Grantee Research Project Results
Final Report: Chemical Induced Changes in Gene Expression Patterns Along the HPG-axis at Different Organizational Levels Using a Small Animal Model (Japanese medaka)
EPA Grant Number: R831846Title: Chemical Induced Changes in Gene Expression Patterns Along the HPG-axis at Different Organizational Levels Using a Small Animal Model (Japanese medaka)
Investigators: Giesy, John P. , Jones, Paul D. , Newsted, John L. , Hecker, Markus
Institution: Michigan State University
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2004 through August 31, 2007
Project Amount: $749,904
RFA: Computational Toxicology and Endocrine Disruptors: Use of Systems Biology in Hazard Identification and Risk Assessment (2004) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Endocrine Disruptors , Computational Toxicology , Human Health
Objective:
Much of the recent concern and energy around endocrine disrupting compounds has been focused on chemicals that are hormone direct agonists or antagonists, especially those that interact with the estrogen receptor (ER). Effects consistent with exposure to ER agonists have been observed in fish exposed to natural hormones and some synthetic chemicals such as nonylphenol (NP), nonylphenol polyethoxylates (NPEs), and octylphenol (OP). Because chemicals can cause both direct (receptor-mediated) and indirect effects through changes in signal transduction pathways, methods are needed that permit the screening of multiple effects. Furthermore, methods are needed that can screen for these effects simultaneously in a number of tissues, including during critical windows of development, when tissues may be small and the amount of material available for testing is small and difficult to remove from the organism. We proposed to develop technologies using molecular techniques such as in situ hybridization and a Q RT PCR array to characterize and map effects of chemicals on the hypothalamic-pituitary-gonadal (HPG) axis with a special emphasis on steroidogenic pathways and hormonal control mechanisms along the HPG-axis in the Japanese medaka.
Specific Objectives:
- Development of methods that will permit the identification of changes in the expression profiles of genes that are associated with key aspects along the HPG-axis such as hormone receptors, gonadotropins, steroidogenic enzymes, etc. in the Japanese medaka, as a small animal model.
- Application of these techniques to develop gene expression profiles for “model” compounds of specific modes of endocrine actions (e.g. estrogenic, androgenic).
- Map molecular changes along the HPG as a function of tissue, cellular and sub-cellular structures.
- Compare changes in gene expression patterns to biologically relevant endpoints such as tissue morphology and reproductive performance.
Summary/Accomplishments (Outputs/Outcomes):
Two core technologies were developed in this study to investigate and characterize effects if EDCs along the HPG axis using the Japanese medaka (Oryzias latipes); whole mount in situ hybridization (ISH) and a Q RT PCR array. The purpose of this combinatory approach was to combine the strength of each system to bridge the gaps and uncertainties of the other assay.
In Situ Hybridization
The whole mount ISH methodology was developed after exploring a series of approaches utilizing different sectioning and visualization techniques. Based on the results obtained during these studies it was concluded that the cryosectioning methods without tape-transfer systems were not feasible for the use in ISH, and it was decided to use sections of paraffin embedded sections because they permit the preservation of tissue morphologies in comparison to cry-sectioning techniques, and because they were shown to produce reproducible results when subjected to in situ hybridization. Furthermore, it was decided that the use of DIG-labeled ISH methods was not feasible for the use with Japanese medaka as utilized in our studies due to the great variability between runs and the relatively great background signal. While an alternative approach using 35S-labeled riboprobes allowed spatial resolution of gene expression on whole mount sections of medaka, as well as the ability to analyze cellular morphological detail in the same organism, it was limited due to relatively poor resolution and its semi-quantitative nature (Tompsett et al. 2008). It was concluded that refinement of this approach either utilizing silver grain emulsion or fluorescence technologies had the potential to significantly improve both resolution and potential for quantification. It was decided to further pursue methods involving fluorophore-based detections system due to prohibitively expensive nature of silver grain emulsion approaches. One of the key issues encountered during the development of the fluorophore based ISH (FISH) technologies was auto-fluorescence of tissues or components of the ISH procedure. To overcome these issues we applied a combination of chemical treatment (sodium borohydride [SB]) and confocal laser scanning microscopy (CLSM) image analysis. The thus optimized FISH protocols resulted in the development of a test system that allows for the identification of gene expression in paraffin embedded whole mount sections of small fish with relatively great sensitivity and spatial specificity. Furthermore, the utilization of FISH with whole mount sections of medaka detected patterns of the expression of certain genes in tissues and cell types that had not been reported previously, and that could not have been revealed by the use of other commonly used molecular techniques such as RT PCR, Northern blotting, or RNAse protection assays. When this FISH method was applied to investigate the effects of a series of model endocrine disruptors with know modes of action (the synthetic estrogen ethinylestradiol, the anabolic androgen trenbolone and the aromatase inhibitor fadrozole) it was found to not only reveal changes in tissue specific patterns of gene expression but also to have the capacity to associate these changes with cellular or sub-cellular structures allowing to link molecular patterns with pathologies.
Q RT PCR Array
A Japanese medaka HPG PCR array was developed with the aim to combine the quantitative performance of real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of genes associated with endocrine pathways in brain, liver and gonad. Specifically, SYBR® Green-based technologies were utilized to validate the performance of the Japanese medaka HPG PCR array by examining effects of five model compounds, 17α-ethinylestradiol (EE2), 17β-trenbolone (TRB), fadrozole (FAD), and the imidozole-type fungicides, prochloraz (PCZ) and ketoconazole (KTC) on Japanese medaka. This system allowed for the successful determination of both spatial and temporal changes in gene expression patterns in response to the exposure with these model chemicals at the tissue or gland level of organization. These studies verified the reliability of the measurements of molecular pathways by the Japanese medaka HPG axis PCR array, and comparison with other studies also demonstrated that the responses of the HPG axis pathway were similar among teleosts. Changes in gene expression were plotted using a novel systems mapping approach consisting of so called “striped view” charts displaying dose-dependent effects for each gene among the different compartments of the endocrine system analyzed. This approach allowed the determination and comparison of changes in gene expression patterns among chemicals, exposure time, and gender. The performance of the PCR array system developed in this study is limited to evaluating the effects of chemicals at the tissue level due to problems associated with availability of tissue that is inherent with the use of small animal models. Thus, while it allows the mapping of gene expression patterns among tissues it does not permit the analysis of interactions between tissue compartments, cells, or sub-cellular structures.
Conclusions:
The technologies developed as part of this project allowed for screening of multiple effects in different tissues in whole fish, even at points in development when the tissues are too small to be accurately dissected for use in more traditional molecular techniques. In addition, they allowed separating effects by tissue and cell type, and to identify potential interactions between different organs in response to chemical exposure. Finally, molecular effects could be successfully linked with changes at the physiological and organismal level. To fully explore the potential of these methods, however, they should be used in combination with other techniques. The combination of the ISH and the Q RT PCR array technologies as done in this project can be used to not only map changes in gene expression among tissues, but they can also be used to link these to processes at the cellular and sub-cellular level, and that then ultimately aid in understanding the molecular mechanisms that are associated or lead to pathologies. This combination will provide essential information that will further our understanding of chemical interaction with whole organismal systems, and thus, provide valuable information for chemical risk assessment. Finally, the here developed technologies do not only allow one to characterize chemical induced effects but also represent a powerful tool for the elucidation of basic biological processes such as association of the expression of certain genes with tissues, cellular and sub-cellular structures that are important for the development of baseline information to be used in risk assessments.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 31 publications | 3 publications in selected types | All 3 journal articles |
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Park JW, Tompsett AR, Zhang X, Newsted JL, Jones PD, Au DW, Kong R, Wu RS, Giesy JP, Hecker M. Advanced fluorescence in situ hybridization to localize and quantify gene expression in Japanese medaka (Oryzias latipes) exposed to endocrine-disrupting compounds. Environmental Toxicology and Chemistry 2009;28(9):1951-1962. |
R831846 (Final) |
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Tompsett AR, Park JW, Zhang X, Jones PD, Newsted JL, Au DWT, Chen EXH, Yu R, Wu RSS, Kong RYC, Giesy JP, Hecker M. In situ hybridization to detect spatial gene expression in medaka. Ecotoxicology and Environmental Safety 2009;72(4):1257-1264. |
R831846 (Final) |
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Zhang X, Hecker M, Park JW, Tompsett AR, Newsted J, Nakayama K, Jones PD, Au D, Kong R, Wu RS, Giesy JP. Real-time PCR array to study effects of chemicals on the Hypothalamic-Pituitary-Gonadal axis of the Japanese medaka. Aquatic Toxicology 2008;88(3):173-182. |
R831846 (Final) |
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Supplemental Keywords:
Small fish model, molecular mapping, fluorescence in situ hybridization (FISH), Q RT PCR array, histology, pathology,, RFA, Health, Scientific Discipline, Ecology, Genetics, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Biology, Endocrine Disruptors - Human Health, altered gene expression, endocrine disruptor screening program, fish, altered sexual development, EDCs, endocrine disrupting chemicals, Japanese medaka, developmental biology, rapid genetic screening tool, animal models, gene expression, biochemistry, biological effectsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.