Copper Binding to Fish Gill Cell Lines: Towards Determination of Biotic Ligand Model Parameters

In aquatic environments, copper is a potentially toxic element that is also an essential nutrient, aiding in the growth and development of all aerobic organisms. In small quantities, copper acts as a micronutrient, playing a large role in many enzymatic reactions, however, at increased concentrations, copper is a potential aquatic pollutant that can cause metal toxicity. The biotic ligand model (BLM) is a useful tool that is often used for metals risk assessment and helps to establish protective water criteria guidelines. Previously, the BLM has been used to model metal complexation directly at the biotic ligand (e.g., gill) for both whole organisms and primary gill cells but has not yet been used to model metal complexation directly for gill cells from a continuous cell culture. Cell viability curves, using two fluorescence indicator dyes, Alamar Blue and CFDA-AM, were generated using both the RTgill-W1 and LSgill-e cell lines to determine what concentrations of copper (II) sulfate induces cell toxicity. These tests confirmed the titratable range for both cell lines in order to maintain viability was 0-300 µM in L-15, a specific culture media. For this study, graphite furnace atomic absorption spectroscopy (GFAAS) was used to directly characterize copper binding to both a rainbow trout gill cell line (RTgill-W1) and a lake sturgeon gill cell line (LSgill-e) in a monolayer, in suspension, and in cell culture inserts where copper titrations were performed at non-toxic concentrations. Both cell lines showed weaker binding than primary rainbow trout gill pavement cells and up to an order of magnitude less binding than primary rainbow trout gill mitochondrial rich cells. Results suggest that the interaction of copper with cells derived from a continuous cell line is much weaker than interactions with cells from a primary culture. Based on research performed, these results suggest the use of RTgill-W1, or LSgill-e cell lines would most likely not suffice as an alternative to whole organism and primary cell testing for viability and toxicity assays