17alpha-ethynylestradiol-induced vitellogenin gene transcription quantified in livers of adult males, larvae, and gills of fathead minnows (Pimephales promelas).
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 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 concentrations as low as 2 ng/L of the endocrine-disrupting compound 17alpha-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 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 that 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.
Lattier
David L
DL
National Exposure Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Cincinnati, Ohio 45268 , USA. lattier.david@epa.gov
Reddy
Tirumuru V
TV
Gordon
Denise A
DA
Lazorchak
James M
JM
Smith
Mark E
ME
Williams
Daniel E
DE
Wiechman
Barry
B
Flick
Robert W
RW
Miracle
Ann L
AL
Toth
Gregory P
GP
D000818:Animals; D003530:Cyprinidae / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004784:Environmental Monitoring / Q000379:methods*; D004997:Ethinyl Estradiol / Q000633:toxicity*; D005880:Gills; D007814:Larva; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D014819:Vitellogenins / Q000096:biosynthesis* / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D012333:RNA, Messenger; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D004997:Ethinyl Estradiol