No abstract

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...

No abstract

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91 phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47 phox . gp91 phox (Nox 2) and p47 phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47 phox , which was sensitive to inhibitors of sphingomyelinase and protein kinase C (PKC). These inhibitors and p47 phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKC are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKC, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr 845 and Tyr 1173 but not at Tyr 1045 . Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fasassociated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKC, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47 phox , Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase-and PKC-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.

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.

Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy.

Ultraviolet-B (UVB) (290 -320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiationinduced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40 -45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.