Effects of Benzo[k]fluoranthene at Two Temperatures on Viability, Structure, and Detoxification-Related Genes in Rainbow Trout RTL-W1 Cell Spheroids

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 (<i>CYP</i>)<i>1A</i>, <i>CYP3A27</i>, aryl hydrocarbon receptor (<i>AhR</i>), glutathione S-transferase (<i>GST</i>), uridine diphosphate-glucuronosyltransferase (<i>UGT</i>), catalase (<i>CAT</i>), multidrug resistance-associated protein 2 (<i>MRP2</i>) and bile salt export protein (<i>BSEP</i>). 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 <i>CYP1A</i> was augmented with both BkF concentrations, while <i>AhR</i>'s increased at the higher concentration. The CYP1A protein showed a dose-dependent increase. Temperature and BkF concurrently modelled <i>UGT</i>'s expression, which increased in the 100 nM condition at 23 °C. Conversely, <i>CYP3A27</i>, <i>MRP2</i>, and <i>BSEP</i> expressions lowered at 23 °C. <i>CAT</i> and <i>GST</i> 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.