ADVANCING IN VITRO TECHNIQUES FOR ECOTOXCIOLOGICAL APPLICATION: Evaluation of a novel fish cell line co-culture system through PFAS-induced transcriptional responses

Abstract

Ecotoxicology is increasingly challenged by the demand for toxicological data on a growing range of environmental contaminants. At the same time, continued reliance on laboratory animals is limited by ethical considerations and regulatory principles. The development of novel cell-based methodologies, replacing and reducing the need for animal-based assays are therefore of vital importance for the continued progress and efficacy of ecotoxicological research and regulatory work. In this study a co-culture model combining the two rainbow trout cell lines RTgutGC and RTL-W1 has been established in a dual compartment system. The aim was to evaluate this novel co-culture model’s utility for future ecotoxicological applications. Following the establishment the model was exposed to perfluoroalkyl acids, most notably perfluorooctanoic acid (PFOA), and the transcriptional responses were recorded by real time quantitative PCR (qPCR). Additionally, a feeding regime in which rainbow trout was feed PFOA-spiked food was developed to enable comparisons to in vivo data. The study revealed both promising features as well as limitations for the in vitro model PFOA exposure induced significant transcriptional responses related to lipid metabolism and xenobiotic detoxification processes, both in the co-culture model and in monocultures of RTL-W1. However, more pronounced treatment related effects were found for the monoculture, suggesting a lower sensitivity in the co-cultured cells. The absence of transcriptional responses for RTL-W1 in the basolateral compartment indicated low toxicant exposure, likely explained by low permeability of the gut cell barrier above. Comparisons to the dietary exposure data was proven challenging, highlighting the complexity of in vitro to in vivo extrapolations. Interestingly, an elevated basal expression of CYP1A1 in the co-cultured RTL-W1 cells imply enhanced xenobiotic metabolic competence potentially triggered by the dual cell culturing environment. This finding shows potential for the model’s applicability as an improved in vitro model for mechanistic studies of toxicological processes related to biotransformation reactions.