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: Klieforth, Barbara I
Project Period: October 1, 2004 through September 30, 2006 (Extended to September 30, 2008)
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 objective of the proposed research is to develop a rapid, sensitive, biologically-integrated screening assay to identify endocrine-disrupting chemicals (EDCs) using a sensitive medaka (Oryzias latipes) fish model. Specifically, the proposed research will focus on two identified areas of interest:
- identification and evaluation of endocrine disrupting chemicals (EDCs),
- and categorization of EDCs into estrogenic, anti-estrogenic, androgenic, anti-androgenic, thyroidogenic, and anti-thyroidogenic activity.
In the first year, we propose to create a medaka cDNA library from pooled livers, gonads, craniums (a source of brain, pituitary, and thyroid gland) of adult medaka exposed to 6 groups of endocrine disruptors (estrogenic, anti-estrogenic, androgenic, anti-androgenic, thyroidogenic, and anti-thyroidogenic). This approach will provide us with a cDNA library that is enriched in transcripts specifically upregulated by these prototypical EDCs. 5000 individual clones from this library will be sequenced and unique Expressed Sequence Tags (ESTs) will be polymerase chain reaction (PCR) amplified, and used to create the glass slide microarrays. Once developed, predictive gene sets will be identified by exposing medaka to three additional compounds per endocrine disruptor group and evaluating the resulting microarray gene expression patterns with standard statistical tools. In the second and third years, we will test the predictive ability of the microarray gene chip with:
- a suite of known EDCs representative of each category of EDC,
- substances previously identified as lacking endocrine activity,
- and substances not previously characterized as to their endocrine activity.
We will screen these compounds and compare their gene expression profiles or "signatures" to the prototypic EDC profiles and classify the chemicals as:
- not an EDC,
- low probability of endocrine disrupting (ED) activity,
- moderate probability of ED activity,
- and high probability of ED activity.
Once each compound has been classified, one chemical from each category will be tested on 1-week-old larvae to confirm the scoring system. A subgroup of the larvae will be allowed to grow to maturity, and growth, sexual abnormalities, and histopathology will be determined to confirm endocrine disruption.
Our screening assay will enable us to accurately identify gene expression patterns that are both causative and/or predictive of endocrine disruption. Development of a medaka microarray chip will provide a powerful screening tool that can simultaneously yield information on not only the parent chemical's ED activity but also any active metabolites. The basic screening system is also directly amenable to the future characterization of organ-specific responses to EDCs, as well as to characterizing differences in EDC gene-expression profiles associated with exposure of different life stages. Furthermore, assessment of complex contaminant mixtures of EDCs will be possible with this novel method and could greatly facilitate future aquatic environmental EDCs identification and evaluation.