GENERATION OF REACTIVE OXYGEN SPECIES BY THE REDOX CYCLING OF NITROPRUSSIDE

The formation of reactive oxygen species during the redox cycling of s odium nitroprusside by rat liver microsomes and by chemical reductants was evaluated. The reduction of sodium nitroprusside by ascorbate and glutathione results in formation of the nitroprusside nitroxide radic al which, on freezing at 77 K, results exclusively in the tetracyano [ Fe(CN4)NO](2-) and the pentacyano [Fe(CN5)NO](3-) forms of nitroxide r adicals, respectively. The role of reducing agents on the inter-conver sion of these two forms of nitroxide radical is discussed. The NADH an d NADPH dependent microsomal reduction of nitroprusside results in the production of nitroprusside nitroxide radical, which in the presence of oxygen undergoes redox cycling to generate superoxide radical, and eventually hydroxyl radical is formed by a Fenton-type of reaction. St udies on the effect of several biologically or toxicologically relevan t iron chelators on NADPH-dependent microsomal reduction of nitropruss ide and subsequent formation of hydroxyl radical indicate that certain iron chelators such as isocitrate act as hydroxyl radical scavengers (depending on its concentration), but other chelators such as EDTA and DTPA function as good catalysts for the generation of hydroxyl radica ls. The NADH and nitroprusside dependent microsomal production of hydr oxyl radical is better in the presence of ATP, or equal in the presenc e of acetate, or diminished in the presence of DTPA when compared to t he NADPH- and nitroprusside-dependent microsomal production of hydroxy l radicals, The effect of these chelates on the redox cycling of iron and nitroprusside by microsomes is discussed. Rat liver sub-mitochondr ial particles and human hepatoblastoma cells (HepG2 cell line) also ge nerated superoxide and hydroxyl radicals during the redox cycling of n itroprusside. These results provide direct evidence for the production of reactive oxygen species during the redox cycling of nitroprusside. The use of nitroprusside as a nitric oxide donor in biological system s may be complicated by the necessity to consider the generation of re active oxygen species due to redox cycling of this compound by cellula r reductases and low-molecular weight reductants.