IN-VITRO STUDIES OF INTERACTIONS OF NO-CENTER-DOT DONOR DRUGS WITH SUPEROXIDE AND HYDROXYL RADICALS

Nitric oxide (NO .) is a free radical characterized by a high spontane ous chemical reactivity with many other molecules including the supero xide radical (O-2 .(-)). This complex interaction may generate a perox ynitrite anion (ONOO-), which behaves as an important mediator of oxid ative stress in many pathological states. In the present study, in vit ro experiments were performed to assess directly the O-2 .(-) and hydr oxyl (. 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 me dium containing the different NO . donor drugs were measured by the as say based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate ( MGD) with NO . that yields a stable spin-adduct measured by electron p aramagnetic resonance (EPR). O-2 .(-) and . OH generation was characte rized by EPR spin trapping techniques, using the spin trap 5,5-dimethy l-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer ad justed at pH 7.4, 7 and 6. Under these experimental conditions, SNP ex hibited the strongest superoxide scavenging properties, characterized by IC50 values expressed in the mu molar range, which decreased at low pH. Addition of SNP (800 mu M) to solution containing MGD and Fe2+ (5 :1) at pH 7 4 produced a three line EPR spectrum which is identified t o [(MGD)(2)-Fe2+-NO]. In control experiments no EPR signal was observe d. We obtained the same results with NaNO2 and an augmentation of the spin-adduct level was noted with the prolongation of the incubation pe riod. In return, molsidomine (2 mM) did not produce, in our conditions , a detectable production of NO .. NaNO2 displayed a significant super oxide scavenging effect only at pH 6, whilst neither molsidomine nor S IN 1 had any effect. Therefore, the superoxide scavenging properties o f SNP, NaNO2, and molsidomine appeared to be closely related to their potential for NO . release, which partially depends on the pH conditio ns. The behaviour of SW 1 is more complicated, the speed of oxygen dif fusion probably acting as a limiting factor in NO . formation in our c onditions. The production of NO . was detected in presence of SIN 1. T he intensity of the complex is comparable with the signal founded with NaNO2. By contrast, all molecules exhibited hydroxyl radical scavengi ng properties, highlighting the capacity of . OH to react with a wide range of molecules. In conclusion, considering the poor chemical react ivity of O-2 .(-), the NO . donor drugs/O-2 .(-) interactions suggest a special relationship between these two radical species, which, in ce rtain pathological states, could lead to the generation of cytotoxic e nd-products with strong oxidizing properties.