Cellular toxicity of sulfamethoxazole reactive metabolites—I: Inhibition of intracellular esterase activity prior to cell death

Abstract

Reactive metabolites produced by oxidative metabolism of the parent compound are considered responsible for the toxicity of a number of drugs, including idiosyncratic reactions to sulfonamide antibiotics. Using sulfamethoxazole hydroxylamine (SMX-HA) as a model compound, we report the use of a pH-sensitive fluorescent probe, 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), to identify early subcellular targets of chemically synthesized, toxic drug metabolites in peripheral blood mononuclear cells. When toxicity was assessed with this probe immediately after a 2-hr drug challenge, SMX-HA produced a concentration-dependent decrease in cellular fluorescence which was not accompanied by the development of compromised cell membrane integrity until 18 hr later. Dissipation of pH gradients across the cell membrane with nigericin and monensin demonstrated that decreased intracellular pH was only a small component of SMX-HA-induced toxicity. Loading cells with BCECF 30 min prior to SMX-HA challenge produced only a 3% decrease in cellular fluorescence at an SMX-HA concentration of 1 mM, whereas addition of BCECF after drug challenge resulted in a 71% decrease in fluorescence, consistent with a direct drug effect on cellular esterase activity. This was confirmed by monitoring BCECF cleavage in cell lysates in the presence and absence of SMX-HA. These studies demonstrate that inhibition of cellular esterase activity accounted for the observed loss of cellular fluorescence after drug exposure. Since changes in cellular fluorescence at 2 hr correlated well with cell death at 18 hr, we conclude that SMX-HA inhibition of intracellular esterase activity is an early event in the process that terminates in metabolite-induced cell death.