Effects of the polycyclic aromatic hydrocarbon benzo[k]fluoranthene at two temperatures in 3D-cultured rainbow trout RTL-W1 cells

Freshwater habitats are affected by prevalent pollutants, like polycyclic aromatic hydrocarbons (PAHs), and by increasing temperatures due to global warming. Monolayer (2D) cultures of fish cell lines like the liver-derived RTL-W1 are commonly used to investigate toxicological effects. However, 3D systems reproduce more efficiently in vivo responses and may be more valuable in ecotoxicological research.

An RTL-W1 3D model (60,000 cells/well) was generated using 96-well ultra-low attachment (ULA) plates. Mature, 10-day-old spheroids were exposed to control, solvent control (0.1% DMSO), and 10 nM and 100 nM of benzo[k]fluoranthene (BkF) at 18 ºC and 23 ºC. After a 4-day exposure, spheroids were assessed for viability (lactate dehydrogenase (LDH) and Alamar Blue assays), biometry, histology (optical and electron microscopy), and mRNA expression of target genes related to xenobiotic metabolism (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). Protein expression of CYP1A was also assessed by immunocytochemistry (ICC).

Temperature and BkF exposure did not alter the spheroids’ viability or biometry. Nevertheless, in electron microscopy, more aspects of cell degeneration were noticed at 23 ºC. CYP1A expression increased in both BkF conditions and AhR only in the higher concentration. Regarding UGT, temperature and BkF interactively modelled its expression, which markedly increased in the 100 nM condition at 23 ºC. In contrast, CYP3A27, MRP2, and BSEP expression were lower at 23 ºC. CAT and GST were not affected by temperature or BkF. ICC for CYP1A showed a dose-dependent increase.

The results suggest that RTL-W1 spheroids may be a handy novel model for in vitro research on liver effects of BkF/PAHs and temperature, namely on detoxifying genes.

Funding: Master Degree in Environmental Contamination and Toxicology, U.Porto; FCT strategic funding UIDB/04423/2020 and UIDP/04423/2020 to CIIMAR/CIMAR.