Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology

Abstract

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4 °C) but replicated at high temperatures (30 °C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Keywords: 3,4 DCA; Australasian snapper; Chinook salmon; EROD; Epithelial; Toxicology.

Figure 1:

Gill cell lines CAgill1PFR and OTgill1PFR originated from (A) Chrysophrys auratus and (B) Oncorhynchus tshawytscha, respectively. Mechanical disruption of snapper gill tissue resulted in attachment of partial lamellae, with (C) epithelial-like cells or (D) fibroblast-like cells emerging. Enzymatic digestion of salmon gill tissue resulted initially in (E) attachment of single cells with various morphologies, which became primarily epithelial-like in early primary culture (F). Subconfluent (G) CAgill1PFR and (H) OTgill1PFR under routine culture conditions. Magnified culture in insert. Scale bars in C-H represent 100 µm.

Figure 2:

The impact of (A, D) basal media formulation, (B, E) temperature, and (C, F) FBS concentration on the metabolic activity of CAgill1PFR (A–C) and OTgill1PFR (D–F) was monitored via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), as a metric of cell proliferation, assay over a 10-day period. The data represent the means ± SEM of three independent experiments, each with three technical replicates.

Figure 3:

Immunocytochemistry targeting (A) pan cytokeratin showed fibrous cytoplasmic expression in both (i) CAgill1PFR and (ii) OTgill1PFR. (B) E-cadherin was detected at cell–cell junctions in (i) CAgill1PFR and (ii) OTgill1PFR. (C) Vimentin expression in (i) CAgill1PFR was visible as fibrous networks whereas expression in (ii) OTgill1PFR was more diffuse through the cytoplasm. Scale bars represent 100 µm.

Figure 4:

OTgill1PFR and CAgill1PFR were treated with increasing concentrations (0–100 mg/ L) of 3,4-dichloroaniline (3,4 DCA) for 24 h after which (A) cell metabolism was measured via Alamar blue, (B) membrane integrity was measured via CFDA-AM, and (C) lysosomal membrane integrity was measured by neutral red. The data represent the means ± SEM of three independent experiments, each with three technical replicates.

Figure 5:

Induction of CYP1A by beta-naphthoflavone in cultures of (A) CAgill1PFR and (B) OTgill1PFR. Cultures were exposed for 24 h to increasing beta-naphthoflavone concentrations (1 to 100 nM) and CYP1A levels measured as EROD activity. The data represent the means ± SD of three independent experiments, each with three technical replicates. DMSO, the carrier for beta-naphthoflavone, acts as a control. P < 0.05 by one-way ANOVA followed by Tukey’s least Significant Difference test. Means that do not share a letter are significantly different.