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The Use of Gene Arrays to Determine Temporal Gene Induction in Sheepshead Minnows Exposed to E2
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| Abstract: | Gene arrays provide a means to study differential gene expression in fish exposed to environmental estrogens by providing a "snapshot" of the genes expressed at a given time. Such array data may also uncover previously unknown biochemical pathways affected by estrogenic compounds. To study the temporal induction of certain estrogen responsive genes, we have expanded an existing cDNA gene array {Larkin, 2002 #1342; Larkin, 2003 #1340} developed for the sheepshead minnow (SHM) (Cyprinodon variegates) to include over 200 additional genes that may be differentially regulated by EDCs. These genes were obtained from SHM liver cDNA libraries, suppressive subtractive hybridization libraries of liver mRNA from SHM exposed to known EDCs (methoxychlor, nonylphenol) or from differential display analyses. The gene arrays were used to examine temporal gene induction in livers of adult male SHM exposed to 100 ng/L and 500 ng/L 17 -estradiol for 6, 12, 24 or 48 hr. The estrogen receptor alpha gene was induced beginning at 6 hr, but the level of induction did not increase with longer exposure times. The estrogen responsive genes (vitellogenins [VTG 1, VTG2] and zona radiata [ZRP2, ZRP3]) were upregulated beginning 12 hr after exposure with the induction level increasing as exposure time increased. VTG1 was expressed at a 10-fold higher level than VTG2 after 48 hr. Other genes that appear to be upregulated over 48 hr include ATP synthase 6, serum amyloid protein, tryptophan 2,3 dioxygenase, a secretory phospholipase precursor and several unidentified genes. Several genes appear to be downregulated including fibrinogen, cytochrome b and cytochrome c oxidase. A number of genes increase after 6 and 12 hr of exposure, but are down-regulated to levels below those of unexposed fish after 48 hr. Those genes include chitinase, a scavenger receptor, C type lectin S and several unidentified genes. Quantitative real-time PCR (TaqMan) was used to validate the array results for VTG1, VTG2, ZRP2 and ZRP3. Through the use of gene arrays, temporal patterns of altered gene expression can be determined for key genes involved in metabolic pathways, and these changes can be quantified by real-time PCR. |
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| Citation: | Knoebl, I., J. L. Blum, and N. Denslow. The Use of Gene Arrays to Determine Temporal Gene Induction in Sheepshead Minnows Exposed to E2. Presented at Society of Environmental Toxicology & Chemistry, Portland, OR, November 14-18, 2004. |
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| Contact: |
Linda Ransick - (513) 569-7395 or ransick.linda@epa.gov
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| Division: |
Ecological Exposure Research Division |
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| Branch: |
Molecular Ecology Research Branch |
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| Product Type: |
Abstrct/Oral |
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| Presented: |
11/15/2004 |
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