Singlet Oxygen Mediates the UVA-induced Generation of the Photoaging-associated Mitochondrial Common Deletion

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...

Real-time PCR analysis of a 3895 bp mitochondrial DNA deletion in nonmelanoma skin cancer and its use as a quantitative marker for sunlight exposure in human skin

“…Furthermore, we and others have shown that mtDNA deletions can be induced in cultured human skin cells by sublethal repetitive doses of UVA (Berneburg et al, 1999;Krishnan et al, 2004). In agreement with observations of mtDNA damage in other tumours (Birch-Machin, 2006;Czarnecka et al, 2006;Parr et al, 2006) we have previously identified somatic mtDNA changes in human skin as a result of the first detailed study of mtDNA damage in NMSC (Durham et al, 2002).…”

Sequence-specific DNA damage by reactive oxygen species: Implications for carcinogenesis and aging

“…formation can cause DNA misreplication that may lead to mutation such as G•C→T•A transversion, and carcinogenesis (36,50).…”

“…Increasing evidence demonstrates that UVA, as well as UVB, contributes to photoaging (36). The molecular mechanisms of photodamage by UVA, the sunlight's major ultraviolet constituent, are poorly understood.…”

Photoaging of human skin

“…To explain the generation of these largescale deletions in mtDNA, a modified slip-replication mechanism and a central role of ROS have been postulated (39)(40)(41). Recent work has been able to provide a possible link for the involvement of ROS in the generation of the most frequent mtDNA deletion, the so-called common deletion (42). Employing an in vitro model system, it has been possible to demonstrate that normal human fibroblasts when repetitively exposed for 3 weeks to sublethal doses of UVA light exhibit a time-and dose-dependent increase of the common deletion.…”