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 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
"Endocrine-disrupting" compounds have been defined as exogenous agents that interfere with the "synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior". Much of the recent concern and energy has been focused on compounds 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), 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 propose to develop a screening method to use molecular techniques such as in situ hybridization, in situ RT-PCR and immuno-histochemical staining (IHCS) to screen for 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. The proposed method will allow for screening of multiple effects in multiple tissues, even at points in development when the tissues are too small to be accurately dissected for use in more traditional molecular techniques. The proposed methods will be applied to a set of "model" and "test" compounds for a set of target genes. Once the methods have been developed and validated, they can be adapted for use with other genes and/or species of interest and used to efficiently and completely screen for endocrine disruptor effects beyond simple receptor binding.
To investigate the tissue-specific expression of genes in the most efficient manner a variety of in situ molecular techniques will be used to visualize and quantify mRNA and/or gene products. To maximize sensitivity and permit multiplexed gene expression quantification methods will be based on in situ hybridization and a variety of in situ PCR techniques. The aim of these investigations is to produce the simplest and most 'transportable' techniques so the use of a common technique for all genes investigated will be a priority for the final protocol.
The proposed research program will establish an integrative and quantitative small fish model to identify and evaluate the modes of action, key target sites, and biological relevance of EDCs acting along the HPG-axis. To achieve this we will apply state-of-the-art molecular techniques in a whole animal systems approach (see approach section). The studies will be conducted in two phases. In the first phase normal physiological processes along the HPG-axis will be described in order to establish a natural basis for the evaluation of EDC effects. Information on the natural variability, reproductive cycling, diurnal pattern, needs, etc. will be used to optimize the proposed test system. Thus, we will establish a general model system that can be used in the assessment of single chemical and complex mixture effects on reproductive endocrinology. In the second phase we will use this system to detect and assess changes in tissue-specific gene expression patterns (GEPs) caused by the exposure to endocrine active "model" compounds, and their mixtures. We will identify key genes for each individual exposure scenario and establish a quantitative RT-PCR system as a rapid screening tool for these genes.
A model will be developed to predict the biological relevance of the observed changes in GEPs. By identifying the systemic target sites, and the series of biological events from gene expression to the manifestation of an adverse outcome (e.g., reproductive performance), we will identify thresholds at the molecular level that are indicative of effects on the fitness of the individual, including survival, growth and reproduction (fertility and fecundity as well as survival of the offspring). The proposed model test system will provide a new and powerful approach in hazard identification and toxicological risk assessment. Understanding the potential of individual chemicals and complex environmental mixtures to interfere with molecular pathways of concern will enhance our understanding of the basic mechanisms of toxicities, and thus, will have the potential to develop better focused, more rapid and cost effective models for quantitative risk assessment. The bioassay can be used to screen for a wider range of endocrine disruptor effects.