2005 Progress 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: R831846
Title: 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. , Hecker, Markus , Jones, Paul D. , Newsted, John L.
Institution: Michigan State University
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2004 through August 31, 2007
Project Period Covered by this Report: September 1, 2004 through August 31, 2005
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: Health , Safer Chemicals , Endocrine Disruptors , Computational Toxicology , Health Effects
The objectives of this research project are to: (1) develop methods that will permit the identification of changes in the expression profiles of genes that are associated with key aspects along the hypothalamic-pituitary-gonadal (HPG) axis such as hormone receptors, gonadotropins, and steroidogenic enzymes in the Japanese medaka, as a small animal model; (2) apply these techniques to develop gene expression profiles for “model” compounds of specific modes of endocrine actions (e.g., estrogenic, androgenic), and compare these changes to biologically relevant endpoints such as reproductive performance; (3) apply multivariate statistical pattern recognition techniques to differentiate the effects of different classes of chemicals; and (4) test the hypothesis that the identified responses can be used to classify endocrine disrupting “test” chemicals into groups to facilitate risk assessments of multiple chemicals and development of quantitative predictive models based on structure activity relationships (QSARs).
A subset of genes has been selected for the development of whole fish in situ hybridization methods. Beta-actin was selected as the housekeeping gene, and two isoforms of the aromatase gene, CYP19A and B, were selected as initial target genes due to the fact that their expression is highly tissue specific and responsive to endocrine disruptors. cDNA and riboprobes have been successfully developed for these genes and currently are being tested and optimized using cryosectioned tissues and parafin-embedded sections for whole fish mounts. The in situ hybridization procedures are being optimized in the laboratory. The methods have demonstrated a very high level of staining for all probes tested. Although some of the staining seems to be of nonspecific background, the sense and antisense probes do exhibit the expected binding profiles (see Figure 1). Certain factors that may decrease the nonspecific binding are in the process of being tested, including high stringency posthybridization washes, high temperature hybridization washes, and high temperature posthybridization washes. At this point, the probes in use are digoxygenin-labeled. They are visualized using standard protocols.
Figure 1. Medaka Brain Stained With CYP19B Sense (A; negative control) and Antisense (B) Probe
Future activities include the optimization of both sectioning and hybridization procedures, including the development of probes for additional genes. In addition, fluorescent-labeled probes will be developed as a means to increase sensitivity and reduce background. The optimization of the sectioning methods will focus on a modification of the cryosectioning techniques utilizing a tape transfer system that showed great promise for whole body sectioning of various species in other projects. Currently, preparations are underway for an initial chemical exposure study with adult medaka. This study will consist of 10 male and 10 female fish that will be exposed to a single high dose of a model chemical and a control group. The purpose of this study is to evaluate the in situ hybridization protocols using the CYP19 probes and β-actin probe in medaka exposed to fadrozole, a well-known aromatase inhibitor. The rationale for selecting fadrozole is that it will demonstrate that our model and selected endpoints will act in accordance with that observed in a different fish model, fathead minnow. Results from in situ hybridization slides will be confirmed by reverse transcription polymerase chain reaction (RT-PCR), which will be conducted on a subset of samples. These results will be used to verify the utility of this in situ hybridization model and to prioritize future research activities.