SUPEROXIDE SCAVENGING ACTIVITY IN LEUKOCYTES AND ABSENCE OF CELLULAR TOXICITY OF A SERIES OF COUMARINS

Sixteen synthetic or plant-derived coumarins of dietary importance wit h different patterns of substitution were tested for their capacity to scavenge superoxide and for their cytotoxicity. Superoxide was genera ted by human polymorphonuclear leukocytes stimulated by phorbol myrist ate acetate and was measured using the reduction of ferricytochrome c or of nitroblue tetrazolium (NBT). Eleven of the coumarins, all lackin g dihydroxy substitution, did not scavenge superoxide. Of the remainin g five, the most potent scavenger was fraxetin (7,8-dihydroxy-6-methox ycoumarin) with an IC50 (concentration producing 50% inhibition) of 2. 3 mu M in the cytochrome assay and 5.8 mu M using NBT. The other four coumarins (all containing ortho-dihydroxy catechol functions, and foun d previously to be pro-oxidant in cell-free systems by virtue of reduc tion of ferric to ferrous ions), themselves rapidly reduced cytochrome c. Therefore their effects on superoxide were measured using NBT, yie lding IC50 values in the range 8.5 to 82.0 mu M. Fraxetin and the othe r active and inactive coumarins were not directly cytotoxic at 100 mu M to leukocytes or to erythrocytes, as shown by their failure to cause release of cytosolic lactate dehydrogenase or to cause haemolysis, re spectively. However, all five dihydroxylated pro-oxidant coumarins wer e toxic to NS20Y neuroblastoma cells in 24 hr culture, whereas the oth er eleven coumarins were nontoxic. We conclude that 7,8-dihydroxylated coumarins such as fraxetin are agents which are not themselves direct ly cytotoxic and are capable of direct scavenging of superoxide anion radicals, an action which might be protective at sites of leukocyte ac tivation during inflammation. However, in the presence of free ferric ions they may exert potentially damaging pro-oxidant actions, includin g cytotoxicity. This series of compounds provides a useful basis for s tructure-activity studies designed to achieve separation or combinatio n of these properties.