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

Abstract

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.

Keywords: PAHs; benzo(k)fluoranthene; global warming; in vitro models; spheroids.

Figure 1

alamarBlue and lactate dehydrogenase (LDH) assay data for RTL-W1 spheroids after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF): C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF at 18 °C and 23 °C. Data in boxplots correspond to the median, mean (+), interquartile range, minimum and maximum, displayed by condition on the left panel and grouped by temperature on the right. Relative fluorescence units (RFU) and absorbance values (490 nm) are shown against each condition and temperature. Different letters (a and b) illustrate significant temperature differences according to a two-way ANOVA followed by Tukey’s test (p < 0.05). n = 5 independent assays per temperature.

Figure 2

Equivalent diameter (µm), area (µm²) and sphericity of RTL-W1 spheroids after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF): C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF at 18 °C and 23 °C. Data correspond to the median, mean (+), interquartile range, minimum and maximum, displayed by condition on the left panel and grouped by temperature on the right. n = 5 independent assays per temperature.

Figure 3

Hematoxylin and eosin-stained histological sections of RTL-W1 spheroids after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF) at 18 °C and 23 °C: C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF.

Figure 4

Semithin sections of RTL-W1 spheroids after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF) at 18 °C and 23 °C: C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF. A—pyknotic nuclei; EC—elongated cells; S—senescent cells; V—vacuoles.

Figure 5

Ultrathin sections of RTL-W1 spheroids after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF) at 18 °C and 23 °C: C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF. (A)—C (18 °C); (B)—C (23 °C); (C)—SC (18 °C); (D)—SC (23 °C); (E)—10 nM BkF (18 °C); (F)—10 nM BkF (23 °C); (G)—100 nM BkF (18 °C); (H)—100 nM BkF (23 °C). DB—dense bodies; EDL—electron-dense lipids; F—intermediate filaments; G—Golgi apparatus; L—electron-lucent lipids; M—mitochondria; MP—membrane protrusions; RE—endoplasmic reticulum.

Figure 6

Grading of the frequency of selected ultrastructural aspects of RTL-W1 spheroids after a 4-day exposure to benzo(k)fluoranthene (BkF): C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF. Values range from 0 (undetected) to 3 (very frequent). Data are presented in boxplots as median, interquartile range, minimum and maximum. Different letters (a vs. b) illustrate significant differences between control and treated conditions (joined under horizontal brackets) according to a one-way ANOVA (Kruskal–Wallis) followed by Dwass–Steel–Critchlow–Fligner pairwise comparisons (p < 0.05).

Figure 7

Immunocytochemistry labelling of RTL-W1 spheroids with anti-CYP1A antibody after a 4-day exposure (from day 10 to day 14 in culture) to benzo(k)fluoranthene (BkF) at 18 °C and 23 °C.

Figure 8

Relative gene expression of detoxification target genes (CYP1A, CYP3A27, AhR, CAT, GST, UGT, MRP2, and BSEP) in RTL-W1 spheroids after 4 days of exposure (between days 10 and 14 in culture) to benzo(k)fluoranthene (BkF) at 18 °C and 23 °C: C—control; SC—solvent control; BkF10—10 nM of BkF; BkF100—100 nM of BkF. Data are presented in boxplots as median, mean (+), interquartile range, minimum and maximum, displayed by condition on the left panel and grouped by temperature on the right. Conditions (joined under horizontal brackets) not sharing a common letter (a or b) differ significantly according to a two-way ANOVA followed by Tukey’s test (p < 0.05). AhR—aryl hydrocarbon receptor; BSEP—bile salt export pump; CAT—catalase; CYP1A—cytochrome P450 1A; CYP3A27—cytochrome P450 3A27; GST—glutathione S-transferase; MRP2—multidrug resistance-associated protein; UGT—uridinediphosphate (UDP)–glucuronosyltransferase.