Singlet Oxygen Mediates the UVA-induced Generation of the
Photoaging-associated Mitochondrial Common Deletion*
Mark
Berneburg
,
Susanne
Grether-Beck
,
Viola
Kürten
,
Thomas
Ruzicka
,
Karlis
Briviba§,
Helmut
Sies§, and
Jean
Krutmann
¶
From the
Clinical and Experimental Photodermatology,
Department of Dermatology, § Institute for Physiological
Chemistry I, Heinrich-Heine-University of Düsseldorf,
Moorenstrasse 5, D-40225 Düsseldorf, Germany
 |
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.
 |
INTRODUCTION |
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).

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Scheme 1.
Model for generation of common deletion via
UVA-induced singlet oxygen (modified from Shoffner et
al. (9)). A, replication of mtDNA commences
at the origin of the heavy strand (OH)
separating the light from the heavy strand. Abbreviations used are:
DR1, direct repeat 1 (empty box); DR2,
direct repeat 2 (solid box); OL,
origin of light strand replication. B, DR1 of the heavy
strand and DR2 of the light strand misanneal generating a downstream
loop of the heavy strand. Due to a high content of guanosines it is
particularly susceptible to strand breaks generated by UVA irradiation
or other sources of singlet oxygen. C, after
degradation of the excluded loop and ligation of the free ends of the
heavy strand, replication can be completed, leading to a normal
(left) and a deleted (right) mtDNA
molecule.
|
|
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 responsible for the increased frequency of mtDNA mutations in
photoaged human skin (14, 20-22). Evidence for a direct link between
reactive oxygen species and the generation of large deletions of the
mitochondrial genome, however, has not yet been provided.
In the present study we demonstrate that the common deletion can be
generated under defined conditions in human dermal fibroblasts through
repetitive UVA irradiation. By employing this in vitro model
we provide evidence that UVA radiation-induced mtDNA deletions are
caused by the generation of singlet oxygen.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Human normal skin fibroblasts were cultured in
Eagle's minimum essential medium (Life Technologies GmbH, Eggenstein,
Germany) containing 15% fetal calf serum (Greiner, Frickenhausen,
Germany), 0.1% L-glutamine, 2.5% NaHCO3, and
1% streptomycin/amphotericin B in a humidified atmosphere containing
5% CO2. Cells were kept in 10-cm culture dishes for
culture and irradiation.
Generation of the Common Deletion by UVA Irradiation--
For
UVA irradiation, medium was replaced by PBS, lids removed, and cells
were exposed to radiation from a UVASUN 5000 Biomed irradiation device
(Mutzhas, Munich, Germany). The emission was filtered with UVACRYL
(Mutzhas) and UG1 (Schott Glaswerke, Munich, Germany) and consisted of
wavelengths greater than 340 nm. The UVA output was determined with a
UVAMETER (Mutzhas) and found to be approximately 70 milliwatts/cm2 UVA at a tube-to-target distance of 30 cm.
In order to generate the common deletion, cells were irradiated three
times daily with 8 J/cm2 UVA for 4 consecutive days and
checked for viability by trypan blue exclusion. Cells were then
aliquoted 1:1 with one aliquot stored at
80 °C until extraction of
mtDNA. The other aliquot was plated to a 10-cm culture dish for ongoing
culture and irradiation as indicated.
Chemical Treatments and Singlet Oxygen Generation--
All
chemicals were purchased from Sigma except for sodium azide (Merck) and
applied as described previously (23). Vitamin E (as
-tocopheryl
succinate) was dissolved in ethanol and added to cells 24 h before
irradiation at a concentration of 25 µM. For irradiation
in the presence of heavy water, deuterium oxide (99.9 atom % 2H) was used at a final concentration of 95% in PBS (24).
Singlet oxygen was generated by thermal decomposition of the
endoperoxide of the disodium salt of
3,3'-(1,4-naphthylidene)dipropionate (NDPO2). Cells
were incubated with 0.3 mM NDPO2 in PBS for
1 h in the dark at 37 °C, which yielded singlet molecular
oxygen and 3,3'-(1,4-naphthylidene)dipropionate (NDP) (25).
This singlet oxygen system was shown to be well suited for use in cell
cultures, because it is water-soluble and nontoxic for these cells up
to 40 mM for 1-h incubation (24). Infrared emission of
singlet oxygen was measured with a liquid nitrogen-cooled germanium
photodiode detector (model EO-817L, North Coast Scientific, Santa Rosa,
CA) as described previously (25). The rate of singlet oxygen generation
was monitored by the formation of NDP. Fifteen minutes after addition
of NDPO2, the rate of singlet oxygen generation was 1 µM/min.
DNA Extraction--
Total cellular DNA was extracted from normal
human fibroblasts employing the QIAamp Tissue Kit (Qiagen, Hilden, Germany).
PCR Analysis--
For estimation of mtDNA content, PCR with
primers C1 and C2 was carried out, amplifying a product of 247 base
pairs in length. Amplification of fragments representing the common
deletion was carried out as described previously (20, 26). In brief,
primer oligonucleotides (A1/A2) were designed to anneal outside the
common deletion. During DNA amplification the polymerase extension time was designed to be too short for the amplification of wild-type PCR-products only allowing the efficient amplification of the shorter
and deleted mtDNA-fragments. To increase sensitivity and specificity a
secondary, nested PCR was performed (B1/B2) from the primary PCR product.
Linear amplification conditions for each primer pair used were
determined as described in detail previously (27). Amplification was
found to be linear up to 31 cycles for primers C1/C2 and A1/A2 and up
to 30 cycles for B1/B2. Therefore primary PCR (primers A1/A2 and C1/C2)
was carried out in 100-µl reaction volume with 0.1-0.3 µg of
genomic DNA and 0.5 unit of Taq polymerase. Primer and
nucleotide concentrations were 1 and 400 µM,
respectively. For PCR, initial denaturation (94 °C, 4 min) was
followed by 28 cycles of denaturation (94 °C, 1 min), annealing
(58 °C, 1 min), and extension (72 °C, 45 s). A 2-µl
aliquot of the primary reaction was entered into reamplification
(B1/B2) with the same PCR conditions except concentration of dNTPs (40 µM/dNTP), an annealing temperature of 68 °C, and a
further decrease in the extension time to 30 s. Polymerase chain
reaction was carried out in a Perkin-Elmer DNA Thermocycler 480 (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany), primer
oligonucleotides were generated by MWG-Biotech (Ebersberg, Germany).
Primer Oligonucleotides--
The following sequences and
nucleotide positions (shown in parentheses) are according to Anderson
et al. (28): A1, 5' GCA GTA ATA TTA ATAATT TTC ATG 3'
(7293-7316); A2, 5' CTA GGG TAG AAT CCG AGT ATG TTG 3' (13928-13905);
B1, 5' TGA ACC TAC GAG TAC ACC GA 3' (7901-7920); B2, 5' GGG GAA GCG
AGG TTG ACC TG 3' (13650-13631); C1, 5' ATG CTT GTA GGA CAT AAT AA 3'
(219-238); C2, 5' AGT GGG AGG GGA AAA TAA TA3' (466-447).
Amplification products were visualized in a 1% agarose gel stained
with ethidium bromide (0.25 µg/ml).
Quantification by Ion-Exchange Chromatography--
Products were
quantified by ion-exchange chromatography connected to an on-line
ultraviolet spectrophotometer (Gynkotek, Germering, Germany), which
allowed exact quantification of amplification products at 260 nm (27,
29), and quantification of deleted mtDNA was carried out as described
previously (20). The amplification product generated by primer pair C1
and C2 represents a segment of the mtDNA (30), carrying no known
mutations of the mtDNA. Thus, it served as a reference fragment
representing the overall amount of mtDNA molecules present in the
cultured cells. Values determined for PCR products representing the
common deletion were normalized to values of the reference fragments of
the same cell lines.
Restriction Enzyme Analysis--
To confirm their identity, PCR
products were subjected to diagnostic digestion with the restriction
enzyme XbaI (New England Biolabs GmbH, Schwalbach, Germany).
 |
RESULTS |
Generation of the Common Deletion in Normal Human Fibroblasts by
Repetitive UVA Irradiation--
In order to determine the maximal UVA
dose that allows sublethal repetitive irradiation, normal human
fibroblasts were exposed to UVA doses of 0, 4, 8, and 16 J/cm2 three times daily (Fig.
1). Repetitive irradiation with 16 J/cm2 induced cell death, whereas irradiation with doses of
0, 4, and 8 J/cm2 had no effect on cell viability. In the
subsequent experiments cells were therefore exposed to repetitive doses
of 8 J/cm2.

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Fig. 1.
Viability of normal human fibroblasts after
repetitive UVA irradiation. Normal human fibroblasts were
irradiated for 12, 24, and 36 times at 0 ( ), 4 ( ), 8 ( ), and
16 ( ) J/cm2. After each interval cell viability was
assessed by trypan blue exclusion before and after irradiation and
compared with sham-irradiated cells. Cell numbers ranged between 1.5 and 5.0 × 106/dish in all samples and only decreased
significantly when irradiated at 16 J/cm2.
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Amplification of total DNA extracts from normal human fibroblasts with
primer oligonucleotides C1/C2 by PCR yielded the expected fragment of
247 base pairs in all experiments. The amplification of this fragment
confirmed the effective extraction of mtDNA and served as a reference
fragment for subsequent quantification (Fig. 2a). Nested PCR of fibroblasts
exposed to repetitive UVA irradiations followed by ion-exchange
chromatography showed no signal for the common deletion after 12 irradiations. Exposure of cells to 24 irradiations yielded the first
detectable signal (Fig. 2, b and c), and maximal
induction was observed after an irradiation of cells for 36 times.
Sham-irradiated control cells showed no increase of the common deletion
at any of the irradiation intervals. Digestion of PCR products with the
restriction enzyme XbaI confirmed the identity of the
amplified fragments (data not shown).

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Fig. 2.
UVA radiation generates the common deletion
in human fibroblasts. Cells were irradiated with 8 J/cm2 of UVA 12, 24, and 36 times. Total cellular DNA was
extracted and subjected to PCR, amplifying either the reference
fragment representing the total mitochondrial genome or the common
deletion. Positive control represents a sample of a patient with known
disease caused by the common deletion. Sham-irradiated cells were
treated identically with regard to medium change and passaging. Data
are given as mean ± S.D. of relative content of the common
deletion of three experiments and plotted in arbitrary units.
a, representative agarose gel of PCR amplifications of the
reference fragment. b, representative agarose gel of PCR
products representing the common deletion. c,
quantification by ion-exchange chromatography. Values of deleted mtDNA
molecules were normalized for total amount of mtDNA molecules
(reference fragment).
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|
Role of Singlet Oxygen in the Generation of the Common
Deletion--
The role of singlet oxygen as a mediator of UVA
radiation effects has been shown for several systems (31, 32).
Therefore, in the present study, reagents capable of quenching (sodium
azide, vitamin E) or enhancing (deuterium oxide) singlet oxygen effects were examined for their capacity to affect UVA radiation-induced generation of the common deletion. As shown in Fig.
3, repetitive irradiation of fibroblasts
in the presence of sodium azide significantly suppressed the induction
of the common deletion in a dose-dependent manner.
Conversely, suppression of UVA-mediated mtDNA mutagenesis was also
observed when cells were treated with vitamin E during UVA radiation
exposure. Moreover, irradiation of normal fibroblasts in the presence
of deuterium oxide (D2O) resulted in a slight but
consistent increase of signal intensity for the common deletion when
compared with cells exposed to UVA radiation alone (Fig. 4).

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Fig. 3.
Suppression of UVA-mediated mtDNA mutagenesis
by sodium azide or vitamin E. Cells were exposed to 8 J/cm2 of UVA for 12, 24, and 36 times in the absence or
presence of NaN3 (20 and 50 mM) and vitamin E
(25 µM). In order to assure diffusion into cells and
hydrolysis by esterases, cells were pretreated with vitamin E 24 h
prior to UVA irradiation. For subsequent irradiations vitamin E was
present continuously. The positive control represents a sample of a
patient with known disease caused by the common deletion.
Sham-irradiated cells were treated identically with regard to medium
change and passaging. Data are given as mean ± S.D. of relative
content of the common deletion of three experiments and are plotted in
arbitrary units. a, representative agarose gel of PCR
products representing the common deletion. b, quantification
by ion-exchange chromatography.
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Fig. 4.
Singlet oxygen mediation of UVA effect.
Coincubation of irradiated cells with D2O: for UVA
irradiation of fibroblasts, in the presence of heavy water, medium was
replaced with deuterium oxide (99.9 atom % 2H) at a final
concentration of 95% in PBS. After irradiation, D2O was
removed again, and cells were kept in normal medium. Generation of
singlet oxygen by NDPO2: unirradiated cells were treated with singlet
oxygen generated by thermal decomposition of the endoperoxide of the
disodium salt of NDPO2, 0.3 mM in PBS for
1 h in the dark at 37 °C, and yielded singlet molecular oxygen
and NDP. The rate of singlet oxygen generation was monitored by the
formation of NDP. Fifteen min after addition of NDPO2, the
steady-state rate of singlet oxygen generation was 1 µM/min. Control cells were stimulated with NDP, which had
been generated by thermal decomposition from the same batch of
NDPO2 used in these same experiments. Data are given as
mean ± S.D. of relative content of the common deletion of three
experiments, and error bars are given where applicable.
a, representative agarose gel of PCR products
representing the common deletion. b, quantification by
ion-exchange chromatography.
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|
The capacity of sodium azide and vitamin E to suppress and of deuterium
oxide to enhance the UVA radiation-induced generation of the common
deletion indicated a prominent role for singlet oxygen in this system.
Therefore it was assessed whether these UVA radiation-induced effects
could be mimicked by stimulating unirradiated cells with singlet oxygen
generated via thermal decomposition of NDPO2 (23, 25). As
shown in Fig. 4, incubation of cells with NDPO2 led to the
induction of the common deletion in normal human fibroblasts parallel
to the observed UVA induced mutagenesis, whereas incubation with NDP
did not generate a signal for the common deletion.
 |
DISCUSSION |
Generation of the Common Deletion in Vitro with Physiological Doses
of UVA--
Large scale deletions of mitochondrial DNA such as the
common deletion are thought to play a central role in aging of human tissues. Analysis of the mechanism by which the common deletion is
generated in human cells during the normal aging process would require
an assay system allowing the generation of mitochondrial mutations
under standardized conditions and in a reproducible manner by exposing
relevant target cells to physiologically occurring oxidative stress. In
the present study we have therefore assessed whether it is possible to
generate mitochondrial DNA mutations in cultured human dermal
fibroblasts by exposing them in vitro to UVA radiation.
The assay system is based on the following. Chronically sun-exposed
human skin has a higher content of mtDNA mutations than sun-protected
skin, and a role for mtDNA mutations in photoaging of human skin has
been proposed (14, 20, 22). Comparative analysis of the epidermal
versus dermal compartment of photoaged skin revealed that
the common deletion is almost exclusively present in dermal cells (14).
Thus, in the present study, dermal fibroblasts were used and exposed to
UVA radiation, which can penetrate into the dermis at significant dose
levels. The UVA radiation doses employed for in vitro
irradiation of fibroblasts are of physiological relevance, since they
are similar to those administered to human skin during the course of a
15-30-min sun exposure on a summer day at noon at the northern
latitude of 30-35° (33). Human cells are capable of repairing damage
to mtDNA induced by reactive oxygen species (34, 35), and therefore an
irradiation regimen has been developed that shifts the steady-state in
cells between irradiation-induced damage and ongoing repair toward the
damage side. By employing this protocol, it has indeed been possible to
reproducibly generate the common deletion in human fibroblasts under
standard conditions (Fig. 2).
A possible explanation for this could be that cells harboring the
common deletion show a selective growth advantage, thus leading to
increasing amounts of this deletion. It has been described, however,
that there are selection mechanisms in cells containing large scale
mtDNA deletions that lead to a growth disadvantage rather than an a
growth advantage (26, 36). Therefore it is very unlikely that the
observed increase of the common deletion is simply due to differences
in growth. Furthermore, the induction of the common deletion was a
function of the number of UVA radiation exposures given to cells,
indicating that it resulted from cumulative photodamage. We therefore
propose that UVA radiation-induced generation of the common deletion in
human dermal fibroblasts, as described here, represents a novel
in vitro model to study the mechanism (i) by which the
common deletion is generated in human cells in general and (ii) by
which UVA radiation contributes to aging in particular.
Single Strand Breaks by Singlet Oxygen--
It has been proposed
that oxidative stress is responsible for the generation of
mitochondrial DNA mutations in human cells (5), but a direct link
between reactive oxygen species and large scale deletions of mtDNA has
thus far been elusive. Here we have demonstrated that UVA radiation
causes the generation of the common deletion in human fibroblasts
through an oxidative mechanism that depends on the generation of
singlet oxygen. Evidence for the involvement of singlet oxygen in
biological processes is based on the use of quenchers to diminish
singlet oxygen-mediated effects, on strategies that allow extension of
the half-life of singlet oxygen to enhance singlet oxygen-mediated
effects, and on the use of singlet oxygen-generating systems. By
employing these three different strategies we here provide evidence
that generation of the common deletion in human dermal fibroblasts through repetitive UVA irradiation critically depends on the generation of singlet oxygen. Formation of mitochondrial DNA mutations is thought
to occur by misannealing of direct repeats situated in the
D-loop (Scheme 1) during replication of mtDNA, thereby
leading to excluded loops (9). In order to complete the deletion
process, single strand breaks need to be generated to permit
exonucleolytic degradation of the looped-out DNA. Based on the present
study we propose a model in which singlet oxygen generation is
responsible for base damage and subsequent strand break formation. In
DNA, guanosines represent the prime targets for modification by UVA, and singlet oxygen (37, 38) and G stretches are extremely susceptible
to modification (39-41). In mtDNA, replication is started at the
replication origin of the heavy strand (OH) generating the
D-loop (Scheme 1), which after misannealing leads to the
excluded loop (9). The heavy strand contains several guanosine
stretches of 3 to 5 bases of which many are in direct proximity to the
direct repeats. Therefore it is tempting to speculate that the required strand breaks are indeed generated in this excluded loop via singlet oxygen.
Relevance for Photoaging--
Our studies identify a previously
unrecognized biological function of singlet oxygen. In addition, they
demonstrate that oxidative stress is indeed responsible for the
generation of large scale deletions of mitochondrial DNA in human cells
that have been exposed to UVA radiation, which induces tissue aging
under normally occurring conditions. In previous studies, singlet
oxygen was found to mediate other UVA radiation-induced biological
effects as well. In particular, UVA radiation-induced expression of
metalloproteinases I, II, and III expression in human dermal
fibroblasts was mediated through the generation of singlet oxygen (24).
The increased expression of matrix metalloproteinases in human skin
fibroblasts is thought to be partially responsible for the decreased
content of collagen fibers in photoaged human skin. Furthermore recent
findings indicate that singlet oxygen is linked with the in
vivo UVA action spectrum, which is responsible for photoaging of
mouse skin (42). Taken together with the present observation that UVA
radiation-induced singlet oxygen is capable of generating mitochondrial
DNA mutations in UVA-irradiated dermal fibroblasts, it is possible that
the generation of singlet oxygen in human skin is of central importance for photoaging. Singlet oxygen quenching may thus represent an effective strategy to protect human skin from photoaging.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Alan Lehmann, Prof. Bryn
Bridges, and Dr. Norbert Gattermann for valuable comments.
 |
FOOTNOTES |
*
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft, SFB 503, project B2 and B1, and the
Biomedizinische Forschungszentrum of the Heinrich-Heine-University,
Düsseldorf, Germany.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-211-811-7627; Fax: 49-211-811-8830; E-mail:
krutmann{at}rz.uni-duesseldorf.de.
 |
ABBREVIATIONS |
The abbreviations used are:
mtDNA, mitochondrial
DNA;
PCR, polymerase chain reaction;
PBS, phosphate-buffered saline;
NDP, 3,3'-(1,4-naphthylidene)dipropionate.
 |
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