2004 Progress Report: A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting ChemicalsEPA Grant Number: R831301
Title: A High Throughput Zebrafish Embryo Gene Expression System for Screening Endocrine Disrupting Chemicals
Investigators: Callard, Gloria V.
Institution: Boston University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2003 through September 30, 2007
Project Period Covered by this Report: October 1, 2003 through September 30, 2004
Project Amount: $400,000
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Health , Safer Chemicals , Children's Health , Endocrine Disruptors
Few of the estimated greater than 87,000 chemicals added to the environment by anthropomorphic activities actually have been tested for endocrine disrupting activity. Thus, there is an urgent need to develop screening methods for identifying environmental endocrine disrupting chemicals (EDCs). The overall objective of this research project is to investigate the proposition that perturbations in the normal amount or timing of a hormone-regulated gene product can be taken as evidence of chemical exposure and used as an endpoint in a screening assay to detect EDCs.
The specific objective of this research project is to demonstrate that quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of expressed genes in living zebrafish embryos is an excellent in vitro screening system for detecting and characterizing: (1) multiple subclasses of EDCs; (2) multiple gene targets and tissue targets of a given EDC subclass; and (3) EDCs that target genes both upstream and downstream of receptor binding. Two major classes of persistent environmental pollutants, with potential endocrine disrupting effects, are estrogenic chemicals that interact with estrogen receptors (ERs) and dioxinlike chemicals that interact with arylhydrocarbon receptors (AhRs). This research project focuses on specific molecular markers of ER- and AhR-mediated EDC actions and effects in the brain, ovary, and liver. In addition, this research project is viewed as a proof-of-principle study to develop approaches and protocols with universal applicability for screening chemicals with effects on any other nuclear receptor signaling pathway of interest and to establish the feasibility of procedures and endpoints that are amenable to high-throughput modifications.
Specifically, qRT-PCR analysis of gene expression in living zebrafish embryos was applied to detect and characterize the effects of: (1) EDCs that act via ERs to induce brain P450 aromatase (P450aromB) and hepatic vitellogenin (vtg) expression; (2) EDCs that act via AhRs to reduce gonadal aromatase (P450aromA) and increase hepatic P4501A1 expression; (3) EDCs that interact directly with preformed aromatase enzymes to block aromatization; and (4) EDCs that perturb ER and AhR expression.
Primer Design and Optimization of qRT-PCR Conditions
P450aromB and -A. Assay development and validation in embryonic, larval, and adult male and female zebrafish has been completed (Sawyer and Callard, in preparation, 2004). Results show that the assay is sensitive and precise (linear between 1 x 10-6 and 1.0 ng mRNA). Reproducibility is excellent within and across operators when tested in multiple experiments and at multiple starting points within a single experiment (e.g., RNA extraction, RT, PCR)(intra- and interassay coefficient of variation, 2-5%). Resultant data from staged embryos confirm and extend our published results using the much more labor intensive semiquantitative RT-PCR/Southern transfer and hybridization method but has the added advantage that expressed levels of the two mRNAs (after correction for amplification efficiency) can be compared directly at a single time point, as a function of the stage of development, and in response to estradiol treatment. Significantly, the response to estrogen as measured by QRT-PCR (> 150-fold at 96 hpf) is much more robust than that measured by the earlier semiquantitative assay methods (~ fivefold).
ER-alpha, -beta A, and -beta B. Specific primers that target each of the three zebrafish ERs were designed. To assess gene specificity, each primer set was tested by semiquantitative RT-PCR using both embryonic RNA and authentic ER plasmids. Each primer pair gave a single product of expected size for its target RNA but gave no product for the other two ERs, indicating specificity. Amplification conditions for each of the three ERs using the newly designed primer sets were optimized using an ABI Prism 7900HT sequence detection system and the SYBR green fluorescent labeling system in the core facility at Boston University. As described above, the assays were sensitive, precise, gene specific, and had a low intra- and interassay coefficient of variation. Resultant data confirmed the developmental program obtained by semiquantitative RT-PCR/Southern transfer analysis of staged embryos/larvae. Experiments testing ER responses to estradiol, diethylstilbestrol, and bisphenol A between 24 and 96 hours of development are in progress.
vtg. Primer pairs that target zebrafish vtg RNA were designed and tested using semiquantitative RT-PCR and embryonic RNA to demonstrate a single amplicon of predicted size. Conditions were optimized for qRT-PCR as described above. Further validation of the assay using embryos is in progress.
AhR2 and cyp1A1. Primer sets specific for QPCR analysis of these genes were obtained from Dr. Mark Hahn (Woods Hole Oceanographic Institution) and validated and optimized for our experimental conditions as described above. Embryo experiments are in progress.
Splicing Isoforms of P450aromB. Recently, we observed that an intron-retained form of P450aromB is developmentally programmed, downregulated by estradiol, and upregulated by dioxin and sediment elutriates from contaminated environments (Novillo, et al., in preparation, 2004). The variant mRNA predicts either rapid mRNA degradation (nonsense-mediated decay) or a biologically inert aromatase enzyme. This serendipitous observation underlines the importance of primer design and exhaustive validation procedures for each gene of interest, if results of chemical screening are to be interpreted correctly. Currently, we are attempting to design primers that will allow us to discriminate between the variant and normal P450arom mRNAs in the same sample.
Characterization and Optimization of Embryo Treatment Protocols
To determine the embryonic period of maximum sensitivity and responsiveness to each EDC class of interest, the following experiments were performed:
Estrogen Effects. Using authentic estradiol, cumulative (24-96 hpf) dose-response (0.0001, 0.001, 0.01, 0.1, 1, and 10 μM) effects were tested by qRT-PCR analysis of P450aromB, -A, ER-alpha, -betaA, -betaB, vtg, and actin (as a control) RNAs. Results showed that the embryonic estrogen response system is gene specific as measured by magnitude (vtg > P450aromB > ER-alpha); sensitivity (P450aromB > vtg > ER-alpha); and specificity (ER-betaA, -betaB, and actin = no change) of the estrogenic response. The same six mRNAs are being assayed to evaluate dose-response effects at different developmental stages (24-48, 48-72, 72-96, and 96-120 hpf).
Dioxin Effects. Using 2,3,7,8-tetrachlorodibenzo-p-dioxin as an authentic AhR ligand, cumulative (24-96 hpf) dose-response (0.001, 0.01, 0.1, and 1 nM) effects were tested by qRT-PCR analysis of P450aromB, -A, ER-alpha, -betaA, -betaB, vtg, and actin (as a control) RNAs and of AHR1, AHR2, and cyp1A1 mRNAs. The AhR response system as measured by the direction and magnitude of the response was: cyp1A1 (200X, up) greater than P450aromA (15X, down) greater than AhR2 (2X, up); however, sensitivity was similar for all three genes (threshold: 0.001-0.01 nM). P450aromB and actin were unresponsive. Analysis of other gene targets is in progress.
Estrogen-Dioxin Interactions. To predict how mixtures of chemicals in different amounts and ratios could affect assay results, experiments were carried out to test possible interactions between the ER and AhR response systems. Analyses of target genes for these two experiments are in progress.
Screening of Environmental Samples. The same gene targets were used to assay for the presence of estrogen-like and dioxin-like chemicals in environmental samples. Water and sediment were collected from polluted and reference ponds in the vicinity of the Massachusetts Military Reservation on Cape Cod. Impacted ponds were identified by a report describing turtles with reproductive abnormalities (endocrine disruption?) in the same pond. The zebrafish embryo assay detected both estrogen-like and dioxin-like chemicals in the impacted but not the reference pond and in sediment elutriates but not water. These results demonstrate the utility of the zebrafish embryo bioassay when EDCs are present as mixtures in the real environment and indicate the potential of the bioassay for identifying the actual contaminants.
In the next reporting period, we will:
- Complete and submit the manuscripts listed above.
- Complete qRT-PCR assays described above (optimization, validation) and their applicability to analysis of embryo experiments.
- Initiate routine screening of representative chemicals (and mixtures) from different EDC classes.
- Collect and test additional environmental samples from polluted and unpolluted environments (e.g., New Bedford Harbor). This objective is viewed as an important link between laboratory testing and the actual environment.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 54 publications||7 publications in selected types||All 7 journal articles|
||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.||