Primary hepatocytes from Arctic char (Salvelinus alpinus) as a relevant Arctic in vitro model for screening contaminants and environmental extracts

Abstract

Contaminants find their way to the Arctic through long-range atmospheric transport, transport via ocean currents, and through increased anthropogenic activity. Some of the typical pollutants reaching the Arctic (PAHs, PCBs) are known to induce cytochrome P450 1a (CYP1A) protein expression and ethoxyresorufin-O-deethylase (EROD) activity through the aryl hydrocarbon receptor (AhR). In addition, some endocrine disrupting chemicals (EDCs) such as estrogen mimics (xenoestrogens) have been documented in Arctic areas and they may interfere with natural sexual development and reproduction. In vitro assays that are capable of detecting effects of such pollutants, covering multiple endpoints, are generally based on mammalian or temperate species and there are currently no well-characterized cell-based in vitro assays for effect assessment from Arctic fish species. The present study aimed to develop a high-throughput and multi-endpoint in vitro assay from Arctic char (Salvelinus alpinus) to provide a non-animal (alternative) testing method for an ecologically relevant Arctic species. A method for isolation and exposure of primary hepatocytes from Arctic char for studying the toxic effects and mode of action (MoA) of pollutants was applied and validated. The multi-versatility of the bioassay was assessed by classical biomarker responses such as cell viability (membrane integrity and metabolic activity), phase I detoxification (CYP1A protein expression, EROD activity) and estrogen receptor (ER) mediated vitellogenin (Vtg) protein expression using a selection of model compounds, environmental pollutants and an environmental extract containing a complex mixture of pollutants. Primary hepatocytes from Arctic char were successfully isolated and culture conditions optimized to identify the most optimal assay conditions for covering multiple endpoints. The hepatocytes responded with concentration-dependent responses to all of the model compounds, most of the environmental pollutants and the environmental sample tested. The bioassay response and sensitivity of the hepatocytes from Arctic char differed slightly from closely related salmonid species, thus highlighting the need for developing in vitro assays relevant for Arctic species. The present multi-endpoint in vitro assay offer a highly versatile tool to screen potential effects of pollutants and complex samples relevant for Arctic exposure scenarios.

Keywords: Arctic char; CYP1A; EROD; In vitro; Primary hepatocytes; Vitellogenin.