Monochloramine Impairs Caspase-3 Through Thiol Oxidation and Zn²⁺ Release

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 Zn²⁺. We explored the effects of the thiol oxidant monochloramine (NH₂Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH₂Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn²⁺ 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 NH₂Cl 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 NH₂Cl up to 100 μm, 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, NH₂Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH₂Cl-induced accumulation of Zn²⁺ in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH₂Cl, impair host pathways of apoptosis through release of Zn²⁺ from endogenous pools.