title: Rapid ecotoxicological testing using transformed BF-2 cells incorporating a luminescent reporter gene.
abstract: Since there is an ethical need to minimise the experimental use of higher organisms such as fish, especially those used in acute toxicity testing, fish cells are considered to be useful surrogates for fish in toxicity screening. The use of fish cell lines in conventional bioassays such as neutral red retention assays is however labour intensive, lengthy and costly. The use of luminescent reporter genes has been explored in our laboratory. In this study, a transfected BF-2 cell line (BF-2/luc1) was used for rapid toxicity testing on selected chemicals and results were compared with those obtained with in vivo fish testing and in vitro fish cell neutral red retention assays. The effect of temperature on the sensitivity of BF-2/luc1 was also investigated. BF-2/luc1 cells were harvested and suspended in PBS at 2.5-3.0x10(6)cells/ml. Individual aliquots of the suspended cells (40 microl each) were incubated for either 0.5 or 6 h at room temperature (22 degrees C) in the presence or absence of the toxicants. Bioluminescence was assayed using 17.5 microl Brightglo luciferase reagent which lysed the cells and provided the substrate luciferin. Luminescence was measured in a luminometer (Turner TD 20/20). The EC50 values obtained from BF-2/luc1 cells (0.5-6 h) generally compared well with the LC50 values (24-96 h) obtained from the in vivo fish tests on a range of species. The present study also showed that BF-2/luc1 cell sensitivity increased significantly when incubation temperature during toxicant exposure increased from 15 to 35 degrees C. The use of luminescent reporter genes in monitoring fish cells offers the possible advantages of increased sensitivity over the neutral red retention assay and a more rapid test to replace stain based bioassays, and provides a rapid screening method that could reduce the need for acute fish toxicity testing.
authors.affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk
authors.forename: T
authors.initials: T
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Wex
authors.forename: D M
authors.initials: DM
authors.lastname: Rawson
authors.forename: R S
authors.initials: RS
authors.lastname: Thompson
mesh_terms: D000818:Animals; D002460:Cell Line; D004396:Coloring Agents; D005399:Fishes / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics*; D007928:Lethal Dose 50; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D009499:Neutral Red; D010473:Perciformes; D010575:Pesticides / Q000633:toxicity; D013696:Temperature; D018675:Toxicity Tests / Q000379:methods*; D014162:Transfection; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D004396:Coloring Agents; D010575:Pesticides; D014874:Water Pollutants, Chemical; D009499:Neutral Red; D008156:Luciferases