title: Targeted salmon gene array (SalArray): a toxicogenomic tool for gene expression profiling of interactions between estrogen and aryl hydrocarbon receptor signalling pathways.
abstract: In toxicogenomics, gene arrays are valuable tools in the identification of differentially expressed genes and potentially identify new gene biomarkers altered by exposure of organisms to xenobiotic compounds, either singly or as complex mixtures. In this study, we investigated the mechanisms of interaction between estrogen receptor (ER) and aryl hydrocarbon receptor (Ah receptor or AhR) signalling pathways using toxicogenomic approaches. First, we generated cDNA libraries using suppressive subtractive hybridization (SSH) of clones containing differentially expressed genes from Atlantic salmon (Salmo salar) separately exposed to ER and AhR agonists. Second, a targeted gene array (SalArray) was developed based on true-positive differentially expressed genes. In the experimental setup, primary cultures of salmon hepatocytes isolated by a two-step perfusion method were exposed for 48 h to nonylphenol (NP; 5 microM) and 3,3',4,4'-tetrachlorobiphenyl (TCB; 1 microM), singly and combined, in the absence or presence of antagonists. Using a targeted SalArray, we demonstrate that exposure of salmon to NP singly or in combination with TCB produced differential gene expression patterns in salmon liver. Array analysis showed that exposure of hepatocytes to NP mainly altered genes involved in the estrogenic pathway, including genes for steroid hormone synthesis and metabolism. The anti-estrogenic properties of TCB were demonstrated in the array analysis as genes induced by NP were decreased by TCB. To study the effects of TCB on ER-mediated transcription, hepatocytes were treated for 48 h with tamoxifen (Tam; 1 microM) and ICI182,780 (ICI; 1 microM). The effect of AhR on ER-mediated transcription was investigated by blocking AhR activity with alpha-naphthoflavone (ANF; 0.1 and 1 microM). Quantitative real-time polymerase chain reactions confirmed the changes in expression of ERalpha, ERbeta, vitellogenin (Vtg), zona radiata protein (Zr-protein), and vigilin for the ER pathway and AhRalpha, AhRbeta, AhRR, ARNT, CYP1A1, UDPGT, and a 20S proteasome beta-subunit for the AhR pathway. We found that exposure to NP and TCB both singly and in combination produced gene expression patterns that were negatively influenced by individual receptor antagonists. TCB caused decreased ER-mediated gene expression, and NP caused decreased AhR-mediated responses. Inhibition of AhR with ANF did not reverse the effect of TCB on ER-mediated transcription suggesting that AhRs do not have a direct role on TCB-mediated decreases of ER-mediated responses. In contrast, the inhibition of ER with Tam and ICI reversed the transcription of AhR-mediated responses (except AhRR). Taken together, the findings in the present study demonstrate a complex mode of ER-AhR interaction, possibly involving competition for common cofactors. This complex mode of interaction is further supported by the observation that the presence of ER antagonists potentiated the transcription of AhR isoforms and their mediated responses when TCB was given alone (more so for AhRbeta). Thus, the inhibitory ER-AhR interactions can be used to further investigate specific genes found to be affected in our targeted SalArray chip that are important for the reproductive effects of endocrine disruptors.
authors.affiliation: Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, 7491 Trondheim, Norway.
authors.forename: Anne Skjetne
authors.initials: AS
authors.lastname: Mortensen
authors.forename: Augustine
authors.initials: A
authors.lastname: Arukwe
mesh_terms: D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D017364:Collagenases / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004967:Estrogens / Q000502:physiology*; D020869:Gene Expression Profiling / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020239:Receptor Cross-Talk / Q000187:drug effects; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000502:physiology*; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020317:Salmo salar / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D043922:Toxicogenetics / Q000379:methods*
chemical_list: D001571:Benzoflavones; D018076:DNA, Complementary; D004967:Estrogens; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; C011512:alpha-naphthoflavone; D017364:Collagenases