2004 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, 2004 through September 30, 2005
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 are to:
- develop a microarray chip designed to identify EDCs;
- identify gene expression profiles associated with all the categories of endocrine disrupting activity;
- conduct a statistical analysis of gene expression profiles to develop response criteria that identify patterns predictive of endocrine disruption;
- and 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.
To date, our research has focused on the first objective of the project. An initial set of 1,236 DNA sequences with endocrine-related functions were identified and annotated from genomic databases for medaka. A subset of 165 Expressed Sequence Tags (ESTs) and cDNA sequences was selected for cloning and inclusion in the microarray gene chip to identify EDCs.
We conducted a literature review to identify agonist/antagonist pairs of chemicals for each of the three-receptor systems of interest to the project: the estrogen, the androgen, and thyroid hormone receptors. Range-finding studies with 1-week old Qurt medaka larvae have been conducted to determine the effective concentrations of the six selected compounds with endocrine disrupting activity:
- 17-β-estradiol—an estrogen;
- faslode—an antiestrogen;
- 11-ketotestosterone—an androgen;
- flutamide—an antiandrogen;
- T3—a thyroidogen;
- andbisphenol A—a thyroid hormone receptor antagonist.
The endpoints for these studies included: incidence of intersex, sex ratios, health indicators (i.e., body weight, body length, condition index), and histopathological biomarkers. Acute toxicity studies were completed and most of the experimental fish were grown out to determine sublethal effects. There were no significant mortalities in any of the treatment groups after 96-hour exposure. As a result, we have identified an exposure range that will not yield acute toxic effects and is thus ideal for the study of sublethal effects. Gender-specific effects on length, weight, and condition index have been observed. For instance, the antiestrogen Faslodex presented a nonmonotonic effect on female size. Specifically, females at the lowest concentration presented a significantly (p = 0.000) smaller size compared to the rest of the treatments. This type of response at low dosages is common in EDC research. Occurrence of intersex gonads and sex ratios of larvae exposed to EDCs currently is being evaluated by examination of histological samples. A Mendelian 1:1 sex ratio has been verified for control Qurt medaka embryos. We achieved 100 percent accuracy in sexing embryos by visual inspection based on subsequent evaluation of adult fish whose phenotypic sex was verified by gonad examination.
RNA isolation is a crucial step in the development of DNA microarrays. Therefore, seven experiments were conducted to optimize the yield and quality of RNA to be used for Suppression Subtractive Hybridization (SSH) and microarray hybridization experiments. The main objectives of these experiments were to:
- determine the number of Qurt medaka larvae necessary to obtain an adequate yield of mRNA for microarray analysis;
- and optimize the methods utilized for total RNA and mRNA isolation.
We concluded that groups of 60 to 100 larvae and 300 to 400 larvae per sample ensure adequate yields of total RNA and mRNA, respectively. High yields of total RNA have been consistently isolated. The practice of pooling the identified numbers of medaka larvae will reduce the biological source of variation of this assay. In addition, consistency in RNA isolation is desirable for this high-throughput screening technique to be both reproducible and reliable.
Working with staff scientists from Combimatrix Corporation, we identified and selected approximately 9500 medaka gene sequences that Combimatrix Corporation used as the basis of a custom oligonucleotide array. Our immediate goal is to use this array as a screening tool to characterize the timing of maximum alteration in gene expression in medaka after EDC exposure. We also hope to use this array to identify specific sequences whose expression is affected by exposure to EDCs, and eventually incorporate these sequences into our final array. This work is ongoing.
Our future research activities include:
- conducting Suppression Subtractive Hybridization (SSH) to identify additional EDC-responsive genes at the exposure conditions identified in the range-finding studies;
- performing additional time-series experiments to determine the optimal exposure conditions for the EDC screening experiments;
- completing the array-based evaluation of the period of maximum alteration in gene expression induced by EDC exposure;
- constructing the microarray chip;
- and validating the microarray chip using a set of chemicals with known EDC action, as well as chemicals with previously undefined endocrine activity.
We will be giving the following presentations:
Teh SJ. Session: TS01—from gene expression to ecological processes. To be presented at the American Society of Limnology and Oceanography 2005 Aquatic Sciences Meeting, Salt Lake City, UT, February 20-25, 2005.
Teh SJ. From gene expression to population sex ratios: integrating biomarkers to reveal antiandrogen-specific profiles in medaka fish (Oryzias latipes). Poster to be presented February 22-24, 2005.
Teh SJ. Effect of timing of exposure on the optimal gene expression of medaka fish exposed to the antiandrogen flutamide. To be presented at the Northern California Regional Chapter of the Society of Environmental Toxicology and Chemistry 15th Annual Meeting, University of California–Berkeley, CA, May 3-4, 2005.