Gene expression patterns in estrogen (nonylphenol) and aryl hydrocarbon receptor agonists (PCB-77) interaction using rainbow trout (Oncorhynchus Mykiss) primary hepatocyte culture.
It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3',4,4'-tetrachlorobiphenyl, PCB-77) and estrogen receptor agonist (ER; nonylphenol, NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 microM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 microM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERalpha, ERbeta, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRalpha mRNA (except 1 microM PCB-77, which caused the induction of AhRalpha mRNA levels). AhRbeta mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 microM NP restored the PCB-77-inhibited AhRbeta mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.
Mortensen
Anne Skjetne
AS
Department of Biology, Norwegian University of Science and Technology (NTNU), Trondheim.
Tolfsen
Christina C
CC
Arukwe
Augustine
A
D000818:Animals; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism; D020847:Estrogen Receptor Modulators / Q000633:toxicity*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D017686:Oncorhynchus mykiss; D010636:Phenols / Q000633:toxicity*; D011078:Polychlorinated Biphenyls / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D014819:Vitellogenins / Q000235:genetics / Q000378:metabolism
D004527:Egg Proteins; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D010636:Phenols; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014819:Vitellogenins; C423083:zona radiata protein, fish; C025256:nonylphenol; D011078:Polychlorinated Biphenyls; D019363:Cytochrome P-450 CYP1A1; C028451:3,4,3',4'-tetrachlorobiphenyl