PARENT SESSION
PT2 - Endocrine Disruption
Tuesday, 19 November 2002
8:00 AM to 6:30 PM
Exhibit Hall
(P577) Differential Display of Trenbolone and DEHP Induced Gene Transcript Patterns in Fathead Minnow Liver.
Reddy, Tirumuru*,1, Miracle, Ann1, Flick, Robert1, Lazorchak, James1, Smith, Mark2, Lattier, David1, Toth, Gregory1, 1 U.S. EPA, Ecological Exposure Research Division, MERB, Cincinnati, OH, USA2 SoBran, Environmental Inc. c/o U.S. EPA/EERD, Cincinnati, OH, USA
ABSTRACT- The endocrine disruptor risk assessment process is being delayed without more robust data on the estrogenic and androgenic activity of chemicals found in the environment such as trenbolone and di(2-ethylhexyl) phthalate (DEHP). Trenbolone is an androgenic compound known to reduce vitellogenin (Vtg) blood protein levels in female fathead minnows. Our preliminary results showed that the plasticizer di(2-ethylhexyl) phthalate (DEHP) induced vitellogenin gene transcription in the livers of adult male fathead minnows (Pimephales Promelas). In this study, we used fluoro differential display to investigate the global gene expression pattern in the liver of male and female fathead minnows exposed to trenbolone or DEHP. Adult (8-10 months old) male and female fathead minnows were continuously exposed to moderately hard reconstituted water (MHRW) as control, and MHRW with 50 ppt trenbolone or 10 ppb of DEHP, for one week. Total liver RNA was isolated, and reverse transcribed using an anchored oligo-dT primer. The cDNAs were subsequently amplified in a PCR reaction using fluorescent TMR-labeled anchored oligo-dT primers, in conjunction with four separate arbitrary decamers. The PCR products were separated on a 5.6% polyacrylamide gel, and visualized with a fluorescent scanner. Several bands were identified that exhibited differential expression among fathead minnows exposed to trenbolone, DEHP and controls. These bands were excised from the gel, and the cDNAs were eluted into buffer, re-amplified, and sequenced using an MJ Research BaseStation sequencer. Acquired nucleotide sequences were compared to sequences in GenBank using Blastn and Blastx programs to identify fathead minnow gene homologs.
Key words: Vitellogenin, Gene Induction, Differential Display, Fathead Minnows