A combination of fluorescent probes for evaluation of cytotoxicity and toxic mechanisms in isolated rainbow trout hepatocytes.
The toxicity of 17 chemicals to freshly isolated rainbow trout hepatocytes was determined using four different fluorescent probes measuring different endpoints. Calcein was used to determine viability of the cells, rhodamine 123 and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) to monitor mitochondrial activity and 5-chloromethylfluorescein diacetate (CMFDA) to monitor intracellular glutathione (GSH) levels. An EC(50) value 10 times lower than that obtained with the calcein assay was taken to indicate a specific action. Two sets of test chemicals were used. The first set contained five chemicals with a known specific toxic action and two chemicals without a known main specific action at a cellular level, and this set was used to examine the specificity of the probes. The second set consisted of 10 chemicals from the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) chemical list and included only one chemical with a known main specific action at a cellular level. In the first set of chemicals the CMFDA assay detected all chemicals with known GSH-depleting action, and the results obtained with the second set of chemicals indicated that none of the chemicals act mainly by GSH depletion. However, the CMFDA assay showed one false positive (antimycin-A). The results obtained with the rhodamine 123 and the JC-1 assay were less clear. The rhodamine 123 assay recognized the three chemicals in set 1 interfering with mitochondrial function but gave one false positive (ethanol) in this set, whereas the JC-1 assay recognized only two of the chemicals and gave one false positive (menadione). Both assays failed to detect the only chemical in set 2 with a known action on mitochondria (KCN). We conclude that calcein and CMFDA are good candidates as fluorometric probes for detection of cell viability and depletion of GSH, respectively. However, rhodamine 123 and JC-1, on the other hand, do not appear to be good probes for screening of mitochondrial activity in rainbow trout hepatocytes.
Lilius
H
H
Department of Biology, Abo Akademi University, BioCity, Artillerigatan 6, FIN-20520 Turku/Abo, Finland.
Hästbacka
T
T
Isomaa
B
B