Ethinylestradiol Exposure of Primary Culture Brown Trout Hepatocytes Induce Morphological Changes in Peroxisomes

Estrogens are often found as aquatic pollutants and they are able to influence liver peroxisome (Px) dynamics in fish. Our group reported a seasonal pattern of morphofunctional changing in Px that is coincident with different stages of the reproductive cycle in brown trout (Salmo trutta f. fario). In vivo experiments showed that Px volume and Px enzymes activities were lower when female brown trout had higher levels of estradiol [1,2]. The mechanisms and toxicological implications are unknown, so herein we aimed to mimic the above mentioned in vivo scenario by using brown trout primary hepatocyte cultures exposed to ethinylestradiol (EE2), as an alternative methodology to infer the potential Px morphological alterations. Sexually immature brown trouts were used for hepatocyte isolation by means of two steps perfusion technique with collagenase type IV. Cells were plated at 1x106 cell/mL in 24 well plates using L-15 medium without phenol red and supplemented with fetal bovine serum charcoal. No additional O2/CO2 was used and the temperature was maintained at 19 °C. Cells were exposed to: 10 and 50 μM of EE2 and L15 medium with 0.1% of ethanol (solvent control). After 96 h of exposure, hepatocytes were collected and processed for transmission electron microscopy. Catalase cytochemistry technique was used in order to better differentiate the peroxisomes. Ultrathin sections were observed and photographed in a JEOL 100CXII electron microscope. Peroxisome and nucleus volume densities were estimated applying manual stereological techniques.