Monochloramine impairs caspase-3 through thiol oxidation and Zn2+ release.

Abstract

BACKGROUND: Caspase-3, a pro-apoptotic enzyme, represents a class of proteins in which the active site contains reduced thiol (S-H) groups and is modulated by heavy metal cations, such as Zn(2+). We explored the effects of the thiol oxidant monochloramine (NH(2)Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH(2)Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn(2+) within the cytoplasm. MATERIALS AND METHODS: Isolated gastric glands were prepared from rabbit mucosa by collagenase digestion. Caspase-3 activity was measured colorimetrically in suspensions of healthy rabbit gastric glands, following exposure to various concentrations of NH(2)Cl with or without the zinc chelator TPEN [tetrakis-(2-pyridylmethyl)ethylene diamine] for 1 h, and re-equilibration in Ringer's solution for 5 h. Conversion of procaspase-3 to active caspase-3 was monitored by Western blot. RESULTS: Monochloramine inhibited caspase-3 activity in a dose-dependent fashion. At concentrations of NH(2)Cl up to 100 microM, these effects were prevented if TPEN was given concurrently and were partly reversed if TPEN was given 1 h later. Caspase-3 activity was preserved by concurrent treatment with a thiol-reducing agent, dithiothreitol. CONCLUSIONS: At pathologically relevant concentrations, NH(2)Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH(2)Cl-induced accumulation of Zn(2+) in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH(2)Cl, impair host pathways of apoptosis through release of Zn(2+) from endogenous pools.