risk. They all indicated that the river sampling locations were relatively clean. Chemical-based monitoring resulted for many substances in measurements below detection limit and could only explain <20% of the observed in vitro toxicity. Effect-based monitoring yielded more informative conclusions as it allowed for ranking the sampling sites and for estimating a margin-of-exposure towards chronic effect ranges. Effect-based monitoring was also cheaper and more cost-effective (i.e. yielding more information per euro spent). Based on its identified strengths, weaknesses, opportunities, and threats (SWOT), a future strategy for effect-based monitoring has been proposed.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis.

Humic substances alleviate the aquatic toxicity of polyvinylpyrrolidone-coated silver nanoparticles to organisms of different trophic levels.

The present study investigated how humic substances (HS) modify the aquatic toxicity of silver nanoparticles (AgNPs) as these particles agglomerate in water and interact with HS. An alga species (Raphidocelis subcapitata), a cladoceran species (Chydorus sphaericus), and a freshwater fish larva (Danio rerio), representing organisms of different trophic levels, were exposed to colloids of the polyvinylpyrrolidone-coated AgNPs in the presence and absence of HS. Results show that the presence of HS alleviated the aquatic toxicity of the AgNP colloids to all the organisms in a dose-dependent manner. The particle size distribution of the AgNPs' colloidal particles shifted to lower values due to the presence of HS, implying that the decrease in the toxicity of the AgNP colloids cannot be explained by the variation of agglomeration size. The surface charge of the AgNPs was found to be more negative in the presence of high concentrations of HS, suggesting an electrostatic barrier by which HS might limit interactions between particles and algae cells; indeed, this effect reduced the algae toxicity. Observations on silver ions (Ag(+)) release show that HS inhibit AgNP dissolution, depending on the concentrations of HS. When toxic effects were expressed as a function of each Ag-species, toxicity of the free Ag(+) was found to be much higher than that of the agglomerated particles.

Gene expression markers in the zebrafish embryo reflect a hepatotoxic response in animal models and humans.

The zebrafish embryo (ZFE) is a promising non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatotoxic responses. Here, we hypothesize that the detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of new compounds and to the reduction of rodents used for screening. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of cholestasis, steatosis and necrosis, and two non-hepatotoxic controls. Histopathology revealed various specific morphological changes in the ZFE hepatocytes indicative of cell injury. Gene expression profiles of the individual compounds were generated using microarrays. Regulation of single genes and of pathways could be linked to hepatotoxic responses in general, but phenotype-specific responses could not be distinguished. Hepatotoxicity-associated pathways included xenobiotic metabolism and oxidoreduction related pathways. Overall analysis of gene expression identified a limited set of potential biomarkers specific for a common hepatotoxicity response. This set included several cytochrome P450 genes (cyp2k19, cyp4v7, cyp2aa3), genes related to liver development (pklr) and genes important in oxidoreduction processes (zgc:163022, zgc:158614, zgc:101858 and sqrdl). In conclusion, the ZFE model allows for identification of hepatotoxicants, without discrimination into specific phenotypes.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Transcriptomic analysis in the developing zebrafish embryo after compound exposure: individual gene expression and pathway regulation.

The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, d-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity.

Triazole-induced gene expression changes in the zebrafish embryo.

The zebrafish embryo is considered to provide a promising alternative test model for developmental toxicity testing. Most systems use morphological assessment of the embryos, however, microarray analyses may increase sensitivity and predictability of the test by detecting more subtle and detailed responses. In this study, we investigated the possibility of relating gene expression profiles of structurally similar chemicals tested in a single concentration, to a complete transcriptomic concentration-response of flusilazole (FLU). We tested five other triazoles, hexaconazole (HEX), cyproconazole (CYP), triadimefon (TDF), myclobutanil (MYC), and triticonazole (TTC) at equipotent concentrations based on morphological evaluation. Results showed that every compound had a different degree of regulation within their anti-fungal and developmental toxicity pathways, steroid biosynthesis and retinol metabolism, respectively. Assuming that the ratio between these pathways is relevant for efficacy compared to developmental toxicity, we found TTC was more efficient and CYP was more toxic compared to the other triazoles. With the approach used in this study we demonstrated that gene expression data allow more comprehensive assessment of compound effects by discriminating relative potencies using these specific gene sets. The zebrafish embryo model can therefore be considered a useful vertebrate model providing information of relevant pathways related to anti-fungal mechanism of action and toxicological activity.

Concentration-response analysis of differential gene expression in the zebrafish embryotoxicity test following flusilazole exposure.

The zebrafish embryotoxicity test (ZET) is considered a promising alternative model in predictive toxicology. Currently, morphological assessment of the embryo is the main readout for this assay. However, implementation of transcriptomics may help to detect more subtle effects, which may increase the sensitivity and predictability of the test. In this study, we tested a concentration response of flusilazole in the ZET. After exposure for 24 h postfertilization, microarray analysis revealed a number of processes to be regulated in a concentration-dependent way. We identified development related processes, retinol metabolism and transcription, as well as processes corresponding to the antifungal mechanism of action, steroid biosynthesis, and fatty acid metabolism, to be differentially regulated. Retinol metabolism and transcription were already significantly altered at concentrations that were not inducing morphological effects. Differential expression of genes related to steroid biosynthesis and fatty acid metabolism showed a concentration response similar to morphological response. An increase in concentration was also positively associated with an increase in magnitude of expression for individual genes within functional processes. Our study shows that transcriptomics analysis in the ZET is a more sensitive readout of compound-induced effects than morphological assessment. However, the interpretation of differential gene expression in terms of predicting morphological effects is not straightforward and requires further study.

Acute toxicity of poly- and perfluorinated compounds to two cladocerans, Daphnia magna and Chydorus sphaericus.

With their global distribution, environmental persistence, and potential risk to human beings and ecosystems, poly- and perfluorinated compounds (PFCs) are of particular concern for research and regulatory communities. However, insufficient toxicity data are available for most poly- and perfluorinated compounds to assess their possible environmental hazards accurately. Therefore, the acute toxicity of seven poly- and perfluorinated carboxylic acids and alcohols on two cladocerans, Daphnia magna and Chydorus sphaericus, was evaluated in the present study. The adverse effects of these PFCs on these two cladocerans decreased with increasing fluorinated carbon chain length (nC) and quantitative structure-activity relationships were developed to quantify this observation. Because the 50% inhibition effects (EC50) values obtained are far above concentrations typically found in surface water, acute harmful effects of these chemicals to D. magna and C. sphaericus are not expected in the real environment.

Chemical class-specific gene expression changes in the zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals.

The zebrafish embryotoxicity test (ZET) is an alternative test to predict embryotoxicity of substances based on morphological assessment. Implementing transcriptomics may increase sensitivity and objectivity of the test system. We applied the category approach to compare effects of compounds from two chemical classes, the glycol ethers and 1,2,4-triazoles, on the embryo. At 24h post fertilization, microarray analysis revealed several thousands of responsive genes after glycol ether exposure, whereas the triazoles significantly regulated only several hundreds of genes. Principal component analysis of the genes commonly regulated per chemical class demonstrated that the two classes can be distinguished. Gene set enrichment analysis showed that after glycol ether exposure mainly gene sets related to development were downregulated. After triazole exposure, gene sets corresponding to previously described mechanisms of action, such as glycolysis and fatty acid metabolism were regulated. Our results demonstrate that transcriptomics in the ZET provides a more sensitive endpoint than standard morphological assessment. In addition, information about mechanisms of action of substances may become available, thereby facilitating the extrapolation of findings to mammalian species including man.

Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies.

The zebrafish embryotoxicity test (ZET) is a fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. We developed a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system. For teratogenic effects a separate scoring list was developed. The relative effects of eight glycol ethers and six 1,2,4-triazole anti-fungals were evaluated in this system and results were compared with in vivo developmental toxicity potencies. Methoxyacetic acid and ethoxyacetic acid appeared as the most potent glycol ether metabolites, inducing growth retardation and malformations. Other glycol ethers showed no developmental toxicity. Flusilazole appeared the most potent triazole, followed by hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole, respectively. In general, the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking. In conclusion, the ZET with the GMS system appears an efficient and useful test system for screening embryotoxic properties of chemicals within the classes of compounds tested. This alternative test method may also be useful for the detection of embryotoxic properties of other classes of chemicals.

Fish embryo toxicity of carbamazepine, diclofenac and metoprolol.

Frequently measured pharmaceuticals in environmental samples were tested in fish embryo toxicity (FET) tests with Danio rerio, based on the draft OECD test protocol. In this FET test 2-h-old zebrafish embryos were exposed for 72 h to carbamazepine, diclofenac and metoprolol to observe effects on embryo mortality, gastrulation, somite formation, tail movement and detachment, pigmentation, heartbeat, malformation of head, otoliths and heart, scoliosis, deformity of yolk, and hatching success at 24, 48 and 72 h. We found specific effects on growth retardation above 30.6 mg/l for carbamazepine, on hatching, yolk sac and tail deformation above 1.5mg/l for diclofenac, and on scoliosis and growth retardation above 12.6 mg/l for metoprolol. Scoring all effect parameters, the 72-h-EC(50) values were: for carbamazepine 86.5mg/l, for diclofenac 5.3mg/l and for metoprolol 31.0mg/l (mean measured concentrations). In conclusion, our results for carbamazepine and metoprolol are in agreement with other findings for aquatic toxicity, and also fish embryos responded in much the same way as rat embryos did. For diclofenac, the FET test performs comparably to Early Life Stage testing.

Comparing the sensitivity of algal, cyanobacterial and bacterial bioassays to different groups of antibiotics.

Antibiotics may affect both primary producers and decomposers, potentially disrupting ecosystem processes. Hence, it is essential to assess the impact of antibiotics on aquatic ecosystems. The aim of the present study was therefore to evaluate the potential of a recently developed test for detecting antibiotics in animal tissue, the Nouws Antibiotic Test (NAT), as a sensitive bioassay to assess the effects of antibiotics in water. To this purpose, we determined the toxicity of sulphamethoxazole, trimethoprim, flumequine, tylosin, streptomycin, and oxytetracycline, using the NAT adapted for water exposure. The sensitivity of the NAT was compared to that of bioassays with bacteria (Microtox), cyanobacteria and green algae. In the Microtox test with Vibrio fischeri as test organism, no effects were observed for any of the test compounds. For three of the six antibiotics tested, the cyanobacteria were more vulnerable than the green algae when using photosynthetic efficiency as an endpoint. The lowest EC50 values for four out of six tested antibiotics were obtained using the NAT bacterial bioassay. The bacterial plate system responded to antibiotics at concentrations in the microgL(-1) and lower mgL(-1) range and, moreover, each plate proved to be specifically sensitive to the antibiotics group it was designed for. It is concluded that the NAT bioassay adapted for water exposure is a sensitive test to determine the presence of antibiotics in water. The ability of this test to distinguish five major antibiotic groups is a very strong additional value.

Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches.

Tetrabromobisphenol-A (TBBPA) is nowadays one of the most frequently used brominated flame retardants (BFRs) and can be considered as a high production volume chemical. Over the last decade, numerous reports of increasing concentrations of BFRs in the environment and humans have been published. However, the toxicological knowledge on TBBPA, and more specifically its molecular mode of action, is rather fragmentary. In this study two populations of adult zebrafish (Danio rerio) were exposed for 14 days to 0.75 microM and 1.5 microM TBBPA. Subsequently, we employed a combined transcriptomic and proteomic approach to evaluate the molecular effects of TBBPA in zebrafish liver. Oligonucleotide microarrays were used to study the effects on gene expression levels. These results were validated through real-time PCR. The proteome of the liver was analysed by means of differential in-gel electrophoresis (DiGE), an innovative application of traditional 2D-PAGE. Combination of the extracted datasets allowed reassembling of individual molecular responses into a comprehensive overview of affected molecular pathways. Interpretation of the results depicted an interference of thyroid and Vitamin A homeostasis in the exposed zebrafish, TBBPA also elicited responses indicating onset of oxidative stress and general stress responses. Additionally, numerous differentially expressed transcripts could be associated with defence mechanisms or corresponded to metabolizing enzymes. Furthermore, cellular metabolism was clearly affected, illustrated as disturbance of e.g. lipid, carbohydrate, and organic acid metabolic processes. Summarizing, these results enabled us to hypothesize several working mechanisms of TBBPA and demonstrated the potential of a combined genome and proteome approach to generate detailed mechanistic toxicological information.

Toxicity of analytically cleaned pentabromodiphenylether after prolonged exposure in estuarine European flounder (Platichthys flesus), and partial life-cycle exposure in fresh water zebrafish (Danio rerio).

Residues of polybrominated diphenylethers (PBDEs), extensively applied as flame retardants, are widely spread in the aquatic environment and biota. The present study investigates effects of the environmentally relevant lower brominated diphenylethers in two fish species in vivo under controlled laboratory conditions. Euryhaline flounder (Platichthys flesus) and freshwater zebrafish (Danio rerio) were exposed to a range of concentrations of a commercial pentabromodiphenylether mixture, DE-71. Chemical analysis of exposed fish showed a pattern of PBDE congeners that was very similar to that in wild fish. The resulting range included environmentally relevant, as well as higher levels. Animals were investigated histopathologically with emphasis on endocrine and reproductive organs. In zebrafish, hatching of embryos and larval development were assessed. Biochemical parameters were investigated in flounder as markers for suggested dioxin-like activity (ethoxyresorufin-O-deethylase=EROD), and activation of endogenous estrogen synthesis (gonad aromatase activity). Thyroid hormones were analyzed in plasma in both species. Benchmark analysis using internal PBDE concentrations showed a mild dose-dependent decrease of hepatic EROD and ovarian aromatase activities, and plasma thyroxin levels in flounder, and an increase of plasma thyroid hormone levels in zebrafish. These trends did not result in statistically significant differences from control fish, and major histopathological changes were not observed. Reproduction in zebrafish appeared to be the most sensitive parameter with statistically significantly reduced larval survival and non-significant indications for decreased egg production at internal levels that were more than 55 times the highest environmental recordings. The present results indicate limited risk for endocrine or reproductive effects of current environmental PBDE contamination in fish.

Effects of the estrogen agonist 17beta-estradiol and antagonist tamoxifen in a partial life-cycle assay with zebrafish (Danio rerio).

A partial life-cycle assay (PLC) with zebrafish (Danio rerio) was conducted to identify endocrine-disrupting effects of 17beta-estradiol (E2) and tamoxifen (TMX) as reference for estrogen agonist and antagonist activity. Adult zebrafish were exposed for 21 d and offspring for another 42 d, allowing differentiation of gonads in control animals. The assessed end points included reproductive variables (egg production, fertilization, and hatching), gonad differentiation of juveniles, histopathology, and vitellogenin (VTG) expression. With E2, the most sensitive end points were feminization of offspring (at 0.1 nM) and increased VTG production in males (at 0.32 nM). At 1 nM, decreased F, survival, increased F, body length and weight, VTG-related edema and kidney lesions, and inhibited spermatogenesis were observed. Oocyte atresia occurred at even higher concentrations. Exposure to TMX resulted in specific effects at an intermediate test concentration (87 nM), including oocyte atresia with granulosa cell transformation and disturbed spermatogenesis (asynchrony within cysts). In F1, decreased hatching, survival, and body weight and length as well as decreased feminization were observed. Decreased vitellogenesis and egg production in females and clustering of Leydig cells in males occurred at higher concentrations. Toxicological profiles of estrogen agonists and antagonists are complex and specific; a valid and refined characterization of endocrine activity of field samples therefore can be obtained only by using a varied set of end points, including histology, as applied in the presented PLC. Evaluation of only a single end point can easily produce under- or overestimation of the actual hazard.

Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish.

Some ubiquitous pollutants of the aquatic environment, such as PCBs or other polyhalogenated aromatic hydrocarbons, may disrupt the thyroid hormone system. In a partial life cycle assay with zebrafish (Danio rerio), we studied the effects of the reference compound propylthiouracil (PTU) on reproduction, growth and development, histopathology of some target tissues, and plasma thyroid hormone levels. PTU induced a concentration-dependent increase of egg production with a concomitant decrease of mature oocyte size but had no effect on fertilization rate or hatching. In F1, serious dysmorphogenesis was found in 4 dph larvae at the highest PTU level tested (100 mg/L), and there was a dose-dependent decrease in body length and weight at 42 dph (significant at 100 mg/L PTU). At this time, there was also a decreased scale thickness, suggesting inhibited metamorphosis, detectable at 1 mg/L PTU and higher. PTU also induced activation of the thyroid follicles in a concentration-dependent way, in juveniles associated with hyperemia in the thyroid area, and depletion of liver glycogen. Effects in adults were associated with decreased circulating levels of the thyroid hormones T3 and T4. These observations indicate that disruption of the thyroid hormone system may affect the fitness of these aquatic organisms. The zebrafish model may contribute to the identification of thyroid hormone disrupting activity in water samples and also in the interpretation of histological observations in free-ranging fish species.

Vitellogenin expression in zebrafish Danio rerio: evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridisation.

Several histological methods were tested for their potential to detect the in vivo induction of vitellogenin in zebrafish, after exposure to 17beta-oestradiol (E2), and validated by correlating semi-quantitative measurements on digital images to vitellogenin plasma values measured by ELISA and morphological criteria. All methods, except for vitellogenin-specific immunohistochemistry on liver, detected vitellogenin production in male zebrafish at the exposure level of 1 nM E2/l, and correlated well to each other and to ELISA results on plasma, thus indicating their specificity. The level of sensitivity is in the range of the induction of clinical (histopathological) effects, although slightly below the level of sensitivity of the plasma ELISA. Vitellogenin specific in situ mRNA hybridisation on liver appeared laborious and not applicable on routinely prepared material. Vitellogenin specific immunohistochemistry on plasma and basophilia of male liver are cost- and effort-effective detection methods of vitellogenin production, and can be applied routinely on standard histological sections. These methods are, therefore, suitable to evaluate vitellogenin production as an indicator of exposure to compounds with estrogenic activity, at the level of induction of clinical effects. They are a useful tool for hazard identification of endocrine disruption, especially when combined with routine histopathology.