Morphological Alterations Caused by Estrogenic and Anti-Estrogenic Signaling in Peroxisomes of Primary Brown Trout Hepatocytes – Stereological Approach Using Catalase Immunofluorescence

Currently, it is no longer a novelty that the presence of hormones, namely those interfering with the estrogenic pathway, are widely spread along distinct environmental compartments and/or bio-accumulated in exposed organisms, causing disruptive imbalances in many physiological functions, for instance in fish. As supportive background for developing this study it was considered our previous evidences pointing to a negative association between ovary maturation (higher titers of estrogens) and key enzymatic activities in peroxisomes (e.g., catalase), in brown trout (Salmo trutta f. fario) model [1]. Peroxisomes are catalase rich organelles with a high plasticity to deal with a large variety of stimuli, including changes either in size and/or number [2]. In the light of the exposed, our main aim here was to investigate the potential of an agonist (17α-ethynylestradiol – EE2) and an antagonist (fulvestrant – ICI-182, 780) of estrogen receptor, to affect peroxisomes in brown trout. The in vitro hepatocyte primary cell culture was obtained by a two-step collagenase perfusion technique, from immature juvenile trouts. Hepatocytes were plated with a density of 1x106 cells/mL, by using 500 μL of L-15 medium without phenol red. Cells were maintained in an incubation chamber at 19 ºC without additional O2/CO2. Then, two independent experiments were performed. In experiment I, hepatocytes were exposed to: 10 and 50 μM of EE2 and 0.1% of ethanol (solvent control). As to experiment II, the exposure conditions besides control were: 1 μM of EE2 and a mixture of 1 µM EE2 and 10 µM ICI (Mix). After 96 h of exposure, cells were fixed for paraffin and processed accordingly with a previous optimized protocol [3] for catalase immunofluorescence (primary antibody rabbit polyclonal anti-catalase; secondary antibody goat anti-rabbit IgG-CFL 488). Stereological analyses, specifically directed to the volume densities of peroxisomes in relation to the hepatocyte were done in 10 fluorescence photos/treatment for both experiments.