The Critical Role of Hepes in SIN-1 Cytotoxicity, Peroxynitrite Versus Hydrogen Peroxide

The cytotoxicity of the superoxide anion radical- and nitric oxide-releasing compound SIN-1 to L929 cells was studied in Krebs-Henseleit buffer, pH 7.4, in the presence and absence of Hepes. SIN-1 cytotoxicity was significantly higher in the presence of Hepes than in the absence of Hepes. The available amount of peroxynitrite formed from SIN-1, however, was significantly decreased by Hepes as indicated by decreased oxidation of dihydrorhodamine 123. On the other hand, Hepes largely increased the formation of H2O2 from SIN-1. Catalase protected the L929 cells from SIN-1 cytotoxicity in the buffer with Hepes. In the buffer without Hepes catalase did not have any protective effect. In contrast, tyrosine and tryptophan provided significant protection against SIN-1 cytotoxicity independent of the presence of Hepes. These results demonstrate that the immediate toxic agent formed from SIN-1 decisively depends on the presence of Hepes. In its absence cytotoxicity is most likely mediated by peroxynitrite while in the presence of Hepes, cytotoxicity is conveyed by co-operative action of hydrogen peroxide and reactive nitrogen species.