Activation of estrogen receptor signaling by the dioxin-like aryl hydrocarbon receptor agonist, 3,3',4,4',5-pentachlorobiphenyl (PCB126) in salmon in vitro system.
Available toxicological evidence indicates that environmental contaminants with strong affinity to the aryl hydrocarbon receptor (AhR) have anti-estrogenic properties in both mammalian and non-mammalian in vivo and in vitro studies. The primary objective of the present study was to investigate the interactions between the AhR and estrogen receptor (ER) in salmon in vitro system. Two separate experiments were performed and gene expression patterns were analyzed using real-time PCR, while protein analysis was done by immunoblotting. Firstly, salmon primary hepatocytes were exposed to the dioxin-like PCB126 at 1, 10 and 50 nM [corrected] and ER agonist nonylphenol (NP) at 5 and 10 microM, singly or in combination. Our data showed increased levels of ER-mediated gene expression (vitellogenin: Vtg, zona radiata protein: Zr-protein, ERalpha, ERbeta and vigilin) as well as increased cellular ERalpha protein levels after treatment with NP and PCB126, singly or in combination. PCB126 treatment alone produced, as expected, increased transcription of AhR nuclear translocator (Arnt), CYP1A1 and AhR repressor (AhRR) mRNA, and these responses were reduced in the presence of NP concentrations. PCB126 exposure alone did not produce significant effect on AhR2alpha mRNA but increased (at 1 and 50 pM) and decreased (at 10 pM) AhR2beta mRNA below control level. For AhR2delta and AhR2gamma isotypes, PCB126 (at 1 nM) [corrected] produced significant decreases (total inhibition for AhR2gamma) of mRNA levels but was indifferent at 10 and 50 pM, compared to control. NP exposure alone produced concentration-dependent significant decrease of AhR2beta mRNA. In contrast, while 5 microM NP produced an indifferent effect on AhR2delta and AhR2gamma, 10 microM NP produced significant decrease (total inhibition for AhR2gamma) and the presence of NP produced apparent PCB126 concentration-specific modulation of all AhR isotypes. A second experiment was performed to evaluate the involvement of ER isoforms in PCB126 mediated estrogenicity. Here, cells were treated with the different concentrations of PCB126, alone or in combination with ICI182,780 (ICI) and sampled at 12, 24 and 48 h post-exposure. Our data showed that PCB126 produced a time- and concentration-specific increase of ERalpha and Vtg expressions and these responses were decreased in the presence of ICI. In general, these responses show a direct PCB126 induced transcriptional activation of ERalpha and estrogenic responses in the absence of ER agonists. Although not conclusive, our findings represent the first study showing the activation of estrogenic responses by a dioxin-like PCB in fish in vitro system and resemble the "ER-hijacking" hypothesis that was recently proposed. Thus, the direct estrogenic actions of PCB126 observed in the present study add new insight on the mechanisms of ER-AhR cross-talk, prompting a new wave of discussion on whether AhR-mediated anti-estrogenicity is an exception rather than rule of action.
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D004147:Dioxins / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology*; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology; D011078:Polychlorinated Biphenyls / Q000494:pharmacology*; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects*; D011960:Receptors, Estrogen / Q000819:agonists*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020317:Salmo salar / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D004147:Dioxins; D004965:Estrogen Antagonists; D004968:Estrogens, Non-Steroidal; D010636:Phenols; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; C025256:nonylphenol; D011078:Polychlorinated Biphenyls; C023035:3,4,5,3',4'-pentachlorobiphenyl