Testing the trout liver RTL-W1 cell line potential to study the influences of temperature on the effects of endocrine disruptors

Abstract

Global warming is an undeniable reality, and one consequence is a continuous increase in the temperature of different water bodies. This phenomenon affects numerous processes in the aquatic ecosystems, including the impacts of the widely present endocrine-disrupting chemicals (EDCs). Within EDCs, ethinylestradiol (EE2) and progestins, such as levonorgestrel (LEV), are commonly detected in environmental waters. It is thus imperative to study the effects of temperature changes in the toxicology of EDCs to aquatic animals, including fish. Endocrine disruption research often relies on in vitro models, complying with the 3Rs principles. Primary hepatocytes are commonly used but also are specific and well-characterized cell lines, such as the rainbow trout liver-derived RTL-W1. This cell line presents several functional features of differentiated hepatocytes. To test the cell line potential to study the way temperature models the effects of EDCs, we exposed RTL-W1 cells (72h) to EE2, LEV or a mixture of both synthetic hormones (at 10 μM). Exposures were carried out at 18 ºC and 21 ºC, in 24-well plates, 80.000 cells per well. The cells grew at either temperature. At the end of exposures, cells were trypsinized, and the viability measured using the trypan blue exclusion assay. Cell suspensions were then centrifuged at 200 rcf, for 5 min, and pellets were frozen at -80 ºC for gene expression analysis. No differences were found in cell viability among experimental groups. The expression of two CYP P450 enzymes enrolled in xenobiotic metabolism was analyzed by qRT-PCR. The Two-way ANOVA results revealed that CYP1A was affected by temperature as its expression was lower at 18ºC than at 21ºC. As to CYP3A27 expression, it was independent of temperature but was significantly higher in the EE2 + LEV mixture compared with control and solvent control conditions. Given these results, the RTL-W1 cell line seems like a promising model to study the interactions of temperature and exposure effects of EDCs. Studies are undergoing to include genes from other relevant signalling pathways such as estrogenic, progestogenic and lipid metabolism.