Establishing brown trout primary hepatocyte spheroids as a new alternative experimental model-Testing the effects of 5α-dihydrotestosterone on lipid pathways.

Three-dimensional (3D) fish liver cultures mimic the in vivo cellular microenvironment, which is ideal for ecotoxicological research. Despite that, the application of these cultures to evaluate toxic effects in fish is scarce. A 3D model of brown trout (Salmo trutta f. fario) primary hepatocyte spheroids was optimized in this study by using DMEM/F-12 with 15 mM of HEPES, 10 mL/L of an antibiotic and antimycotic solution and FBS 10% (v/v), at 18 °C with ∼100 rpm. The selection of optimal conditions was based on a multiparametric characterization of the spheroids, including biometry, viability, microanatomy and immunohistochemistry. Biometric and morphologic stabilization of spheroids was reached within 12-16 days of culture. To our knowledge, this study is the first to culture and characterize viable spheroids from brown trout primary hepatocytes for over 30 days. Further, the 3D model was tested to explore the androgenic influences on lipidic target genes after 96 h exposures to control, solvent control, 10 and 100 µM of 5α-dihydrotestosterone (DHT), a non-aromatizable androgen. Spheroids exposed to 100 µM of DHT had decreased sphericity. DHT at 100 µM also significantly down-regulated Acox1-3I, PPARγ and fatty acid synthesis targets (i.e., ACC), and significantly up-regulated Fabp1. Acsl1 was significantly up-regulated after exposure to both 10 and 100 µM of DHT. The results support that DHT modulates distinct lipidic pathways in brown trout and show that this 3D model is a new valuable tool for physiological and toxicological mechanistic studies.

Deciphering influences of testosterone and dihydrotestosterone on lipid metabolism genes using brown trout primary hepatocytes.

Despite of physiological and toxicological relevance, the potential of androgens to influence fish lipid metabolism remains poorly explored. Here, brown trout primary hepatocytes were exposed to six concentrations (1 nM to 100 µM) of dihydrotestosterone (DHT) and testosterone (T), to assess changes in the mRNA levels of genes covering diverse lipid metabolic pathways. Acsl1, essential for fatty acid activation, was up-regulated by T and DHT, whereas the lipogenic enzymes FAS and ACC were up-regulated by the highest (100 µM) concentration of T and DHT, respectively. ApoA1, the major component of high-density lipoprotein (HDL), was down-regulated by both androgens. PPARγ, linked to adipogenesis and peroxisomal ß-oxidation, was down-regulated by T and DHT, while Acox1-3I, rate-limiting in peroxisomal ß-oxidation, was down-regulated by T. Fabp1, StAR and LPL were not altered. Our findings suggest that androgens may impact on lipid transport, adipogenesis and fatty acid ß-oxidation and promote lipogenesis in fish liver.