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17-A ETHYNYLESTRADIOL-INDUCED VITELLOGENIN GENE TRANSCRIPTION QUANTIFIED IN LIVERS OF ADULT MALES, LARVAE, AND GILLS OF FATHEAD MINNOW (PIMEPHALES PROMELAS)
Citation:
Impact/Purpose:
The proof of concept for computational toxicology proceeds from ongoing research on endocrine disrupting compounds. This is based on the enhanced level of research spawned by the FY02 redirection of funds for alternative toxicity testing. Current EDC gene expression research focuses on the use of single gene expression analysis methods to study the bioavailability of estrogens and androgens in small fish models in the evaluation of the effectiveness of risk management technologies. Advances in microarray technology are being most readily applied in FY03 as ORD aims to enhance its genomics and computational methods capabilities.
A special emphasis for this subtask is the ORD technology transfer of molecular indicator methods for EDC exposure and regional characterization of EDCs in effluents from significant segments of a State's water drainage (e.g., in California, the opportunity exists for the survey of up to one-half of the State's water drainage.) research includes proof of concept for CompTox with EDCs where linkages are established between environmental chemical transformation pathways (fate and transport) and organismal outcome based on
disrupted dynamic biological systems.
Description:
We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically-induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription (Vg) product, were used in a reverse transcription-polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to 2 ng/L concentrations of the endocrine disrupting compound, 17a-ethynylestradiol, for 24 h. Vitellogenin transcription products were also, surprisingly, detected in gill tissue and in 48 h old post-hatch fathead minnow embryo larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) using 18S rRNA universal primers and Competimers concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those which cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine- disrupting compounds.