Project name: Oncorhynchus mykiss

Description: The goal of this study was to identify a set of hepatic genes regulated by ligand-dependent activation of the estrogen receptor in juvenile rainbow trout (Oncorhynchus mykiss) that can serve as a biomarker of estrogen exposure. A custom rainbow trout oligo DNA microarray, which contains probes targeting approximately 1450 genes relevant to carcinogenesis, toxicology, endocrinology and stress physiology was utilized to identify transcriptional “fingerprints” of in vivo dietary exposure to 17ß-estradiol (E2), tamoxifen (TAM), estradiol + tamoxifen (E2+TAM), diethylstilbestrol (DES), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and cortisol (CORT). Estrogen exposure altered the expression of up to 49 genes involved in reproduction, immune response, cell growth, transcriptional regulation, protein synthesis and modification, drug metabolism, redox regulation and signal transduction. E2, DES and DHEA regulated 18 genes in common, mostly those associated with vitellogenesis, cell proliferation and signal transduction. Interestingly, DHEA uniquely regulated several complement component genes of importance to immune response. While the effect of TAM on E2-induced changes in gene expression was mostly antagonistic, TAM alone increased expression of VTG1 and other genes associated with egg development and immune response. Few genes responded to CORT treatment, and DHT significantly altered expression of only one gene targeted by the OSUrbt array. Hierarchical cluster and principal components analyses revealed distinct patterns of gene expression corresponding to estrogens and non-estrogens, though unique patterns could also be detected for individual chemicals. A set of estrogen-responsive genes has been identified that can serve as a biomarker of environmental exposure to xenoestrogens.Keywords: estrogen transcriptional profile, chemical transcriptional fingerprintOverall design: Eight experimental treatments were selected to examine hepatic gene expression in response to estrogens, a selective estrogen receptor modulator (SERM) and non-estrogens. Treatments and concentrations (calculated with respect to diet wet weight) are as follows: control (CON), 5 ppm 17ß-estradiol (E2), 50 ppm tamoxifen (TAM), 5 ppm E2 + 50 ppm TAM (E2+TAM), 2 ppm diethylstilbestrol (DES), 500 ppm dehydroepiandrosterone (DHEA), 5 ppm dihydrotestosterone (DHT) and 5 ppm cortisol (CORT). Test chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were added directly to the oil portion of the OTD. Experimental treatments were administered for 14 days, and feeding occurred five days per week. Total hepatic RNA was extracted in TRIzol reagent according to supplier’s instructions. Equal amounts of RNA from each individual liver sample were sub-pooled according to tank (four males pooled per tank) resulting in three biological replicates for each treatment. A reference RNA pool was made by combining equal amounts of RNA from all individual male CON liver samples (12 liver samples total). For detection of gene expression on the OSUrbt array, the Genisphere 3DNA Array 350 kit (Hatfield, PA) was used in a dye-swap, reference sample design following the supplier’s protocol. During reverse transcription (SuperScript II, Invitrogen) of total RNA (7 µg) using the supplied Genisphere oligo d(T) primers, each biological sample was tagged separately with a capture sequence for one of two fluorescent dendromer reagents, Cy3 or Cy5. Each reverse transcription reaction also included spiked in mRNA corresponding to the SpotReport Alien Oligo controls. Corresponding reference RNA was reverse transcribed with the capture sequence for the opposite reagent (Cy3 or Cy5). To account for any differences in dye labeling efficiency, technical replicates were performed for two of the three biological replicates in which the capture sequence tags were swapped between the sample and reference. Thus, five arrays per experimental treatment were processed, including the CON treatment.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Agricultural

Release date: 2007-07-13

Last updated: 2007-06-21