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.

Profiling the Physiological Roles in Fish Primary Cell Culture

“…It was reported that benzopyrene had the potential to induce oxidative stress in primary grouper liver cells and the oxidative effects of benzopyrene were assessed as a potential toxicology research technique [4]. Fish liver cell lines were also widely used in vitro models to study the adverse effects of pollutants in the marine environment [40]. The use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout was reported to measure the metabolism of pharmaceuticals using a substrate depletion assay.…”

Characterizing cytotoxic and estrogenic activity of Arctic char tissue extracts in primary Arctic char hepatocytes

“…Arctic char were collected and terminated in November 2015 with a blow to the head and subjected to a two-step liver perfusion as described in Tollefsen et al (2003) with minor modifications for Arctic char as described in Petersen et al (2017). Blood was removed from the liver by perfusion with a calcium free buffer (NaCl 122 mM, KCl 4.8 mM, MgSO4 1.2 mM, Na2HPO4 11mM, NaH2PO4 3.3 mM, NaHCO3 3.7 mM, EGTA 26 µM, 0°C) at 5 ml/min for 10-15 min.…”

“…Expression of Vtg gene and protein in male and juvenile fish has therefore become a suitable biomarker for (xeno)estrogenic compound exposure (Heppell et al, 1995;Mommsen and Walsh, 1988;Purdom et al, 1994). Synthesis of Vtg has also been used as a biomarker in primary cultures of hepatocytes from temperate fish such as common bream (Abramis brama), Siberian sturgeon (Acipenser baeri), Japanese eel (Anguilla japonica), channel catfish (Ictalurus punctatus), common carp (cyprinus carpio), rainbow trout (Oncorhynchus mykiss), Mozambique tilapia (Oreochromis mossambicus) (reviewed by Navas and Segner, 2006), and Atlantic salmon (Salmo salar) (Tollefsen et al, 2003), and in the recently established multi-endpoint and highthroughput in vitro bioassay with Arctic char hepatocytes for screening single chemicals, complex mixtures, and environmental extracts (Petersen et al, 2017).…”

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion

“…Two-step collagenase perfusion was originally described by Berry and Friend [22] for the rat liver, but Birnbaum et al [23] adapted it for the fish liver as well [24]. The authors Baksi and Frazier [22] and Pesonen and Andersson [21] revised the properties and usage of fish primary cell cultures and, even now, a considerable number of studies are still reporting the application of these cultures as a good alternative for the replacement of aquatic organisms [14,19,[25][26][27][28][29]. However, most of these studies used freshwater species models, and there is a lack of information regarding the isolation and culture conditions for marine fish hepatocytes.…”