A comparative study of the toxicity of chemically reactive xenobiotics towards adherent cell cultures: selective attenuation of menadione toxicity by buthionine sulphoximine pretreatment.
Metabolic activation to reactive intermediates is a prerequisite for many forms of chemically-induced toxicity. Hepa 1c1c-9 cells were exposed to varying concentrations of several reactive metabolites implicated in adverse drug reactions and the toxicity of the compounds assessed using applied fluorescence technology. Cytotoxicity was assessed using the fluorescence of 2', 7'-bis-(2-carboxyethyl)-5-(6)-carboxy-fluorescein as an index of cell viability. The role of glutathione in cellular defence against these chemicals was investigated by pretreating the target cells overnight with buthionine sulphoximine, a specific inhibitor of glutathione synthesis. Depletion of intracellular glutathione augmented the toxicity of N-acetyl-p-benzoquinone imine (1.5-3-fold at 100 and 10 microM). Toxicity produced by the hydroxylamine of sulphamethoxazole (500 microM) was dependent entirely on pretreatment of the cells with buthionine sulphoximine (% cell death = 33 +/- 16 compared with 0 +/- 4 in untreated cells, P < 0.05). By contrast, the lethal effects of the model quinone, menadione, were attenuated markedly following glutathione depletion. The data obtained suggest that this assay, previously used with suspension cultures, may be useful in the rapid in-vitro screening of putative reactive intermediates. Moreover, the application of such methodology should prove beneficial for the elucidation of cellular mechanisms of defence and detoxification.
Riley
R J
RJ
Department of Paediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Spielberg
S P
SP
Leeder
J S
JS
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D016227:Benzoquinones / Q000633:toxicity; D019328:Buthionine Sulfoximine; D002448:Cell Adhesion; D002470:Cell Survival / Q000187:drug effects*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005978:Glutathione / Q000378:metabolism*; D007097:Imines / Q000633:toxicity; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000633:toxicity*
D000963:Antimetabolites; D016227:Benzoquinones; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007097:Imines; D014812:Vitamin K; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C028473:N-acetyl-4-benzoquinoneimine; D005978:Glutathione