Uncovering Morphological Interferences Caused by Androgen Inputs in Peroxisomes from Primary Brown Trout Hepatocytes Using Catalase Immunofluorescence

In general, a greater number of studies have investigated the potential toxic effects of estrogenic active compounds, comparing with androgenic substances, particularly in fish species. However, assays using the androgen testosterone showed that it may interfere with functions normally associated with estrogen pathways, as vitellogenin synthesis in hepatocytes [1]. Previous studies performed in trout specimens, showed morphofunctional changes in liver peroxisomes along its reproductive cycle, which seem to be negatively influenced by the higher levels of estrogens [2]. So, herein we aimed to evaluate the potential morphological effects of an androgen (testosterone) in peroxisomes from primary cultures of trout hepatocytes (Salmo trutta f. fario). Immature brown trouts were used for hepatocyte isolation. Hepatocytes were plated at a density of 1×106 cells/mL in 24 well plates in L-15 medium without phenol red. Cells were maintained in an incubation chamber at 19 ºC without additional O2/CO2. After 24 h in culture, hepatocytes were exposed during 96 h to testosterone at 1 and 50 µM. In order to quantitatively evaluate morphological changes, stereological methods were implemented for determining the peroxisome and nucleus volumetric densities, in relation to the hepatocyte, by examining photos after anti-catalase immunofluorescence (example in Fig. 1). All technical procedures have been previously described [3]. Data was qualitatively complemented by analyzing electron microscopy photos after 3,3’-diaminobenzidine cytochemistry.