Célia Lopes

Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout ( Salmo trutta ), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.

Introduction: The liver is a central metabolic regulator responsible not only for energy storage and allocation but also for the production of essential egg proteins. In seasonal breeders such as brown trout, physiological demands increase during spawning, requiring strong metabolic and structural adaptations. Understanding the cellular mechanisms underlying these fluctuations is essential for clarifying how liver function is regulated during reproduction. Objective: The aim of this study was to investigate cellular mechanisms underlying liver remodelling during the reproductive cycle of male and female brown trout by examining proliferation and apoptosis markers at both molecular and protein levels during four distinct reproductive stages: spawning capable, regressing, regenerating, and developing. Methodology: Adult male and female brown trout were sampled at four reproductive stages. Liver gene expression of proliferating cell nuclear antigen (PCNA) and caspase-3 was analysed using qRT-PCR. At the protein level, liver tissue sections were immunolabelled with antibodies against PCNA and caspase-3. Caspase-3 staining intensity was quantified using ImageJ, while PCNA expression was assessed by counting positively stained nuclei. Results: PCNA gene expression was upregulated during the regressing stage, while PCNA-positive nuclei immunolabelling increased during the regressing and spawning-capable stages. Caspase-3 transcript levels showed no significant differences among stages, whereas immunostaining increased during regenerating and developing stages. Conclusions: Overall, the liver undergoes dynamic cellular remodelling throughout the reproductive cycle, balancing proliferation and apoptosis to meet reproductive demands. The results suggest that proliferation is more evident during regenerating stages, while apoptotic activity may be regulated post-transcriptionally. PCNA also indicates that proliferative activity may persist during spawning capable stages, reflecting ongoing mitotic readiness despite reduced overall proliferation.

Introduction: Temperature is a key environmental factor influencing the physiological and biochemical processes of aquatic organisms, including xenobiotic metabolism. Understanding how temperature modulates the toxicological effects of pollutants such as polycyclic aromatic hydrocarbons (PAHs) is crucial in the context of climate change. Among these compounds, benzo[a]pyrene (BaP) and benzo[a]anthracene (BaA) are priority pollutants in aquatic environments, resulting from incomplete combustion. Their relevance is attributed to persistence and metabolic bioactivation potential. Fish primary hepatocyte cultures represent a relevant in vitro model for studying combined effects of thermal stress and chemical exposures, while supporting the 3Rs principles (Replacement, Reduction, and Refinement). Objective: This study aims to assess temperature-dependent effects of BaP and BaA, and their mixtures in brown trout hepatocytes using cytochrome P450 1A (CYP1A) immunohistochemistry as an indicator of xenobiotic metabolism. Methodology: Primary hepatocytes were isolated using a two-step collagenase perfusion method and cultured in 24-well plates at 18 °C and 22 °C. Cells were exposed for 72 h to supplemented L-15 medium (control) or to 0.1% dimethyl sulfoxide in supplemented L-15 medium (solvent control), as well as to single exposures of 1 and 10 µM of BaP and BaA and to equimolar mixtures of both compounds (1 and 10 µM). Viability was assessed using the lactate dehydrogenase (LDH) assay. CYP1A immunostaining was quantified based on cytoplasmic staining intensity relative to background area. Results: No significant effects on cell viability were observed under any condition. Temperature significantly reduced CYP1A expression in single exposures at 22 °C compared to 18 °C. BaP induced a significant dose-dependent increase, while BaA differed from controls only at 10 µM. In mixtures, only treatment- and dose-dependent effects were observed, with no temperature influence detected. Conclusions: Overall, the data highlight temperature as a key modulator of biochemical responses to PAHs, with single and mixed exposures eliciting distinct effects and suggesting potential synergism in mixtures.

Introduction: The temperature of rivers in the Iberian Peninsula has increased due to global warming. In addition, these rivers are polluted by contaminants of emerging concern, such as polycyclic aromatic hydrocarbons (PAHs). Higher temperatures and pollution concurrently impose threats to the Iberian Peninsula’s endemic species, including the brown trout (Salmo trutta), a cold-water species widely used in ecotoxicological studies. Because the liver is the main biotransformation organ, and is particularly sensitive to both chemical and temperature changes, in vitro liver models may represent valuable alternatives for assessing combined stressor effects, complying with the 3Rs principle. Objective: In line with the above, the present study aimed to evaluate the combined effects of a 4 °C temperature increase and the model PAH benzo[k]fluoranthene (B[k]F) on fish liver cells using a primary brown trout hepatocyte culture as a model. Methodology: Primary hepatocytes were seeded in 6-well plates at a density of 1.0 × 10⁶ cells/mL and exposed for 48 h to 1, 10, and 20 µM B[k]F at 18 °C (normothermia) and 22 °C (warming scenario). Cell viability was assessed using trypan blue, alamarBlue, and lactate dehydrogenase (LDH) assays. Cytochrome P450 (CYP)1A was evaluated in terms of its gene expression by RT-qPCR and its protein expression through immunocytochemistry (ICC). The immunostaining was quantified using a score system which considered five intensity staining levels. Results: Exposure to B[k]F and to the higher temperature increased LDH leakage without interaction effects. In contrast, the other viability assays did not show significant differences across conditions. Regarding CYP1A, both gene and protein expression increased with all B[k]F concentrations in relation to the controls, but were not influenced by temperature. Notably, the lowest B[k]F concentration (1 µM) elicited the highest CYP1A gene expression, suggesting a non-monotonic response. Conclusions: Overall, the model was responsive to both temperature (4 °C) increase and to B[k]F, validating its usefulness for assessing liver pollutant effects in the context of global warming. These findings support the application of fish primary hepatocyte models as relevant tools in ecotoxicology under environmentally realistic multi-stressor scenarios.

Introduction: The 3 Rs principle advocates developing alternative, biologically relevant models. Thus, 3D fish liver in vitro models have been increasingly used for ecotoxicological studies. We previously optimized spheroids from the rainbow trout non-tumoral liver cell line RTL-W1 and employed them to assess the effects of aquatic pollutants. Although they demonstrated potential for assessing ecotoxicological effects, further optimization is warranted to enhance their physiological relevance. Incorporating an extracellular matrix (ECM), such as collagen, has been shown to be a promising strategy to improve spheroids’ structural organization and functionality. Objective: This study aimed to optimize 3D culturing conditions of RTL-W1 spheroids by evaluating the effects of collagen supplementation on viability, morphology, and functional response. Methodology: Spheroids from the RTL-W1 cell line (60,000 cells per well) were cultured in 96-well ultra-low attachment (ULA) plates at 18 °C. After spheroids’ formation, rat tail collagen was supplemented at concentrations of 15 (C15), 30 (C30), and 60 (C60) µg/mL at culture days 7, 8, and 9. Spheroids were collected at two sampling days (10 and 14). Viability was assessed using alamarBlue and lactate dehydrogenase (LDH) assays, while morphology was assessed by optical microscopy. Collagen penetration was evaluated using Masson’s trichrome staining technique. Protein expression of cytochrome P450(CYP)1A was assessed by quantifying immunocytochemistry staining using an anti-CYP1A antibody. Results: On day 10, LDH leakage decreased in C15 and C60, compared with the control, whilst C15 spheroids showed lower absorbance levels in the alamarBlue assay. On day 14, LDH showed no significant differences; however, C30 and C60 had higher alamarBlue absorbance, indicating greater metabolic capacity. Spheroid morphology appeared intact in all conditions. Masson trichrome revealed collagen fibrils at the periphery of the spheroids, especially in C30 and C60, indicating that spheroids incorporated collagen. CYP1A immunostain was present in all conditions, localized in the spheroids’ border, and tended to be higher when supplementation occurred in earlier days. Conclusions: Our results suggest that RTL-W1 spheroids interacted with the collagen matrix and appeared to functionally improve. Data suggest that incorporating ECM may increase the complexity and physiological relevance of RTL-W1 spheroids, thereby better supporting mechanistic and ecotoxicological applications.

The brown trout (Salmo trutta) is a commercially and ecologically significant salmonid fish, yet its hepatic cellular and functional dynamics throughout the reproductive cycle remain poorly characterised, particularly in males. This study investigated seasonal and sex-specific liver plasticity across four reproductive stages: spawning capable (December), regressing (March), regenerating (July), and developing (November). We quantified mRNA and protein abundance of key oestrogen-responsive targets—vitellogenin (VtgA) and zona pellucida (ZP) proteins—alongside cell turnover markers, caspase 3 (Casp3) and proliferating cell nuclear antigen (PCNA). These molecular endpoints were integrated with stereological analyses to estimate hepatocyte, nuclear, and cytoplasmic volumes. Results revealed stage-dependent mobilisation and transient hepatic retention of reproductive proteins; females exhibited stronger vitellogenic signatures and more pronounced seasonal shifts than males. Although VtgA and ZP mRNA levels peaked during the developing and spawning-capable stages, males maintained low but consistent levels throughout the cycle, indicating constitutive hepatic oestrogen sensitivity. Regarding cell turnover, PCNA protein data indicated heightened proliferative activity during the spawning-capable and regressing stages. In contrast, while Casp3 mRNA levels remained stable across all stages, protein detection suggested a post-transcriptional increase in apoptotic signalling during the developing phase, consistent with controlled tissue remodelling rather than extensive cell loss. Stereological data confirmed enlarged hepatocyte and nuclear volumes during periods of high secretory and proliferative demand. Overall, these findings demonstrate significant stage-dependent and sex-specific plasticity in brown trout liver, providing a robust reference framework for ecological monitoring, endocrine disruption assessments, and studies of teleost reproductive physiology.

Polycyclic aromatic hydrocarbons (PAHs), prevalent aquatic contaminants, arise from burning fossil fuels, a major source of greenhouse gases driving global warming. PAHs and warmer temperatures individually exert diverse negative effects on aquatic organisms. However, the effects of PAH exposure and/or rising temperature remain largely unknown. Liver in vitro models, like the rainbow trout (Oncorhynchus mykiss) RTL-W1 liver cell line, have been employed to unravel PAH-exposure effects, primarily on cell viability and enzymatic activity. Here, monolayer-cultured (2D) RTL-W1 cells were used to assess the co-exposure effects of temperature (18 and 21 °C) and two PAHs, benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F), at 10 and 100 nM. After a 72 h exposure, the cell density and viability were evaluated using the trypan blue and LDH assays. The mRNA levels of the detoxification-associated genes aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP)1A, CYP3A27, glutathione S-transferase omega 1 (GSTO1), uridine diphosphate–glucuronosyltransferase (UGT), catalase (CAT), and multidrug resistance-associated protein 2 (MRP2) were measured by RT-qPCR. Temperature influenced cell viability and LDH leakage. Both PAHs reduced the cell density and upregulated the mRNA levels of AhR, CYP1A, CYP3A27, and UGT, while GSTO1 and MRP2 were only augmented after the higher B[k]F concentration. Temperature influenced CAT and UGT expression. There was no interaction between temperature and the PAHs. Overall, the results show that B[k]F has more effects on detoxification targets than B[a]P, whereas a temperature increase mildly affects gene expression. The RTL-W1 in 2D seems useful for unravelling not only the liver effects of PAH but also the impact of temperature stress.

Polycyclic aromatic hydrocarbons (PAHs) and global warming impact aquatic ecosystems, eventually interacting. Monolayer (2D) cultures of cell lines, such as the rainbow trout liver RTL-W1, are employed for unveiling toxicological effects in fish. Nonetheless, three-dimensional (3D) models constitute an alternate paradigm, better emulating in vivo responses. Here, ultra-low attachment (ULA) plates were used to generate ten-day-old RTL-W1 spheroids for exposure to a control, a solvent control (0.1% DMSO) and the model PAH benzo[k]fluoranthene (BkF) at 10 and 100 nM and at 18 and 23 °C (thermal stress). After a 4-day exposure, spheroids were analyzed for viability (alamarBlue and lactate dehydrogenase), biometry (area, diameter and sphericity), histocytology (optical and electron microscopy), and mRNA levels of the detoxification-related genes cytochrome P450 (CYP)1A, CYP3A27, aryl hydrocarbon receptor (AhR), glutathione S-transferase (GST), uridine diphosphate–glucuronosyltransferase (UGT), catalase (CAT), multidrug resistance-associated protein 2 (MRP2) and bile salt export protein (BSEP). Immunocytochemistry (ICC) was used to assess CYP1A protein expression. Neither temperature nor BkF exposure altered the spheroids’ viability or biometry. BkF modified the cell’s ultrastructure. The expression of CYP1A was augmented with both BkF concentrations, while AhR’s increased at the higher concentration. The CYP1A protein showed a dose-dependent increase. Temperature and BkF concurrently modelled UGT’s expression, which increased in the 100 nM condition at 23 °C. Conversely, CYP3A27, MRP2, and BSEP expressions lowered at 23 °C. CAT and GST mRNA levels were uninfluenced by either stressor. Overall, BkF and temperature impacted independently or interactively in RTL-W1 spheroids. These seem to be useful novel tools for studying the liver-related effects of temperature and PAHs.

Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.