Kinetics of reaction of nitrogen dioxide with dihydrorhodamine and the reaction of the dihydrorhodamine radical with oxygen: Implications for quantifying peroxynitrite formation in cells

Dihydrorhodamine 123 (RhH2) has been used to detect 'reactive nitrogen species', including peroxynitrite and its radical decomposition products, peroxynitrite probably oxidizing RhH2 to rhodamine (Rh) via radical products rather than directly. In this study, the radical intermediate (RhH center dot) was generated by pulse radiolysis, and shown to react with oxygen with a rate constant k similar to 7 x 10(8) M-1 s(-1). This fast reaction was exploited in experiments observing Rh being formed slowly (k similar to 4-7 x 10(5) M-1 s(-1)) from oxidation of RhH2 by nitrogen dioxide in a rate-limiting step, >1000-fold slower than the corresponding oxidation by carbonate radicals. The time-dependent uptake of RhH2 into mammalian cells was measured, with average intracellular levels reaching only similar to 10 mu M with the protocol used. The combination of low loading and relatively low reactivity of oxidants towards RhH2 compared to competing cellular nucleophiles suggests rather a small fraction of peroxynitrite-derived radicals (mainly CO3 center dot-) may be scavenged intracellularly by RhH2. (C) 2008 Elsevier Inc. All rights reserved.