Atlantic Salmon Gill Epithelial Cell Line (ASG-10) as a Suitable Model for Xenobiotic Biotransformation

Abstract

Fish are exposed to xenobiotics in the water. Uptake occurs mainly through the gills, which function as an exchange point with the environment. The gills’ ability to detoxify harmful compounds by biotransformation is an essential protection mechanism. The enormous numbers of waterborne xenobiotics requiring ecotoxicological assessment makes it necessary to replace in vivo fish studies with predictive in vitro models. Here, we have characterized the metabolic capacity of the ASG-10 gill epithelial cell line from Atlantic salmon. Inducible CYP1A expression was confirmed by enzymatic assays and immunoblotting. The activities of important cytochrome P450 (CYP) and uridine 5’-diphospho-glucuronosyltransferase (UGT) enzymes were established using specific substrates and metabolite analysis by liquid chromatography (LC) triple quadrupole mass spectrometry (TQMS). Metabolism of the fish anesthetic benzocaine (BZ) in ASG-10 confirmed esterase and acetyl transferase activities through the production of N-acetylbenzocaine (AcBZ), p-aminobenzoic acid (PABA) and p-acetaminobenzoic acid (AcPABA). Moreover, we were able to determine hydroxylamine benzocaine (BZOH), benzocaine glucuronide (BZGlcA) and hydroxylamine benzocaine glucuronide (BZ(O)GlcA) by LC high-resolution tandem mass spectrometry (HRMS/MS) fragment pattern analysis for the first time. Comparison to metabolite profiles in hepatic fractions, and in plasma of BZ-euthanized salmon, confirmed the suitability of the ASG-10 cell line for investigating biotransformation in gills.

Functional and molecular characterization of the Atlantic salmon gill epithelium cell line ASG-10; a tool for in vitro gill research

“…Clearly, the gill, having been sourced from a living organism producing a variety of metabolites and exposed to various environmental stressors had a different cyp expression profile compared with a cell line grown in stable conditions (FBS supplemented media). For example, while the cyp1A1 gene was expressed at a considerably lower level in the cell line (Log2fc −4.4), exposure of the ASG-10 cells to β-naphthoflavone, a known inducer of CYP1A ( Heinrich et al, 2014 ), induced both CYP1A protein expression and activity ( Ivanova et al, 2023 ). Also, the gill cell line LG-1 from Atlantic Lumpfish ( Cycloperus lumpus L) was found to have inducible CYP1A activity ( Sindre et al, 2021 ), while the gill cell line from rainbow trout (RTgill-W1) and Waking catfish ( Clarias batrachus ), GB1, had no detectable CYP1A activity after β-naphthoflavone treatment ( Franco et al, 2018 ).…”