THE CRITICAL ROLE OF HEPES IN SIN-1 CYTOTOXICITY, PEROXYNITRITE VERSUS HYDROGEN-PEROXIDE

The cytotoxicity of the superoxide anion radical-and nitric oxide-rele asing compound SIN-I to L929 cells was studied in Krebs-Henseleit buff er, pH 7.4, in the presence and absence of Hepes. SIN-I cytotoxicity w as significantly higher in the presence of Hepes than in the absence o f Hepes. The available amount of peroxynitrite formed from SIN-1, howe ver, was significantly decreased by Hepes as indicated by decreased ox idation of dihydrorhodamine 123. On the other hand, Hepes largely incr eased the formation of H2O2 from SIN-1. Catalase protected the L929 ce lls from SIN-1 cytotoxicity in the buffer with Hepes. In the buffer wi thout Hepes catalase did not have any protective effect. In contrast, tyrosine and tryptophan provided significant protection against SIN-I cytotoxicity independent of the presence of Hepes. These results demon strate that the immediate toxic agent formed from SIN-1 decisively dep ends on the presence of Hepes. In its absence cytotoxicity is most lik ely mediated by peroxynitrite while in the presence of Hepes, cytotoxi city is conveyed by co-operative action of hydrogen peroxide and react ive nitrogen species. (C) 1998 Elsevier Science Inc.