Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena

Abstract

Although ciliated protozoa such as Tetrahymena have many desirable properties as toxicological test organisms, their attributes would be better realized if multiple cultures could be simultaneously exposed to toxicants, quickly washed to terminate toxicant exposure, and conveniently evaluated for changes in cellular functions. Therefore, multiwell filter plates (MWFPs), manufactured primarily for biochemical applications, were used to expose Tetrahymena thermophila to copper, Triton X-100, and gliotoxin and compared to results of exposure in microcentrifuge tubes (MCTs). For MWFP, removal of toxicant solutions and retention of Tetrahymena in wells was done by placing plates on a manifold and applying pressure with a vacuum pump. Retained cells were resuspended in the same wells and their functions assessed with the fluorescent indicator dyes, Alamar blue to measure energy metabolism, and 5′-carboxyfluorescein diacetate acetoxymethyl ester to evaluate membrane integrity. For MCTs, exposures were terminated by centrifugation, and resuspended Tetrahymena were transferred to conventional multiwell plates for viability assessment with the same fluorescent dyes. Results were measured with a fluorescent multiwell plate reader and dose-response curves were obtained successfully with both procedures. However, MWFPs were much more convenient and rapid, potentially allowing 96 cultures to be processed at a time. Exposing Tetrahymena in MWFPs also allowed the ciliate and a rainbow trout gill cell line, RTgill-W1, to be compared for their sensitivity to toxicants under similar conditions of exposure and by common viability assays. Both cell systems showed toxic responses to Triton X-100 and copper at similar concentrations, but RTgill-W1 was more sensitive to gliotoxin.