In vitro studies of interactions of NO. donor drugs with superoxide and hydroxyl radicals.

ABSTRACT

Nitric oxide (NO.) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O2.-). This complex interaction may generate a peroxynitrite anion (ONOO-), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O2.- and hydroxyl (.OH) radical scavenging effects of various NO. donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO2), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO. in the incubation medium containing the different NO. donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO. that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O2.- and .OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC50 values expressed in the micromolar range, which decreased at low pH. Addition of SNP (800 microM) to solution containing MGD and Fe2+ (51) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)2-Fe2+-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO2 and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO.. NaNO2 displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO2, and molsidomine appeared to be closely related to their potential for NO release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO. formation in our conditions. The production of NO. was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO2. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of .OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O2.-, the NO. donor drugs/O2.- interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.