Cultured rainbow trout gill epithelium as an in vitro method for marine ecosystem toxicological studies

Abstract

Accurate assessment of the toxic potential of waterborne chemicals is vital to pollution control and management in aquatic ecosystems. However, there is a global advocacy for the reduction, replacement, and refinement of the use of whole organisms in chemical screening studies. This has encouraged the development of alternative in vitro and computer-based techniques. In this study we investigated the possibility of optimising cultured rainbow trout gill epithelium to tolerate seawater and its use to assess toxicity of waterborne chemicals. Gill cells were obtained from rainbow trout acclimated to freshwater or to artificial seawater and were cultured in L-15 culture medium supplemented with or without cortisol. Intact gill epithelia were subjected to 20‰, 25‰ or 30‰ artificial seawater for 24 h and cell viability was assessed. The viability of gill cells obtained from freshwater or artificial seawater acclimated fish and grown without cortisol reduced to less than 80% compared to controls. The addition of cortisol to culture medium improved cell viability in seawater with 94%-95% viability compared to controls. The optimised gill cell epithelium was exposed to trace elements at concentrations previously reported as causing 50% response or mortality (EC/LC₅₀) using other cell-based and in vivo studies. Viability of the gill cells were compared to the 50% response or survival reported. The gill cells were found to be more sensitive than other isolated primary seawater-fish cells, having 5%, 16% and 37% survival on exposure to arsenic, cadmium, and lead, respectively. Results from this study has shown that cultured rainbow trout gill epithelia can be optimised to tolerate seawater and can be used in toxicological evaluations of pollutants resuspended in seawater, mimicking marine ecosystem conditions. The optimised gill cell system can serve as a viable in vitro method for marine ecosystem toxicological studies which would facilitate effective pollution control and management.

Keywords: Cell culture; Chemical speciation; Fish gill; In vitro; Marine pollution management; Oncorhynchus mykiss.

Figure 1

Change in Transepithelial Electrical Resistance (TER) of gill epithelium subjected to seawater. FW; Cells from freshwater acclimated fish, SW; Cells from seawater acclimated fish and FW + C; Cells from freshwater acclimated fish cultured in medium supplemented with cortisol. Bars represent the mean ± SD (experiment repeated three times, n = 6). Two-way analysis of variance done using GraphPad Prism 8.0. Asterisks indicate significant difference at p < 0.05 in TER of cells 24 h after being subjected to seawater.

Figure 2

Viability of cells subjected to seawater. FW; Cells from freshwater acclimated fish, SW; Cells from seawater acclimated fish and FW + C; Cells from freshwater acclimated fish cultured in medium supplemented with cortisol. Points represent the mean ± SD (experiment repeated three times, n = 6). Dotted line indicates viability above 90%. FW + C cells had consistent viability above 90% with little variation among replicates.