D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation.
Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.
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D002374:Catalase / Q000494:pharmacology; D002384:Catalysis; D003300:Copper / Q000494:pharmacology*; D004347:Drug Interactions; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D007962:Leukocytes / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000187:drug effects; D009504:Neutrophils / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D010396:Penicillamine / Q000378:metabolism / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism
D007166:Immunosuppressive Agents; D013481:Superoxides; D003300:Copper; D006861:Hydrogen Peroxide; D002374:Catalase; D010396:Penicillamine