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

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 96 h 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 50 mu M; (2) an ER antagonist (ICI 182,780) at 10 and 50 mu M; and (3) mixtures of both (Mix 1-10 mu M EE2 and 50 mu M ICI; Mix II-1 mu M EE2 and 10 mu M ICI and Mix III-1 mu M EE2 and 50 mu M ICI). The mRNA levels of the estrogenic targets (ER alpha, ER beta-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17 beta-hydroxysteroid dehydrogenase 4-17 beta-HSD4, peroxin 11 alpha-Pex11a and PPAR alpha) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 mu M 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 ER alpha mRNA increased after all EE2 treatments, while ER beta-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ER alpha 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 PPAR alpha signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments. (C) 2015 Elsevier B.V. All rights reserved.