The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes.
Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.
Jos
A
A
Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor García González 2, 41012 Seville, Spain. angelesjos@us.es
Cameán
A M
AM
Pflugmacher
S
S
Segner
H
H
D000818:Animals; D000975:Antioxidants / Q000032:analysis / Q000378:metabolism*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002347:Carps; D045744:Cell Line, Tumor; D002470:Cell Survival; D019327:Copper Sulfate / Q000633:toxicity; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity; D016877:Oxidants / Q000633:toxicity*; D010269:Paraquat / Q000633:toxicity; D010473:Perciformes; D011237:Predictive Value of Tests; D011720:Pyrazoles / Q000737:chemistry / Q000378:metabolism
C453681:7-amino-4-chloromethylcoumarin; D000975:Antioxidants; D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016877:Oxidants; D011720:Pyrazoles; C069306:5-chloromethylfluorescein; D019328:Buthionine Sulfoximine; C059597:monochlorobimane; D006861:Hydrogen Peroxide; D005978:Glutathione; D019327:Copper Sulfate; D010269:Paraquat