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.
Keywords: 5α-Dihydrotestosterone; Brown trout; In vitro; Lipidic targets; Primary hepatocytes; Spheroids.
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