Effects of Temperature Increase and Benzo[k]fluoranthene on Viability and CYP1A Response in Brown Trout Hepatocytes ^†

Abstract

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.