2006 Progress Report: Using a Sensitive Japanese Medaka (Oryzias latipes) Fish Model for Endocrine Disruptors ScreeningEPA Grant Number: R831299
Title: Using a Sensitive Japanese Medaka (Oryzias latipes) Fish Model for Endocrine Disruptors Screening
Investigators: Teh, Swee J. , Hall, Linda , Johnson, Michael
Institution: University of California - Davis
EPA Project Officer: Carleton, James N
Project Period: October 1, 2004 through September 30, 2006 (Extended to September 30, 2008)
Project Period Covered by this Report: October 1, 2006 through September 30, 2007
Project Amount: $399,168
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 , Endocrine Disruptors
The overall goal of this research is to develop and validate a high-throughput endocrine disrupting chemical (EDC) screening assay utilizing a microarray gene chip. The four specific objectives of the research project are to: (1) develop a microarray chip designed to identify EDCs, (2) identify gene expression profiles associated with all the categories of endocrine disrupting activity, (3) conduct a statistical analysis of gene expression profiles to develop response criteria that identify patterns predictive of endocrine disruption, and (4) validate the microarray chip using a set of chemicals selected to represent both positive and negative controls, as well as chemicals with previously undefined endocrine activity.
EDCs are believed to act primarily by altering gene transcription. Therefore, microarray-based alterations in gene expression can be used as a marker of exposure. We developed and tested an oligonucleotide microarray to identify genetic activity of six prototypic EDCs. These EDCs were selected to represent pairs of agonist/antagonist for three hormone receptor systems: estrogen/antiestrogen [i.e.,17-β-estradiol/faslodexTM (ICI 182,780)]; androgen/antiandrogen (i.e., 11-ketotestosterone/flutamide); and thyroidogen/antithyroidogen (i.e., thyroid hormone (T3)/amiodarone).
Range-finding experiments with 1-week-old male and female Qurt medaka larvae were conducted to determine the biologically effective concentration (BEC) for each EDC. Groups of fish were exposed to each of several different concentrations of our reference EDCs for 96 hours, grown to sexual maturity (~ 3 months), and sacrificed. Endpoints analyzed for evidence of endocrine-related effects from each set of whole fish samples included: incidence of intersex, sex ratios, health indicators (i.e., body weight, body length, condition index), and histopathological biomarkers. As a result, we have identified a BEC for each of the six reference EDCs on male and female medaka. Subsequently, the identified BECs were used as exposure concentrations for characterizing the microarray-based response to each EDC. In these latter experiments, male and female medaka were exposed (# replicates per gender = 3; # larvae per replicate = 200) to each EDC, sacrificed, and the RNA was isolated for microarray analyses.
After RNA isolation at the University of California at Davis, the genetic material was sent to Combimatrix Corp. in Washington for sample labeling, hybridization, and preliminary data analysis. The custom medaka oligonucleotide array includes approximately 9300 separate genomic probes. The main features of the medaka microarray (or gene chip) include: EDC-affected genes (n = 131), medaka cDNAs (n = 1075), sequences from the medaka genome project (n = 890), and additional sequences extracted from the UniGene database.
Microarray data were analyzed by Dr. Ping-Shi Wu (Lehigh University), a statistician and recent addition to the project team. His analyses identified discrete, statistically significant, gene expression profiles for each of the six EDCs studied to date. Dr. Wu has also completed the first phase of an effort to use a subset of differentially expressed genes from each EDC to discriminate between receptor agonist/antagonists pairs (e.g., 17B estradiol/faslodex).
The medaka microarray is a sensitive tool for detecting endocrine-disrupting activity. There is clear evidence that the medaka microarray can discriminate between the different types of prototypical EDCs.
Future research activities include: (1) seeking funding to perform time-course and dose-response experiments with the prototypical EDCs; (2) developing integrative software to facilitate data analysis; and (3) screening numerous additional chemicals, including additional EDCs and simple mixtures.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other project views:||All 17 publications||2 publications in selected types||All 2 journal articles|
||Leon A, Teh SJ, Hall LC, Teh FC. Androgen disruption of early development in Qurt strain medaka (Oryzias latipes). Aquatic Toxicology 2007;82(3):195-203.||
||Leon A, Wu P-S, Hall LC, Johnson ML, Teh SJ. Global gene expression profiling of androgen disruption in Qurt strain medaka. Environmental Science & Technology 2008;42(3):962-969.||