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.Oxidative phosphorylation in mitochondria is carried out by five protein complexes encoded by both the nuclear DNA and the mitochondrion's own genome, the mitochondrial (mt) 1 DNA. Mutations of mtDNA have been shown previously to play a role in a variety of degenerative diseases mainly affecting muscle and nerve tissues (1-3) as well as diseases such as familial diabetes mellitus (4). Their relevance is not restricted to degenerative diseases, however; e.g. mtDNA mutations are also critically involved in the normal aging process (5-8).The most frequent and best characterized mutation in mtDNA is a deletion of 4,977 base pairs in length, also called the common deletion. This common deletion is considered to be a marker for mutations in the mitochondrial genome, and substantial efforts have been made to elucidate the mechanism by which it is generated. A modified slip-replication mechanism has been proposed (9 -12) involving the misannealing of direct repeats (Scheme 1). Hotspots for the common deletion exhibit structural abnormalities facilitating the misannealing of direct repeats from the light to the heavy strand of the mtDNA (13). This then leads to loop formation of both the heavy and the light strand of mtDNA. The initiation of loop exclusion is thought to be mediated by reactive oxygen species (13,14).Reactive oxygen species can damage mtDNA (15-17), and damage by hydrogen peroxide is more extensive in mtDNA than in nuclear DNA (18). Furthermore it has been shown recently that increased oxidative stress is correlated to an altered mitochondrial function in vivo (19). In addition, oxidative stress induced by solar radiation may also be respon...

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 ؋ 10 6 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 M steady-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 peroxynitritemediated oxidations, as a peroxynitrite reductase.Peroxynitrite is a potent biological oxidant (1) generated, e.g. by endothelial cells, Kupffer cells, neutrophils, and macrophages (see Beckman (2) for review). Peroxynitrite (ONOO Ϫ ) is a relatively stable species compared with free radicals, but peroxynitrous acid (ONOOH) decays with a rate constant of 1.3 s Ϫ1

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation-and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and͞or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

Antioxidant activity of carotenoids in multilamellar liposomes assayed by inhibition of formation of thiobarbituric acid-reactive substances was in the ranking:Mixtures of carotenoids were more effective than the single compounds. This synergistic effect was most pronounced when lycopene or lutein was present. The superior protection of mixtures may be related to specific positioning of different carotenoids in membranes.z 1998 Federation of European Biochemical Societies.

M-3-G is poorly absorbed after a single ingestion of red wine, dealcoholized red wine, or red grape juice and seems to be differentially metabolized as compared to other red grape polyphenols. Our results suggest that not anthocyanins such as M-3-G themselves but rather not yet identified anthocyanin metabolites and/or other polyphenols in red wine might be responsible for the observed antioxidant and health effects in vivo in subjects consuming red wine.