2005 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. , Bartosiewicz, Mathew , Hall, Linda , Johnson, Michael
Current 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, 2005 through September 30, 2006
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 project is to develop and validate a high-throughput endocrine disrupting chemical (EDC) screening assay using a microarray gene chip. The objectives of the 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. To date, our research has focused on developing and testing an oligonucleotide microarray to identify genetic activity of six reference EDCs. These EDCs were selected to represent pairs of agonist/antagonist for three hormone receptor systems: estrogen/anti-estrogen (i.e., estradiol/faslodex); androgen/anti-androgen (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 have been conducted to determine the biologically effective concentration (BEC) for each of these EDCs. Groups of fish were exposed to several different concentrations of our reference EDCs for 96 hours, grown to sexual maturity (approximately 3 months), and sacrificed. Endpoints that were 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 (number of replicates per gender = 3; number of larvae per replicate = 200) to each EDC, sacrificed, and the RNA was isolated for microarray analyses. We have completed all exposures and RNA isolations necessary for microarray analyses.
After RNA isolation at the University of California-Davis, the genetic material was sent to CombiMatrix Corporation in Washington for sample labeling, hybridization, and preliminary data analysis. Staff scientists from CombiMatrix Corporation worked with us early in the project to design a custom medaka oligonucleotide array, which 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=1,075), sequences from the medaka genome project (n=890), and additional sequences extracted from the UniGene database.
Preliminary results of both EDC range-finding and microarray experiments are of significant value and hold great promise for advancing the emerging science of endocrine disruption. Overall, the medaka microarray is a sensitive tool for detecting endocrine-disrupting activity. Results from our first set of microarray experiments appeared to provide clear evidence that the medaka microarray can discriminate between the different types of prototypical (i.e., reference) EDCs under study. An initial assessment of a second series of microarrays, however, apparently yielded negative results (i.e., no statistically significant difference between exposed and control groups). These circumstances delayed our project while we explored possible causes and/or explanations for the disparity between the data in the two series of microarrays. The project biostatistician recently has confirmed that the data analysis conducted by CombiMatrix Corporation relied on an inappropriate normalization technique. A re-analysis of the second data set with proper statistical algorithms revealed significant gene alterations induced by EDC exposures similar to those found in initial microarray trials. At present, all remaining samples of single EDC exposures are properly archived and ready to send for analysis.
Future research activities include: (1) construction of the final microarray chip including only a diagnostic set of genes; (2) completion of the array-based evaluation of the period of maximum alteration in gene expression induced by EDC exposure; and (3) validation of the microarray chip with putative EDCs and environmental mixtures.