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)