There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 × 10⁶ m ⁻¹ s⁻¹. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of “classical” glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 μmsteady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.

Mutations of mitochondrial (mt) DNA accumulate during normal aging. The most frequent mutation is a 4,977-base pair deletion also called the common deletion, which is increased in photoaged skin. Oxidative stress may play a major role in the generation of large scale mtDNA deletions, but direct proof for this has been elusive. We therefore assessed whether the common deletion can be generated in vitro through UV irradiation and whether reactive oxygen species are involved in this process. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion employing a semiquantitative polymerase chain reaction technique. There was a time/dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide evidence for the involvement of reactive oxygen species in the generation of aging-associated mtDNA lesions in human cells and indicate a previously unrecognized role of singlet oxygen in photoaging of human skin.