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Final Report: A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting Chemicals
EPA Grant Number: R831301Title: A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting Chemicals
Investigators: Callard, Gloria V.
Institution: Boston University
EPA Project Officer: Mustra, David
Project Period: October 1, 2003 through September 30, 2007
Project Amount: $400,000
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003)
Research Category: Endocrine Disruptors , Children's Health
Description:
Objective:Of the estimated >87,000 chemicals added to the environment by anthropomorphic activities, few have actually been tested for their ability to disrupt critical hormone-regulated processes of development, reproduction and physiology (termed endocrine disrupting chemicals, EDC). Consequently, there is an urgent need to develop screening methods for identifying EDC. This project rests on the proposition that perturbations in the normal amount or timing of hormone-regulated gene products can be taken as evidence of chemical exposure, and used as end-points for detecting EDC. To address this hypothesis, we reasoned that living zebrafish embryos have the convenience of an in vitro test system with the added value of results from a whole organism in which all tissues and cells are in their normal in vivo context. Also, because endocrine systems and developmental processes are well-conserved in vertebrates, there is a sound scientific basis for extrapolating results from zebrafish to other animals, including humans. Specific objectives were to demonstrate that real time quantitative (q)-PCR analysis of expressed genes can be used to detect and characterize (a) multiple subclasses of EDC; (b) multiple gene- and tissue- targets of a given EDC subclass; (c) EDC that target hormone signaling pathways both upstream and downstream of receptor binding; and (d) EDC present as mixtures in samples from polluted environments. Focus was on known markers of estrogen receptor (ER) and/or arylhydrocarbon receptor (AhR)-mediated actions and effects (n, 9) after exposure of embryos/larvae at successive developmental stages (2 – 120 hr post-fertilization) to authentic ER and AhR ligands and to known or suspected EDC (total, ~30 different chemicals) in detailed dose- and time-course studies. To expand the utility of the assay, and optimize normalization of qPCR readout, additional known markers of development and physiological or reproductive status (n, 18) and a panel of diverse housekeeping genes (n, 8) were characterized in a subset of experiments. Twenty-five previously unknown but potentially informative mRNAs were identified by microarray analysis and verified by qPCR. Results of this project (1) address an urgent need for regulators to better predict which of the chemicals added to the environment have the potential to disrupt hormone-dependent processes of physiology, reproduction and development; (2) provide biologically relevant criteria for prioritizing chemicals for further testing; (3) offer a test platform and database that can be used to define a chemical’s mechanism of action and body-wide effects; and (4) help to interpret observed reproductive and developmental abnormalities in wildlife and humans. This project establishes the feasibility of procedures and endpoints that are amenable to high throughput modifications, and can be viewed as a proof-of-principle study with applicability for screening chemicals that impact any other ligand activated nuclear receptor signaling pathway of interest.
Summary/Accomplishments (Outputs/Outcomes):2.a. Selection of animal model.
For measuring EDC-mediated changes in gene expression, living zebrafish embryos have the convenience of an in vitro test system with the added value of results from a whole organism in which all systems, tissues and cells are in their normal in vivo context.
Although the proposed in vitro assay minimizes animal and chemical use, endocrine disrupting activity and agonist vs. antagonist properties of a chemical can be predicted without a priori knowledge of uptake and accumulation, activating or metabolizing pathways, access to targets, receptor binding and activation, or required coregulators. Also, because endocrine systems and developmental processes are well-conserved in vertebrates, there is a sound scientific basis for extrapolating results from zebrafish to other animals, including humans. To a limited extent (and with support from another project), results obtained with zebrafish embryos have proven to be applicable to killifish living long term in a polluted environment.
2.b. Selection of target genes.
Markers of specific signaling pathways or biological conditions. Estrogen-like (xenoestrogens) and dioxin-like chemicals (DLC) are potential EDC which are commonly found as mixtures at polluted sites. Using PCR cloning and database mining approaches, we have developed primers and real time qPCR assays for 7 zebrafish genes (and 11 additional killifish genes) as specific mechanism-based markers of EDC action and effect. Genes include P450aromB (marker of estrogen biosynthesis and estrogen effect in brain); P450aromA (marker of estrogen biosynthesis in ovary); ERα, -βa, and -βb (markers of estrogen action and effect); vitellogenin (vtg; marker of estrogen effect in liver); cyp1A, AhR1 and AhR2 (markers of dioxin action and effect in liver).
The utility of the zebrafish embryos test system to detect hormone-dependent or –independent chemical effects has been expanded by developing additional qPCR assays for markers known to be linked to fundamental cellular processes (cyclinD, cyclinG1, BAX, Bcl-2), development (Nurr1, Nor1, Nur77, GAP43, a-tubulin, neuropilin, TH1, TH2), and reproduction/ steroidogenesis (17bHSD, 3bHSD, cyp19, cyp11A1, STAR).
Global gene discovery. Additionally, we have used an unbiased genome-wide search (Affymetrix microarray, 17,000 markers) to identify mRNAs differentially expressed in embryos or adults with/without estrogen or dioxin or undergoing experimentally induced neuroregeneration. Of these, a subset of markers were selected for qPCR validation and charactgerization: 25 total (e.g., parvalbumin 4, granulin, IGFI-B).
Housekeeping genes (quality assurance). During the course of these studies, we found that results differed depending on which housekeeping (control) gene was used as an internal normalizer for qPCR. In contrast to mammals, information needed to choose the best control gene for a given condition or experiment was lacking for zebrafish. Accordingly, we systematically characterized 8 different housekeeping genes in embryos during development with/without 10 different chemical additives, and in different tissues of adult males and females. Genes were b-actin, a-tubulin, TBP, GAPDH, G6PD, ELFA, 18S, B2M (see below).
2.c. Optimization of qRT-PCR analysis/quality assurance
Assay development and validation in embryonic/larval and adult male and female zebrafish has been completed for P450 aromatase B and –A mRNA analysis (Sawyer et al., 2006) and serves as a template for assay development of all other targeted mRNAs and to qPCR analysis of the same mRNAs in field-collected killifish adults and embryos (see Greytak et al. 2005). Our assay is sensitive, precise, gene-specific, and has a very low intra- and interassay coefficient of variation (<10%). Reproducibility is excellent within and across operators when tested in multiple experiments, and beginning at different starting points within a single experiment (e.g., RNA extraction, reverse transcription, PCR). Results confirm and extend data reported with earlier methods and reveal much greater estrogen-inducibility (>150-fold) than with semiquantitative RT-PCR. Each gene of interest is normalized to an internal standard (e.g., actin; but see below), after correction for gene-specific differences in amplification efficiency, which is more accurate and cost efficient than multiple idealized external standard curves. Appropriate choice of primers assures specificity with SYBR green, which is more cost effective than TaqMan probes for high-throughput applications. The small amount of input rtRNA (80 ng/microwell) enables us to assay >10 genes in triplicate in a single embryo although, in practice, we pool embryos to minimize biological variation. As well, the assay is applicable.
Choice of housekeeping genes (quality assurance). During the course of these studies, we observed that actin was not consistently acceptable for normalizing PCR data (see above), and that a systematic analysis of a panel of housekeeping genes, currently lacking for zebrafish, would be necessary to validate our zebrafish gene expression bioassay and an important contribution to the community in general. Of 8 housekeeping genes studied, results show that the preferred normalizer is dictated by the nature of the experiment: embryogenesis (18S >> b-actin >> a-tubulin); tissue expression patterns (ELFA > b-actin >> GAPDH); 8 different embryo treatments at a given stage (b-actin > GAPDH >> 18S). Also, in adults, there were unexpected sex differences (TBP in heart, muscle, gonad: F >> M; a-tubulin in liver: M > F).
mRNA splice variants (quality assurance). Approximately 60% of human genes are alternatively spliced, which increases functional versatility but confounds PCR-based methods of expression analysis. Our findings with zebrafish embryos show that certain developmental stages normally express relatively high levels of splice variants of a given target gene (e.g., P450aromB). Likewise, some EDCs (dioxin, estradiol) and environmental mixtures are able to induce splice variants (e.g, P450aromB, ERα). It is also significant that splice variants are site-related in killifish in the natural environment (e.g., ERα). Thus, great care must be given to selection of qPCR primers and their in-assay validation using both authentic plasmids and RNA extracts from exposed embryos, followed by gel electrophoresis and even product isolation and sequencing. This care must be applied gene-by-gene to assure specific and accurate quantitation of normal and variant mRNAs. As regards EDC effects on splicing decisions, this is a heretofore unrecognized mechanism of EDC action that certainly merits further study.
2.d. Selection of test chemicals
Authentic ER and AhR ligands. The majority of experiments were designed to validate and optimize the zebrafish bioassay and therefore used authentic ligands for ER- and AhR-signaling pathways, estradiol and dioxin. These ligands were added to embryos separately in detailed dose-response and time-course studies, and also were used in combination at different ratios to evaluate convergence or overlap of the two pathways on the panel of 7 markers of estrogen action/effect and 4 markers of dioxin action/effect (see above).
Representative chemical classes. To assess the utility (sensitivity, specificity) of these markers to detect interference of ER and AhR signaling by other chemicals, we have now completed dose-response profiles after treating embryos with a wide array of known or suspected EDC, including steroid hormones (e.g., aromatizable and non-aromatizable androgens and their metabolites); drugs known to mimic or block ER or AhR signaling (e.g, diethylstilbestrol, ICI, ATD, anastrazole, fadrozole, tamoxifen, raloxifene; alpha naphthoflavone); phytochemicals (e.g., genistein, zearalenone); industrial byproducts (e.g., bisphenol A, 4-nonylphenol, PCB126); pesticides (e.g., o,p-DDT atrazine, vinclozolin); and cosmetics (tea oil, lavender oil).
Toxicity (quality assurance). In marked contrast to cell-free assays, living zebrafish are sensitive to the toxic effects of added chemicals, and the maximum testable doseage is limited by solubility of a chemical in an acceptable vehicle. Accordingly, viability, mobility and gross anatomic defects are routinely monitored as evidence of toxicity in all embryo experiments. Where toxicity is seen, samples are excluded from the data. For example, concentrations of tea oil that were found to estrogenic in a previous study were highly toxic for zebrafish. Thus, our inability to detect changes in estrogen responsive genes could be due to the low concentrations used.
Vehicles (quality assurance). Routinely, DMSO is used for solubilizing test chemicals. DMSO, as compared to no treatment, does not alter expression of any of our panel of housekeeping genes or other markers. Although ethanol has no effect on housekeeping genes, in our earlier study we found that it upregulated P450aromB.
Environmental samples. Water and sediment were collected from polluted and reference ponds near the Massachusetts Military Reservation (MMR) Superfund site on Cape Cod MA. Impacted ponds had turtles with reproductive abnormalities (endocrine disruption). Samples were processed by filtration, elutriation and centrifugation, and used to replace culture media in increasing percantages. No toxicity effects were observed.
2.e. Baseline, developmentally programmed and treatment-dependent expression.
Developmental profiles. Because fertilized eggs are collected from multiple spawning pairs for each experiment, there is a degree of intra-assay variability in the rate at which embryos develop. Accordingly, results of all studies are based on three independent biological replicates at each stage/treatment group (RNA extraction → qPCR). For each mRNA of interest, we have documented mRNA content of unfertilized eggs (0 hpf) and developmental expression profiles 2 – 120 hr postfertilization (hpf). Predictably, the time of onset and subsequent changes in expression are gene-specific and correspond to known morphogenetic events. For example, the time of onset of P450aromB was much earlier than vtg, corresponding to the earlier development of brain as compared to liver.
Dose-dependent and cumulative treatment effects. To determine effects of dose and exposure duration, different concentrations of E2 (0.0001, 0.001, 0.01, 0.1, 1 mM) or dioxin (0.001, 0.01, 0.1 and 1 nM) were added to media between 24-48 hpf, 24-72 hpf, 24-96 hpf, or 24-96 hpf.. Results indicate that the embryonic estrogen response system is gene specific as measured by magnitude (vtg > P450aromB > ERα); sensitivity (P450aromB > vtg > ERα); and specificity (ERβa, –βb, actin, cyp1A1, AhR2 = no change). The AhR response system was also gene specific: cyp1A1 (200X increase), AhR2 (2X increase) but the ~EC50 was similar for both. P450aromA responses were inconsistent: 2 experiments, 15X downregulation; 2 experiments, no change; one experiment, 1X upregulation. P450aromB, ERα, ERβa, –βb, AhR1, AhR2, vtg and actin were unresponsive. Predictably, fold induction of each gene increased with duration of exposure. In general, however, pre-hatch embryos (<72 hpf) had relatively weak responses when compared to post-hatch larvae (>72 hpf).
Stage-related changes in sensitivity and responsiveness. To further evaluate the ontogeny of estrogen- and dioxin response systems, embryos were treated with a range of E2 (0.0001, 0.001, 0.01, 0.1, 1 μM) or dioxin concentrations (0.001, 0.01, 0.1 and 1 nM) for 24 hr at each of 4 developmental windows: 24-48, 48-72, 72-96, and 96-120 hpf. Results confirmed that prehatch embryos, when compared to later stages, are relatively unresponsive to E2 and dioxin, and also revealed that responses between 72 – 96 hpf were at or near the magnitude of responses at 96 –120 hpf. For routine screening, therefore, a 24 hr treatment period between 72 – 96 hpf was selected as optimal. This paradigm minimizes chemical effects on morphogenesis per se (as opposed to gene regulation), and has the advantage that <96 hpf larvae are less motile than later larvae and hence easier to treat and collect.
Representative chemical classes. Results show that the zebrafish embryo assay faithfully detects chemicals that are estrogen-like in their actions (agonist/antagonist), dioxin-like in their actions (agonist/antagonist), are metabolized in vivo to estrogen-like products (testosterone, androstenedione), block in situ estrogen synthesis (fadrozole), or otherwise interfere with estrogen or dioxin signaling (cyp1A). Comparing E2, with bisphenol A (BPA) and diethylstilbestrol (DES) at different concentrations, estrogen responsive genes were found to be ligand- and gene-specific: P450aromB, DES >E2> BPA; vtg, E2>DES> BPA. The relatively weak agonist, 4-HydroxyPCB51, upregulated estrogen responsive genes at high doses (agonist), but in combination with E2 displayed antagonist effects. To evaluate effects of the pesticides, atrazine, vinclozolin and M2 (a vinclozolin metabolite), embryos were exposed to different doses in a cumulative treatment paradigm. With the exception of cyp1A1, which was induced by 10 μM vinclozolin, no changes in gene expression were observed. Thus, using zebrafish embryos, we cannot confirm reported effects of these compounds on aromatase expression or estrogen signaling. Also, we failed to confirm estrogenic effects of tea oil and lavender oil, but doses that were effective in an earlier cell free assay were toxic in zebrafish.
Environmental samples (see above). Water collected from the impacted pond induced dioxin responsive genes (AhR2, cyp1A1), while the sediment elutriates from the same pond induced estrogen responsive genes (vtg, P450aromB, ERα) and increased the intron-retained P450aromB variant (relative to the normal mRNA form). These results suggest that the zebrafish embryo bioassay has potential for detecting, localizing, and identifying EDC that exist as mixtures in the natural environment. This observation will be followed up as part of a collaborative study. It is also relevant that molecular markers measured in killifish resident in New Bedford Harbor (the most heavily PCB-contaminated site in the US) display evidence of exposure to both estrogen-like and dioxin-like chemicals.
Conclusions:Results of the completed project demonstrate that real time quantitative (q)-PCR analysis of expressed genes in living zebrafish embryos can be used to detect and characterize (1) multiple subclasses of EDC (e.g., estrogen- and dioxin-like); (2) multiple gene- and tissue- targets of a given receptor class (e.g., ER- and AhR-responsive); (3) EDC that target genes upstream of receptor binding (e.g., P450arom); and (4) the presence and nature of EDC in samples from polluted environments. The assay has the power to detect and quantify both agonists and antagonists of a given receptor class, and to detect, localize and identify EDC in complex mixtures collected from polluted environments. Selection of appropriate markers can increase the utility of the zebrafish assay by addressing chemical effects on both hormone- mediated and hormone-independent cellular and developmental processes (e.g., apoptosis: neurodevelopment). Results address quality assurance by demonstrating that choice of primers for qPCR (to exclude or include splice variants), and selection of control (housekeeping) genes for normalizing qPCR results are absolutely critical to the accuracy of the assay. The completed project (1) addresses an urgent need for regulators to better predict which of the estimated 87,000 chemicals in the environment have the potential to disrupt hormone-dependent processes of physiology, reproduction and development; (2) provides biologically relevant criteria for prioritizing chemicals for further testing; (3) offers a platform for studying mechanism and body-wide effects of any EDC of interest; and (3) helps in interpreting reports of reproductive and developmental abnormalities in wildlife and humans.
Reproduction and development in man and animals are essential for survival of species, species diversity, maintenance of ecosystems, and commercial activities. Thus, there is an urgent need for regulators to develop methods to better predict which of the estimated 87,000 chemicals added to the environment have the potential to disrupt hormone-dependent processes of development, physiology and reproduction (EDC, endocrine disrupting chemicals). We have developed an assay using living zebrafish (Danio rerio) embryos/larvae (2 – 120 hr postfertilization) as a whole animal in vitro screening system for simultaneous detection of multiple subsets of EDC: (a) EDC that act directly via estrogen receptors (ER) to induce or block expression of estrogen responsive genes; (b) EDC that act via arylhydrocarbon receptor (AhR) mediated changes in gene expression and attenuate ER-regulated gene expression; (c) EDC that interact directly with preformed aromatase enzyme to block aromatization; (d) EDC that act via estrogen signaling pathways after in vivo metabolism to ER ligands; and (e) EDC that perturb expression of ER, AhR and P450aromatase expression per se. Gene-specific primers and a real-time quantitative reverse transcription-polymerase chain reaction (qPCR) approach were applied to measure multiple targeted mRNAs in a single assay. Characterization of a panel of eight different zebrafish housekeeping genes revealed that selection of normalizers for qPCR analysis are critically affected by developmental stage, treatment, tissue-type and sex. The zebrafish embryo system is a novel alternative to, and extension of, the current EDSP Tier 1 Screening Battery. Although use of zebrafish embryos in vitro minimizes animal and chemical use, it has the added value of an in vivo system for predicting agonist vs. antagonist properties of a chemical without a priori knowledge of uptake and accumulation, activating or metabolizing pathways, access to targets, receptor binding and activation, or required coregulators. The zebrafish embryo gene expression assay has immediate applicability for detecting known and suspected ER- and AhR-acting and interacting EDC, provides biologically relevant criteria for prioritizing chemicals for testing, and offers a platform for further investigating mechanisms and effects of any EDC of interest. The approach and methods described here demonstrate the feasibility of using zebrafish embryos to detect chemicals that impact signaling by other members of the nuclear receptor superfamily.
Journal Articles on this Report: 5 Displayed | Download in RIS Format
| Other project views: | All 55 publications | 6 publications in selected types | All 6 journal articles |
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Burnett KG, Bain LJ, Baldwin WS, Callard GV, Cohen S, Di Giulio RT, Evans DH, Gomez-Chiarri M, Hahn ME, Hoover CA, Karchner SI, Katoh F, MacLatchy DL, Marshall WS, Meyer JN, Nacci DE, Oleksiak MF, Rees BB, Singer TD, Stegeman JJ, Towle DW, Van Veld PA, Vogelbein WK, Whitehead A, Winn RN, Crawford DL. Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 2007;2(4):257-286. |
R831301 (Final) |
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Greytak SR, Champlin D, Callard GV. Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments. Aquatic Toxicology 2005;71(4):371-389. |
R831301 (2006) R831301 (Final) |
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Greytak SR, Callard GV. Cloning of three estrogen receptors (ER) from killifish (Fundulus heteroclitus): differences in populations from polluted and reference environments. General and Comparative Endocrinology 2007;150(1):174-188. |
R831301 (Final) |
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Sawyer SJ, Gerstner KA, Callard GV. Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation. General and Comparative Endocrinology 2006;147(2):108-117. |
R831301 (2004) R831301 (Final) |
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Tarrant AM, Greytak SR, Callard GV, Hahn ME. Estrogen receptor-related receptors in the killifish Fundulus heteroclitus: diversity, expression, and estrogen responsiveness. Journal of Molecular and Cellular Endocrinology 2006;37(1):105-120. |
R831301 (Final) |
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Media: water, groundwater, sediments. Risk Assessment: exposure, risk, risk assessment, effects, health effects, ecological effects, human health, bioavailability, metabolism, dose-response, animal, organism, cellular, enzymes, infants, children, metabolism, sex, cumulative effects); Chemicals, Toxics, Toxic Substances: (chemicals, toxics, PAHs, PNAs, PCBs, Dioxin, metals, heavy metals, solvents, oxidants, organics); Risk Management: pollution prevention; treatment; Scientific Disciplines: environmental chemistry, biology, histology, genetics; Methods/Techniques: analytical, measurement methods; Geographic Areas: northeast, EPA Region 1,
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POLLUTANTS/TOXICS, ENVIRONMENTAL MANAGEMENT, INTERNATIONAL COOPERATION, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Endocrine Disruptors - Environmental Exposure & Risk, Risk Assessment, Risk Assessments, Health Risk Assessment, Physical Processes, endocrine disruptors, Chemicals, Children's Health, Biochemistry, Environmental Policy, Environmental Chemistry, Endocrine Disruptors - Human Health, gene expression, endocrine disruptor screening program, animal models, endocrine disrupting chemicals, health effects, zebrafish embryo gene expression, childhood development, assessment of exposure, zebrafish embryo gene expression system, age-related differences, human health risk, susceptibility, screening assay, biogeochemistry, exposure, environmentally-caused disease, children's vulnerablity, endocrine disrupting chemcials, exposure studies, Human Health Risk Assessment
Progress and Final Reports:
2004 Progress Report
2005 Progress Report
2006 Progress Report
Original Abstract