A Step Forward in the Characterization of Primary Brown Trout Hepatocytic Spheroids as Experimental Models.

Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds.

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.

Fish hepatocyte spheroids - A powerful (though underexplored) alternative in vitro model to study hepatotoxicity.

In vitro fish cell cultures are considered alternative models to in vivo toxicological studies. The two-dimensional (2D) cultures have been used in toxicity testing, but those models have well-known drawbacks, namely in culture longevity and in the maintenance of some in vivo cellular functions. In this context, three-dimensional (3D) systems are now proposed to better mimic in vivo effects. The use of 3D cultures in fish is still limited (e.g., toxicity testing, drug biotransformation and bioaccumulation studies) compared to the number of studies with mammalian cells exploring the potential of these systems. In fish, the liver spheroids have been the most used 3D model, deriving from either liver cell lines or primary cultures of hepatocytes. Because the liver is the main organ for xenobiotic detoxification, hepatocyte spheroids represent a promising alternative to test concentration-responses to xenobiotics and explore mechanistic or ecotoxicological perspectives. Evidence shows that fish hepatocytes cultured in spheroids closely resemble the in vivo counterparts, additionally having higher basal metabolic capacity than hepatocytes cultured in monolayer. This graphical review is an updated critical sum-up of data published with 3D fish hepatocytes and provides background knowledge for the upcoming studies using this model. It further addresses the culture conditions for obtaining fish hepatocyte spheroids and discusses the main factors that can influence the biometry and functionality of spheroids over time in culture and the 2D versus 3D distinct metabolic capacities.

Multi-Parametric Portfolio to Assess the Fitness and Gonadal Maturation in Four Key Reproductive Phases of Brown Trout.

Brown trout is an environmental freshwater sentinel species and is economically important for recreational fishing and aquaculture. Despite that, there is limited knowledge regarding morpho-physiological variations in adults throughout the reproductive cycle. Thus, this study aimed to analyze the fitness and gonadal maturation of cultured adult brown trout in four reproductive phases (spawning capable-December, regressing-March, regenerating-July, and developing-November). The systematic evaluation of males and females was based on biometric, biochemical, and hormonal parameters, along with a histomorphological grading of gonads and the immunophenotype location of key steroidogenic enzymes. The total weight and lengths reached the lowest levels in December. Gonad weights were higher in December and November, while the opposite pattern was found for liver weights. The lowest levels of cholesterol and total protein were also noted during those stages. The 11-ketotestosterone (11-KT) and testosterone (T) for males, and estradiol (E2) and T for females, mostly explained the hormonal variations. The immunohistochemistry of cytochrome P450c17 (CYP17-I), aromatase (CYP19), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) showed sex and site-specific patterns in the distinct reproductive phases. The sex- and season-specific changes generated discriminative multi-parameter profiles, serving as a tool for environmental and aquaculture surveys.

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.

Disruption of classical estrogenic targets in brown trout primary hepatocytes by the model androgens testosterone and dihydrotestosterone.

Estrogenic effects triggered by androgens have been previously shown in a few studies. Aromatization and direct binding to estrogen receptors (ERs) are the most proposed mechanisms. For example, previously, a modulation of vitellogenin A (VtgA) by testosterone (T), an aromatizable androgen, was reported in brown trout primary hepatocytes. The effect was reversed by an ER antagonist. In this study, using the same model the disruption caused by T and by the non-aromatizable androgen - dihydrotestosterone (DHT), was assessed in selected estrogenic targets. Hepatocytes were exposed (96 h) to six concentrations of each androgen. The estrogenic targets were VtgA, ERα, ERß1 and two zona pellucida genes, ZP2.5 and ZP3a.2. The aromatase CYP19a1 gene and the androgen receptor (AR) were also included. Modulation of estrogenic targets was studied by quantitative real-time PCR and immunohistochemistry, using an HScore system. VtgA and ERα were up-regulated by DHT (1, 10, 100 µM) and T (10, 100 µM). In contrast, ERß1 was down-regulated by DHT (10, 100 µM), and T (100 µM). ZP2.5 mRNA levels were increased by DHT and T (1, 10, 100 µM), while ZP3a.2 was up-regulated by DHT (100 µM) and T (10, 100 µM). Positive correlations were found between VtgA and ERα mRNA levels and ZPs and ERα, after exposure to both androgens. The mRNA levels of CYP19a1 were not changed, while AR expression tended to increase after micromolar DHT exposures. HScores for Vtg and ZPs corroborated the molecular findings. Both androgens triggered estrogen signaling through direct binding to ERs, most probably ERα.

Testosterone-induced modulation of peroxisomal morphology and peroxisome-related gene expression in brown trout (Salmo trutta f. fario) primary hepatocytes.

Disruption of androgenic signaling has been linked to possible cross-modulation with other hormone-mediated pathways. Therefore, our objective was to explore effects caused by testosterone - T (1, 10 and 50µM) in peroxisomal signaling of brown trout hepatocytes. To study the underlying paths involved, several co-exposure conditions were tested, with flutamide - F (anti-androgen) and ICI 182,780 - ICI (anti-estrogen). Molecular and morphological approaches were both evaluated. Peroxisome proliferator-activated receptor alpha (PPARα), catalase and urate oxidase were the selected targets for gene expression analysis. The vitellogenin A gene was also included as a biomarker of estrogenicity. Peroxisome relative volumes were estimated by immunofluorescence, and transmission electron microscopy was used for qualitative morphological control. The single exposures of T caused a significant down-regulation of urate oxidase (10 and 50µM) and a general up-regulation of vitellogenin. A significant reduction of peroxisome relative volumes and smaller peroxisome profiles were observed at 50µM. Co-administration of T and ICI reversed the morphological modifications and vitellogenin levels. The simultaneous exposure of T and F caused a significant and concentration-dependent diminishing in vitellogenin expression. Together, the findings suggest that in the tested model, T acted via both androgen and estrogen receptors to shape the peroxisomal related targets.