The effects of fluid shear stress, reduced serum supplementation, and a selection of chemicals on the Rainbow Trout gill cell line RTgill-W1

Abstract

Static, cell-based toxicity testing with the Rainbow trout gill cell-line, RTgill-W1 is a regulator-accepted proxy for fish acute toxicity testing. Yet, the static tests provide effect concentration data without considering fluid shear stress (FSS) present at the gill in vivo, or the adverse outcome pathway (AOP) of the test chemical. Additionally, pressure to cut use of foetal bovine serum in toxicity testing in line with reduction and replacement targets necessitates refinement of methodologies to include use of little or no animal derived serum. Fluid shear stress mediates mechanosensitive pathways responsible for uptake and resistance to toxicants. The first of two experimental phases in this study utilised custom 3D printed chambers housing cell culture inserts for the application of FSS (0.2 dynes cm2) over the cells in the presence and absence of (Cu). Expression of copper stress response genes (metallothionine a and b, atp7a, mtf-1, atox-1, sod1, sod2) and mechanosensitive cation channel, peizo1 together with reactive oxygen species production were measured for comparison of static and FSS exposure. The second study phase comprised measuring gene expression (vitellogenin, chop, znt1, cyp1a1) under benzo(a)pyrene, cadmium and clotrimazole exposures of 24 hours. In addition, RTgill-W1 cultures reduced to 1% FBS were tested against standard 5% cultures in 3,4-dichloroaniline (DCA) toxicity curves. Fluid shear stress increased the metabolic sensitivity of RTgill-W1 to copper and altered the metal responsive gene expression pattern. FSS significantly increased atp7a, peizo1 and sod expression together with ROS production. Gene expression analysis in RTgill-W1 alone was not predictive of adverse outcome pathways, particularly for PAHs owing to the lack of cyp1a1 activity in the cell line. However, responses of RTgill-W1 cultured in 1% FBS to DCA testing matched those of standard 5% FBS cultures. Overall, the results show FSS affects RTgill-W1 responses both alone and in the presence of copper and should be considered in future fish toxicity test replacement methodologies. The preliminary results from 1% FBS RTgill-W1 cultures indicate possibility of fast, simple reduction of animal product use in RTgill-W1 based toxicity tests, but further work is required to characterise the FBS-reduced cultures. Moreover, the addition of more cell lines and assays is necessary for a successful AOP prediction suite.

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ORCID for Nic Bury: orcid.org/0000-0001-6048-6338

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