Creatine supplementation affords cytoprotection in oxidatively injured cultured mammalian cells via direct antioxidant activity

A growing body of evidence suggests that creatine (Cr) might exert protective effects in a variety of pathologies where oxidative stress plays a concausal etiologic role; furthermore, it has been recently reported that Cr displays direct antioxidant activity in a cell-free setting. However, at present, no research has been specifically aimed to directly test the antioxidant potential of Cr on oxidatively injured cultured cells. Here, the effects of Cr were studied using cultured human promonocytic (U937) and endothelial (HUVEC) cells, and murine myoblasts (C2C12) exposed to H2O2, tertbutylhydroperoxide (tB-0014) and, in the case of U937 cells, peroxynitrite. Cr (0.1-10 rnM) attenuated the cytotoxic effects caused by the oxidants in all the cell lines; under our conditions, cytoprotection was invariably associated with elevation of the intracellular fraction of Cr but it seemed to be unrelated to the levels of Cr phosphate (CrP); Cr did not affect the activity of catalase (CAT) and glutathione peroxidase (GpX), but it prevented H2O2- or tB-OOH-induced consumption of the nonprotein sulfhydryl (NPSH) pool in U937 and HUVEC cells; mass spectrometry experiments showed that a 136 MW molecule, which is likely to represent an oxidation by-product of Cr, formed in reaction buffers containing Cr and H2O2 as well as in cellular extracts from H2O2- or tB-OOH- treated Cr-preloaded U937 cells; finally, Cr cytoprotection appeared to be unrelated to chelation of Fe2+. In conclusion, it is suggested that Cr exerts a mild, although significant, antioxidant activity in living cells, via a mechanism depending on direct scavenging of reactive oxygen (in particular hydroxyl radical) and nitrogen species.