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.

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.

A continuous myofibroblast precursor cell line from the tail muscle of Australasian snapper (Chrysophrys auratus) that responds to transforming growth factor beta and fibroblast growth factor.

Chrysophrys auratus (Australasian snapper) is one of the largest and most valuable finfish from capture fisheries in New Zealand, yet no cell lines from this species are reported in the scientific literature. Here, we describe a muscle-derived cell line initiated from the tail of a juvenile snapper which has been designated CAtmus1PFR (Chrysophrys auratus, tail muscle, Plant & Food Research). The cell line has been passaged over 100 times in 3 years and is considered immortal. Cells are reliant on serum supplementation for proliferation and exhibit a broad thermal profile comparable to the eurythermic nature of C. auratus in vivo. The impact of exogenous growth factors, including insulin-like growth factors I and II (IGF-I and IGF-II), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGFß), on cell morphology and proliferation was investigated. Insulin-like growth factors acted as mitogens and had minimal effect on cell morphology. TGFß exposure resulted in CAtmus1PFR exhibiting a myofibroblast morphology becoming enlarged with actin bundling. This differentiation was confirmed through the expression of smooth muscle actin (sma), an increase in type 1 collagen (col1a) expression, and a loss of motility. Expression of col1a and sma was decreased when cells were exposed to bFGF, and no actin bundling was observed. These data indicate that CAtmus1PFR may be myofibroblastic precursor cells descending from mesenchymal progenitor cells present in the tail muscle myosepta.

The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration.

Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.

Effects of dredging in Göteborg harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus).

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Göteborg harbor situated at the river Göta alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Göteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Göteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Göta alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.