Interactions between N-acetyl-p-benzoquinone imine and fluorescent calcium probes: implications for mechanistic toxicology.
Intracellular free calcium ([Ca2+]i) homeostasis has been implicated as an early target in both cellular necrosis and apoptosis. In this study, we have used peripheral blood mononuclear cells (PBMC) as target cells to investigate the effects of several reactive metabolites associated with drug toxicity on [Ca2+]i in order to delineate further early events in cytotoxicity. Compounds implicated in both drug-induced necrosis (N-acetyl-p-benzoquinone imine; NAPQI) and drug hypersensitivity (sulfamethoxazole hydroxylamine; SMX-HA) were examined and their effects on [Ca2+]i compared with those of the T cell mitogen phytohemagglutinin (PHA; 1.5 micrograms/ml) and the calcium ionophore ionomycin (2.5 microM). PHA and ionomycin produced characteristic elevations in [Ca2+]i as monitored by an increase in the fluorescence of fluo-3-loaded cells. SMX-HA did not significantly affect [Ca2+]i at concentrations previously shown to be cytotoxic to PBMC (100 and 500 microM), suggesting that Ca2+ homeostasis is not an early target for SMX-HA toxicity. Addition of NAPQI (250 microM) to fluo-3-loaded cells produced a marked decrease in fluorescence which was not reversed by ionomycin. Conversely, addition of NAPQI to cells loaded with indo-1 resulted in a rapid increase in fluorescence. This effect, however, was found to be attributable to NAPQI addition per se rather than to an increase in [Ca2+]i. HPLC and fluorescence analysis of samples generated from the decomposition of NAPQI revealed the presence of several products which fluoresced intensely at the excitation/emission wavelength pairs of a number of fluorescent probes commonly used to monitor [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
Riley
R J
RJ
Division of Clinical Pharmacology/Toxicology, Hospital for Sick Children, Toronto, Ontario, Canada.
Leeder
J S
JS
Dosch
H M
HM
Spielberg
S P
SP
D000814:Aniline Compounds; D016227:Benzoquinones / Q000378:metabolism* / Q000633:toxicity; D002118:Calcium / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007097:Imines / Q000378:metabolism* / Q000633:toxicity; D007211:Indoles; D007476:Ionophores; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008934:Mitogens; D013420:Sulfamethoxazole / Q000031:analogs & derivatives / Q000378:metabolism / Q000633:toxicity; D014966:Xanthenes
D000814:Aniline Compounds; D016227:Benzoquinones; D005456:Fluorescent Dyes; D007097:Imines; D007211:Indoles; D007476:Ionophores; D008934:Mitogens; D014966:Xanthenes; C059873:sulfamethoxazole hydroxylamine; C059715:Fluo-3; C028473:N-acetyl-4-benzoquinoneimine; D013420:Sulfamethoxazole; C048960:indo-1; D002118:Calcium