title: Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.
abstract: Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50μM; (2) an ER antagonist (ICI 182,780) at 10 and 50μM; and (3) mixtures of both (Mix I-10μM EE2 and 50μM ICI; Mix II-1μM EE2 and 10μM ICI and Mix III-1μM EE2 and 50μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17β-hydroxysteroid dehydrogenase 4-17β-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal. Electronic address: tvmadureira@icbas.up.pt.
authors.forename: Tânia Vieira
authors.initials: TV
authors.lastname: Madureira
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Malhão
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal.
authors.forename: Ivone
authors.initials: I
authors.lastname: Pinheiro
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal.
authors.forename: Célia
authors.initials: C
authors.lastname: Lopes
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal.
authors.forename: Nádia
authors.initials: N
authors.lastname: Ferreira
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal.
authors.forename: Ralph
authors.initials: R
authors.lastname: Urbatzka
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Faculty of Sciences (FCUP), U.Porto-University of Porto, Department of Biology, Rua do Campo Alegre, P 4169-007 Porto, Portugal.
authors.forename: L Filipe C
authors.initials: LF
authors.lastname: Castro
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto-University of Porto, Rua dos Bragas 289, P 4050-123 Porto, Portugal; Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)-University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto, Portugal.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Rocha
mesh_terms: D000818:Animals; D004958:Estradiol / Q000031:analogs & derivatives / Q000633:toxicity; D065171:Estrogen Receptor Antagonists / Q000633:toxicity; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000235:genetics / Q000633:toxicity; D004997:Ethinyl Estradiol / Q000633:toxicity; D000077267:Fulvestrant; D005786:Gene Expression Regulation / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects*; D020675:Peroxisomes / Q000187:drug effects / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014337:Trout / Q000502:physiology*; D014819:Vitellogenins / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D065171:Estrogen Receptor Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D011960:Receptors, Estrogen; D014819:Vitellogenins; D014874:Water Pollutants, Chemical; D000077267:Fulvestrant; D004997:Ethinyl Estradiol; D004958:Estradiol
keywords: Brown trout; Ethinylestradiol; Hepatocytes; ICI 182,780; Peroxisomes