Accepted Manuscript

Title: Oxidation events and skin aging

Author: A. Kammeyer R.M. Luiten

PII: S1568-1637(15)00003-3
DOI: http://dx.doi.org/doi:10.1016/j.arr.2015.01.001
Reference: ARR 561

To appear in: Ageing Research Reviews

Received date: 28-8-2014

Revised date: 15-12-2014
Accepted date: 5-1-2015

Please cite this article as: Kammeyer, A., Luiten, R.M.,Oxidation events and skin aging,
Ageing Research Reviews (2015), http://dx.doi.org/10.1016/j.arr.2015.01.001

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Manuscript

Oxidation events and skin aging

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A. Kammeyer a and R.M. Luiten a
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Department of Dermatology, Academic Medical Center (AMC), University of

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Amsterdam, Amsterdam, The Netherlands

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an
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Corresponding author at: Department of Dermatology, Academic Medical Center,

University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands,
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Tel.: (31)20 5662025, E-mail address: a.kammeyer@amc.uva.nl

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Abstract
The rate of skin aging, or that of tissue in general, is determined by a variable
predominance of tissue degeneration over tissue regeneration. This review discusses
the role of oxidative events of tissue degeneration and aging in general, and for the
skin in particular. The mechanisms involved in intrinsic and extrinsic (photo-) aging
are described. Since photoaging is recognized as an important extrinsic aging factor,
we put special emphasize on the effects of UV exposure on aging, and its variable

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influence according to global location and skin type. We here summarise direct
photochemical effects of UV on DNA, RNA, proteins and vitamin D, the factors

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contributing to UV-induced immunosuppression, which may delay aging, the nature
and origin of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as

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indirect contributors for aging, and the consequences of oxidative events for
extracellular matrix (ECM) degradation, such as that of collagen. We conclude that
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conflicting data on studies investigating the validity of the free radical damage theory
of aging may reflect variations in the level of ROS induction which is difficult to
quantify in vivo, and the lack of targeting of experimental ROS to the relevant cellular
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compartment. Also mitohormesis, an adaptive response, may arise in vivo to
moderate ROS levels, further complicating interpretation of in vivo results. We here
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describes how skin aging is mediated both directly and indirectly by oxidative
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degeneration.This review indicates that skin aging events are initiated and often
propagated by oxidation events, despite recently recognized adaptive responses to
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oxidative stress.
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Keywords:
skin aging, oxidation, ultraviolet (UV), photodamage, mitohormesis, collagen, elastin.

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1. Introduction
1.1. Skin aging
In the twenty-first century, the average age of the population all over the world is still
rising, especially in the industrialized countries (World Population Ageing: 1950-2050,
United Nations Population Division). The consequences of human aging are
predominantly visible in the skin, as increased wrinkling, sagging, and increased
laxity of the skin (Jenkins, 2002). Often considered as undesirable for cosmetic

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reasons, skin aging is also associated with physical disorders of the skin as
discussed in this review. There has always been a fascination for conserving youth.

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People invest much time and money in rejuvenation procedures, of which many lack
proven efficacy (Zouboulis and Makrantonaki, 2011).

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It has been suggested that aged skin has an disturbed barrier function, resulting in a
dry appearance of the skin and an enhanced risk on skin disorders (Hashizume,
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2004). Another health drawback is the enhanced risk on malignancies (Goukassian
and Gilchrest, 2004). Knowledge about the mechanisms of skin aging is important to
develop better skin care products that slow down skin aging and reduce the
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hazardous effects of aging (Farage et al., 2008; Elsner et al., 2011).
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Aging of the skin is induced by both intrinsic, and extrinsic factors (Farage et al.,
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2008; Landau, 2007), all leading to reduced structural integrity and loss of
physiological function (Landau, 2007). Typical extrinsic factors for skin aging are UV-
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exposure and smoking (Farage et al., 2008; Bernhard et al., 2007). This review
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describes the central role of oxidative processes in the initiating - and often
prolonged - events of aging. Most other aging-contributing events, such as
mitochondrial dysfunction, altered intracellular communication, genomic instability,
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cellular senescence and breakdown of the extracellular matrix (ECM) are a

consequence of these oxidative processes.

1.2. Intrinsic aging

Intrinsic aging affects all skin areas. Intrinsically aged skin is thin, transparent and dry,
shows fine wrinkles and irregular hair growth, is unable to sweat sufficiently, and
suffers from loss of subcutaneous fat tissue, leading to hollowed cheeks and eye
sockets, insufficient perspiration, and thinning of nail plates (Sjerobabski-Masnec and
Situm, 2010). These symptoms may vary per body site. It has been observed that the

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Page 3 of 53
intrinsic aging process differs per ethnic group (Davis and Callender, 2011), probably
caused by the degree of pigmentation, and possibly more not yet identified
contributing factors. Skin that is aged only by intrinsic factors does actually not exist.
Individuals that live strictly indoor all their life, may have a close approximation to that
skin status. In general, individuals carry skin that reflects various stages of extrinsic
aging, superimposed on the level of intrinsic aging.

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Reactive oxygen species (ROS) play an important role in skin aging. In the skin,
about 1.5 - 5 % of the consumed oxygen is converted into ROS by intrinsic

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processes (Poljsak et al., 2012). ROS are continuously produced as side products in
the electron transport chain of the aerobic metabolism in the mitochondria, and are

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regarded as the main cause of intrinsic aging (Farage et al., 2008). Keratinocytes
and fibroblasts are the main producers of „mitochondrial‟ ROS in the skin. A ROS that
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is predominantly formed in mitochondria from ground state oxygen, is the reactive
superoxide anion radical (O2-) by the addition of an electron to each oxygen (O 2)
molecule (Fig 1). Abundant generation of superoxide anions, as ROS-particles, may
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harm cellular function. Recent experimental evidence however shows that moderate
levels of ROS have a useful signaling function, especially when superoxide anions
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are converted into hydrogen peroxide (section 5).

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Manganese superoxide dismutase (MnSOD) is the primary mitochondrial neutralizer

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of continuously produced superoxide anions. Other forms of SOD exist outside the
mitochondria, but only MnSOD has been shown to be essential for the survival of
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aerobic life (St Clair and Kasarskis, 2003). Genetic polymorphisms of the human
(Mn)SOD gene and the corresponding alterations in the mature enzyme have been
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identified. The alterations lead to aberrant efficiencies of the Mn-SOD enzyme (St
Clair and Kasarskis, 2003). Consequently, these polymorphisms may largely
determine the natural ability of the cells to neutralize superoxide anions, and the
ability to withstand degeneration of (skin) tissue. The variation in genotypes may
partially explain the differences in aging conditions among individuals of the same
age. The effect of excess of superoxide anions on aging has been shown in Tet-mev-
1 mice, which have mutations in mitochondrial complex II subunits and
overproduction of superoxide anions. These mice show excessive apoptosis, which

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led to low birth weight, growth retardation, precocious aging and other pathologies
(Ishii et al., 2011).

Two other main events associated with intrinsic skin aging are a decrease in
replicative ability of cells and increased degradation of the extracellular matrix. The
replicative ability of all dividing cells decreases with time. In the skin, this particularly
affects keratinocytes, fibroblasts and melanocytes. This process is called cellular

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senescence. Senescent, non-dividing cells are found in higher levels in aged skin
(Dimri et al., 1995). The process of cellular senescence is related to the maximum

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number of cell divisions that somatic cells can undergo. With each division, a small
fragment of the telomere, is lost at the chromosome ends. After 25 to 30 divisions,

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telomeres become critically short, and the DNA loss during subsequent cell divisions
can affect areas of essential genes and lead to loss of somatic cell function. In germ
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cells, the telomerase enzyme prevents this fate by adding six-nucleotide repeats
TTAGGG to the 3‟ end of DNA strands to the telomere region of chromosomes upon
each cell division.
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The other factor involved in skin aging is the increased expression of enzymes that
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degrade the extracellular matrix (ECM) of the dermis upon aging. An increase in
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matrix metalloproteinase (MMP) expression has been shown in senescent fibroblasts

while the expression of the MMP inhibitors (TIMP) is reduced (Millis et al. 1992).
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These events may be triggered by ROS and can be reduced by antioxidants, such
as quercetin (Lee et al., 2013). Moreover, gradual oxidative damage during life to
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DNA bases in genes coding for the dermal matrix components collagen and elastin
leads to their reduced expression in aged skin (Uitto, 1979).
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1.3. Extrinsic aging

Extrinsic aging is caused by environmental oxidative factors, such as solar radiation
(Gilchrest, 1989; Kligman and Kligman, 1986; Wlaschek et al., 2001), cigarette
smoke (Bernhard et al., 2007) or other pollution factors. An epidemiological study
revealed that long term contact with tobacco smoke and UVA-exposure both
independently caused accelerated skin aging (Yin et al., 2001). These factors add to
intrinsic aging. Exposure to UV radiation is the primary factor of extrinsic skin aging,
also referred as to photoaging. The degeneration of the skin by UV radiation is a

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cumulative process and the rate of degeneration depends on the frequency, duration
and intensity of solar exposure and the natural protection by skin pigmentation
(Ortonne, 2002).

Photoaging accounts for as much as 80% of facial aging (Friedman, 2005). Photo-
aged skin is characterized by deep wrinkling, loss of elasticity, dryness, laxity, rough-
textured appearance, teleangiectasies and pigmentation disorders (Fig. 2) and its

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appearance is quite distinct from predominantly intrinsically aged skin (Farage, 2008;
Fisher et al., 2002). The severity of photoaging also depends on the skin type, being

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more prominent in fair skin individuals (skin types I and II) and less noticeable in
people with skin type III or higher (Situm et al., 2010; Davis and Callender, 2011).

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Thus, the severity of photoaging primarily depends on the cumulative dose of UV
exposure received and on the pigmentation status of the skin. The aged appearance
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of photodamaged skin is specifically caused by damage to the structural components
of the connective tissue of the dermis. The connective tissue is produced by
fibroblasts and is composed of three major classes of biomolecules:
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glucosaminoglycans (GAGs), proteoglycans, structural proteins (collagen and elastin)
and special macromolecules (fibrillin, fibronectin, laminin and hyaluronan) (Naylor et
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al., 2011; Uitto, 2008). During the last decades, extensive progress has been made
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understanding the molecular changes in photoaged skin. This review describes the
direct and indirect effects of UV radiation on epidermal and dermal structures in
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relation to aging and the role of oxidative events herein. Although triggered by
different factors, intrinsic aging and extrinsic aging partly involve similar molecular
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pathways (Gilchrest, 1989).

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2. UV radiation and the skin

2.1 Global situation

Solar UV radiation that reaches the earth‟s surface has a wavelength range from 290
to 400 nm. Solar ultraviolet radiation is divided into three categories according to their
wavelength, including UVA (320-400 nm), UVB (290-320 nm) and UVC (200-290 nm).
Considering that solar radiation of wavelengths < 290 nm do virtually not reach the

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earth, 290 nm represents a relevant wavelength limit for UV exposure on earth,
whereas the distinction between UVB and UVA at 320 nm, (some authors refer to

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315 nm) is an artificial subdivision (Parisi and Turner, 2006; van der Leun, 2004) (Fig.
3). UVC radiation causes the formation of mutagenic DNA lesions and greatly

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enhances the risk of developing skin cancer when the skin is exposed to an artificial
UVC-source (Pfeifer et al., 2005). Substantial UVB radiation reaches our skin with
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the sun in overhead position (around noon) and accounts for several types of skin
damage. Exposure to UVB has much more biological impact on the skin than
exposure to UVA, when comparing similar irradiation doses. However, solar UVA
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radiation passes earth‟s atmosphere virtually without being absorbed and is therefore
abundantly present in solar radiation throughout the day. UVA penetrates through the
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epidermis into the dermis during skin exposure (Fig. 4). The UVA/UVB ratio is
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approximately 10/1 at overhead sun position. When the sun leaves its overhead
position, the ratio UVA/UVB becomes larger, as UVB radiation becomes strongly
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absorbed due to the longer path through the atmosphere. Prior to sunset, the
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atmospheric path reaches its maximum length absorbing most shortwave radiation,
including shortwave visible radiation, leading to a red appearance of the sun.
Consequently, UVA exposure of the skin under daily circumstances is much larger
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than UVB.

The radiation intensity (W/m2) of both UVB and UVA depends on several factors,
including the solar zenith angle, reflection by the soil (albedo) (Parisi and Turner,
2006), altitude and the presence of clouds or dust particles in the sky that filter or
scatter the radiation (Diffey, 1998). The irradiation dose (J/m2) is the product of the
radiation intensity multiplied by the exposure time, and predominantly determines the
UV-induced damage to the skin. For example, people living in Northern Australia

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Page 7 of 53
receive roughly twice the UV dose of people living in Mid Europe, due to the location
closer to the equator. In particular outdoor-workers accumulate larger UV doses than
indoor-workers and are therefore more at risk of skin-damaging effects of UV
radiation and the development of skin cancer. This is especially true for body areas
mostly exposed to the sun such as the face, head, neck, and hands (Godar, 2005).

2.2. Penetration of UV radiation into the skin

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The absorption of UV radiation by chromophores in the skin leads to several direct or
indirect chemical interactions, creating direct or indirect damage to biomolecules

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(Svobodova et al., 2006). Photodamage to the skin is initiated, when chemical
interactions lead to alterations of biomolecules in epidermis and dermis. Molecular

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oxygen (O2) does not absorb UVB, nor UVA and is therefore not a direct target of
terrestrial UV radiation. The formation of ROS takes place via indirect ways (section
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3). UVB does not penetrate deeply into the skin, the major part is absorbed in the
epidermis by DNA, aromatic amino acids of proteins, NADH, NADPH, flavins,
quinones, porphyrines, carotenoids, urocanic acid, eu- or pheomelanin and lipids
M
(Svobodova et al., 2006; Young, 1997). UVB is largely responsible for the
development of sunburn. UVA is not directly associated with sunburn because UVA
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is approximately 1000 times less effective than UVB in creating sunburn (Diffey,
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2002). Therefore, UVB but not UVA, was associated with skin photodamage and
photoaging for a long time. However, UVA also plays a substantial role in photoaging
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for two reasons: it penetrates both the epidermis and the dermis (Fig. 4) where it can
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cause damage to the extracellular matrix (ECM), and its solar intensity is much
higher than UVB, as described in section 2.1. Chromophores are able to transform
the energy obtained from UV radiation into other types of energy. Chromophores,
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such as melanin, can transform the absorbed UV radiation into heat, thereby
reducing UV-induced damage to the cells and subsequent photoaging (Ortonne,
2002). The type of melanin, its distribution and density between the keratinocytes
determines the pigmentation grade of the skin. High levels of pigmentation form a
natural protective shield for UV radiation. As a results, dark-skinned people are
approximately 500 times more protected than fair-skinned people against the risk of
non-melanoma skin cancer (Ortonne, 2002).

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Page 8 of 53
2.3. Direct photodamage to DNA, RNA and proteins
UV radiation causes damage to multiple cellular structures both directly and indirectly,
thereby accelerating the aging process. UVB is predominantly responsible for direct
damage. A substantial part of UVB is mostly absorbed in the stratum corneum, but
attenuated UVB radiation intensity also reaches the viable epidermal cells (Hussein,
2005), causing biological damage. DNA damage is most critical type of biological
damage,

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(Pfeifer et al., 2005; Hussein, 2005). Upon absorption of UVB, pyrimidine bases in
DNA can form bonds with adjacent pyrimidines (Goodsell, 2001). This causes the

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formation of cyclobutane-pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4)
photoproducts, also referred as to (6-4)-PP (Fig. 5). CPDs are preferentially

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produced at pyrimidine sites with methylated cytosines (mC). The methylation of
cytosine bases occurs predominantly in 5‟-CG-3‟(CpG) sequences (Tommasi et al.,
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1997). These mutations, preferentially located at methylated CpG sites by solar UV
radiation, are also called “solar-UV signature” (Ikehata and Ono, 2007). CPDs are
mostly found in the DNA of keratinocytes and Langerhans cells (LC) in the epidermis,
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but also in dendritic cells (DC) in the lymph nodes that drain the irradiated skin site,
at least in mice (Sontag et al., 1995).
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The (6-4)-PP are instable and upon absorption of UV wavelengths of around 320 nm,
they are converted into the more stable so-called Dewar valence isomers (Clingen et
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al., 1995) (Fig. 5). CPDs are the major contributor to mutations because they are
less efficiently repaired than (6-4)-PP (Matsumura and Ananthaswamy, 2004). If not
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repaired correctly by the “nucleotide excision repair” (NER) mechanism, these three
premutagenic lesions; CPD, (6-4)-PP, and Dewar, cause UV-specific mutations.
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Thymine-cytosine (T=C) and cytosine-cytosine (C=C) dimers are the most oncogenic
CPDs, since CT single-base and rare CCTT tandem-base mutations are found in
the tumor-suppressor gene p53 in UV-irradiated cancer cells (Brash et al., 1991).
CPD, (6-4)-PP, and Dewar isomers can inhibit polymerase transcription and arrest
replication, which can lead to the activation of the p53 protein. Substantial
accumulation of DNA damage leading to p53 activation may lead to the induction of
apoptosis in healthy keratinocytes. However, other types of mutations stimulate the
growth of the keratinocytes and may lead to the formation of malignancies (Ziegler et
al., 1994). Absorption of UVB by RNA bases can have harmful consequences as well,

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although more transient, during RNA transcription and translation. Mutations in
mRNA can lead to the production of dysfunctional proteins (Ljungman and Zhang,
1996).

To summarize, DNA and RNA bases absorb UVB radiation that can lead to various
mutations and affect cellular protein synthesis. Accumulations of unrepaired
mutations can cause cell cycle arrest and apoptosis. On the other hand, mutations

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can abrogate the apoptotic ability of cells and thereby enhance the formation of
malignancies. The final outcome depends on the skin cell type, the cumulative UV-

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dose and the UV wavelengths.

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In epidermal proteins, UVB is predominantly absorbed by the aromatic amino acids
tryptophan (Trp), tyrosine (Tyr) and cystine, disulfide-bonded cysteine (Watson et al.
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2014). The absorption of UVB can result in excited state species or direct radical
formation as a result of photoionization. This type of direct interaction is referred as to
type I photooxidation. The well-studied, longer lived triplet states of Trp and Tyr have
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been shown to reduce disulphides, such as lipoate and cystine. The resulting
-.
disulphide radical anions (RSSR ) can react with oxygen to form superoxide anions
d

-.
(O2 ) and additionally, the parent disulphide (RSSR) is regenerated (Hoffman and
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Hayon, 1972). Several other reactions with excited Trp and Tyr are possible (Pattison
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and Davies, 2006) that can potentially lead to degradation and oxidative stress to
skin cell constituents. A substantial part of UV-induced protein modifications are
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caused by reactions with singlet oxygen that is generated through UV-excited

(natural or artificial) photosensitizers, referred to as type II photooxidation (Pattison
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and Davies ,2006). Proteins that are rich in Trp and cysteine may be the key targets
of UVA-mediated photodamage, either through type I or type II chemistry (Pattison
and Davies, 2006; Watson et al., 2014), especially the carbonyl proteins (section 3.6).
The modified protein can subsequently lose its function as an enzyme or structural
component. Modified proteins may form aggregates, which pose a serious threat for
the cell. Accumulation of oxidatively damaged proteins is symptomatic for many
diseases and for the aging process. Cells can either attempt to counteract on this
protein damage by several antioxidative or repair responses, or can channel the

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damaged proteins for degradation by the ubiquitin-proteasome system (Kriegenburg
et al., 2011).

2.4. Vitamin D production by UV radiation

Not all photochemical interactions lead to adverse effects. A beneficial effect is the
biosynthesis of vitamin D3 by UVB. The epidermis contains stores of 7-
dehydrocholesterol, an intermediate in cholesterol biosynthesis. This provitamin, or

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prohormone, efficiently absorbs UVB radiation, leading to partial transformation into
vitamin D3 (cholecalciferol) (Holick et al., 1980). The photochemical conversion is

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only induced by short wave UV, mainly UVB radiation. UVB-induced vitamine D3
synthesis thus requires the sun to be in a rather overhead position, around noon.

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Early in the morning and late in the afternoon the UVB content of the solar rays is
neglegible and is insufficient for vitamin D3 formation (Holick, 2001) Vitamin D3
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diffuses from the skin and is transported by the blood circulation to the liver, where it
is converted to calcidiol, 25-hydroxycholecalciferol (25(OH)D). This specific vitamin D
metabolite that can be determined in blood to assess one‟s vitamin D status.
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Ultimately, the hormone-like active form of vitamin D, 1,25-dihydroxycholecalciferol
(1,25(OH)2D) or calcitriol, is formed by further hydroxylation in the kidneys (DeLuca,
d

1988; Holick, 2001).

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Seasonal changes, latitude, social behavior/lifestyle, pigmentation grade and

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sunscreen use can all lead to substantial vitamin D3-insufficiency, which causes
growth retardation and rickets in children and osteoporosis in elderly people. It has
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been shown that aged skin contains less vitamin D3 which contributes to vitamin D
deficiency in elderly people (Reichrath, 2012). In several countries, food is fortified
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with vitamin D3 to compensate for low dietary intake. Vitamin D compounds are
strongly indicated for protection against aging-promoting factors, such as UV
exposure (Reichrath, 2012; Chang et al., 2010).

2.5. UV-induced alterations resulting in immunosuppression

UV exposure of the skin can induce immune suppression and can suppress
inflammation in the skin. This effect of UV radiation does not only occur locally in the
skin, but can also result in systemic immune suppression. Inflammation of the skin is
associated with the influx of immune cells that can produce ROS and degrading

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enzymes that will subsequently damage surrounding tissue. Suppression of
inflammation delays inflammation-induced aging of the skin (Pawelec et al., 2014).
The chromophores in the skin absorb UVB and UVA and induce immunosuppression
in several ways. In general, UV-exposure induces all kinds of photochemical changes
in cells, also in inflammatory cells. Photochemically altered cells, if not necrotic or
apoptotic, will change their immunological behaviour in such a way that the
inflammation may not be sustained, or suppressed. An application is the so-called

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phototherapy with small band UVB exposures of 311 nm, which is widely used for the
treatment of inflammatory skin disorders, such as psoriasis and eczema. In the past

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decades, several chromophoric systems are identified that can contribute to the
observed immunosuppression and may indirectly contribute to the prevention of skin

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aging, as outlined below.

2.5.1 Urocanic acid an

A main photoreceptor in the epidermis that absorbs UVB and - to a lesser extent -
UVA with wavelengths up to 360 nm is trans-urocanic acid (trans-UCA) (Kammeyer
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et al., 1995). This metabolite is formed from the amino acid histidine, derived from
the breakdown of the epidermal protein filaggrin, in the upper layer of the epidermis
d

(Baden and Pathak, 1967). Epidermal trans-UCA has been recognized in the past as
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a natural sunscreen based on being the main UVB absorber in the epidermis
(Zenisek et al., 1955). Trans-UCA also protects epidermal cells against DNA-damage
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(Barresi et al. 2011). A low pigmented epidermis lacks eumelanin as a primary

photoprotector, and trans-UCA may serve as a secondary photoprotector for UVB.
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Upon UV absorption, trans-UCA is photoisomerized to cis-UCA in vivo in the

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epidermis as well as in vitro. Cis-UCA has been demonstrated to have an

immunosuppressive function, partly by enhancing the release of immunosuppressive
mediators such as prostaglandin E2 (Kaneko et al., 2011) and IL-10 (Holan et al.,
1998). UV-damaged antigen presenting cells stimulate antigen-specific T regulatory
cells, which are responsible for the suppression of the immune system by releasing
suppressive mediators such as IL-10 (Schwarz et al., 2000). Cis-UCA can also
mediate immunosuppression by binding to the human serotonine 5-HT2A receptor
that was expressed by insect (Sf9) cells. Cis-UCA-induced activation through the
receptor was demonstrated by a mouse fibroblast line expressing the human 5-HT2A

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receptor (Walterscheid et al. 2006). In summary, cis-UCA can mediate part of the
UV-induced immunosuppressive effects.

Moreover, UCA had been identified as a good hydroxyl radical scavenger

(Kammeyer et al. 1999), possibly providing protection to other epidermal components.
As UCA scavenges, the compound is also converted into UCA photooxidation
products, including 4-substituted imidazoles, such as imidazole-4-carboxylate and

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imidazole-4-acetate, and show comparable, moderate immunosuppressive effects in
a murine model for contact hypersensitivity (Kammeyer et al. 2004; Kammeyer et al.

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2001). UCA has mostly been shown to have a photoprotective role against UVB
effects in the skin . A rather recent study showed UCA-induced reduction of UVB-

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induced DNA damage in vivo in the form of cyclobutane pyrimidine dimers by
comparing wild-type mice versus histidinemic mice, lacking epidermal UCA. Higher
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levels of apoptosis were also detected in the UCA-poor histidinemic mouse epidermis
(Barresi, 2011). However, irradiation of UCA is with UVA in vitro, resulted in the
formation of singlet oxygen (Menon and Morrison, 2002), arguing against a solely
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protective role of UCA. Because of the different skin penetration depths of UVB and
UVA, a distinction between epidermal and dermal radiation effects should be made
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when evaluating the biological effects.

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2.5.2 DNA bases

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DNA bases in epidermal cells are important targets for UV radiation. The pyrimidine
bases C and T can form dimers with a cyclobutane ring in between (C=C, T=T and
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C=T)(section 2.3). The subsequent cellular reactions can lead to immunosuppression,

which was demonstrated in mice, including experiments with T4-endonucleases
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(Kripke et al. 1992). The pyrimidine and purine bases can both be modified by UV-
induced oxidative processes resulting in photoproducts such as 2-hydroxy- and 8-
hydroxyadenine and 8-hydroxyguanine (section 3.4). Similar UV-induced events may
occur among the RNA bases, but are regarded as less important due to the transient
nature of RNA.

2.5.3 Lipid peroxidation

Another UV-induced immunosuppressive mechanism is the UV-induced lipid
peroxidation (LPO) of cell membranes. LPO can form reactive aldehydes that attack

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several inter- and intracellular targets. With enzymatic aid of cyclooxygenase (COX)
several immunomodulating prostaglandines can be formed from C20-fatty acids, such
as arachidonic acid (Basu, 1998). Prostaglandines are an important group of non-
peptide immunomodulators. Prostaglandine-E2 (PG-E2) can be regarded as an
immunosuppressive agent and is generated in the epidermis upon a UVB-induced
sunburn reaction. Sunburn and other types of skin inflammation attract immune cells
with the subsequent rise of oxidative stress that may promote skin aging.

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2.5.4 Tryptophan

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Several photoproducts of the essential amino acid tryptophan (Trp) were identified as
UV-induced immunomodulators in the last two decades. Trp photoproducts can also

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be formed upon UVA exposure, or even upon visible light exposure with the
intervention of excited photosensitizers, such as riboflavin (vitamin B2). Some Trp
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photoproducts are lipophilic and their molecules are rather planar, such as 6-formyl
indolo[3,2-b]carbazole (FICZ). Because of these two properties they bind with high
affinity to the cytosolic aryl hydrocarbon receptor (AHR). Depending on the type of
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Trp-photoproduct, as a ligand for AHR, binding will mediate either pro-inflammatory
or anti-inflammatory reactions of the immune system (Ho and Steinman, 2008; Esser
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et al. 2009). The AHR is predominantly expressed by keratinocytes, Langerhans cells

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in the epidermis, and T helper 17 (Th17) cells (Esser et al., 2009). Ligand-dependent
activation of AHR can enhance the differentiation of Th22 cells that secrete the
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cytokine IL-22 but not IL17 and are believed to play a suppressive role in auto-
immune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA)
ce

(Zhang et al., 2011).

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2.5.5 Vitamin D3
Recent studies have indicated a potential role for vitamin D3, cholecalciferol (section
2.4) in immunosuppression. This UVB-induced form of vitamin D inhibits dendritic cell
maturation and thereby its antigen presenting function. Vitamin D-treated dendritic
cells promote a type of regulatory T cells (Tr1) that secrete anti-inflammatory
cytokines IL-10 and transforming growth factor-β (TGF-β) (van der Aar et al., 2011).
This finding contributes to the understanding of immunosuppression by UV radiation,
but the complete mechanism of action of vitamin D3 is still to be resolved.

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Summarizing, UV radiation, and especially UVB, is needed for sufficient vitamin D
production and induces other immunosuppressive factors. UVB can be beneficial for
reducing symptoms of inflamed skin, such as eczemas, psoriasis and vitiligo.
Perhaps other autoimmune diseases like SLE and MS may also be diminished by
UVB exposure (Pawelec et al., 2014; Gibbs and Norval, 2013). The anti-inflammatory
effects of immunosuppression may delay the skin aging process.

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3. Indirect photodamage to cellular skin components

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3.1. Oxidants

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UV radiation can induce biological damage and accelerate aging through indirect
pathways with endogenous or exogenous photosensitizers that absorb UVA and
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even visible (VIS) wavelengths of radiation from the sun or artificial sources.
Consequences are the generation of singlet oxygen or direct photochemical changes
to biomolecules. UVA and VIS radiation are less absorbed by epidermal components
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and hence penetrate deeper, into the dermis. UVA and VIS can create excited states
of the photosensitizer compounds in this skin compartment (Pattison and Davies,
d

2006). Excited state molecules in the epidermis and the dermis can deal in various
e

ways with the absorbed energy. It can either be transferred into heat (infrared
radiation), but fluorescence or phosphorescence emissions do also occur. Even
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covalent bonds can be broken by passing certain thresholds levels of bond energy,
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resulting in the formation of photoproducts. The photosensitizer in its excited state

can return to ground state, but can also react with other biomolecules, finally
resulting in molecular changes in cells and (connective) tissue composition.
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The cellular damage in the skin by excited photosensitizers takes place in two ways,
depending on the properties of the sensitizers. A type 1 reaction occurs when the
excited photosensitizer and other biomolecules directly interact with each other and
form stable reaction products, or free radicals by electron transfer and hydrogen
abstraction processes. An example is the UVA-induced reaction of psoralen on other
biomolecules, predominantly DNA. This photochemical interaction is used in PUVA
therapy (Parrish et al., 1974). This kind of free radical formation proceeds without
oxygen. A type 2 reaction takes place when the excited photosensitizers transfer

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Page 15 of 53
energy to molecular oxygen (O2), the main source for free radical formation. As a
result, singlet oxygen (1O2) can be generated, a powerful oxidant type of ROS (Fig
6). ROS are very reactive molecules involved in cellular damage and molecular
signaling (Pattison and Davies, 2006). An example of a type 2 reaction is the reaction
of porfyrines with oxygen in the skin when excited after UVA exposure to, or even
visible violet radiation. Because of this feature, porfyrines are used in photodynamic
therapy (PDT) to destroy tumor cells. Trans-UCA is another example of a sensitizer

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that forms singlet oxygen after UVA absorption. The produced singlet oxygen can
attack cell membranes and create additional free radical molecules (McCord, 2000).

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Also, the endogenous photosensitizers riboflavin, tryptophan and porphyrin can
produce ROS upon UVA-exposure (Young , 1997).

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Superoxide dismutase, an antioxidant enzyme, can convert superoxide anions (O2-)
an
into hydrogen peroxide (H2O2), which is a less reactive compound on itself. Moreover,
H2O2 does not have a net electrical charge, which property enables smooth
membrane passage. On the other hand, H2O2 can be converted into various harmful
M
or harmless compounds (Fig. 7), for example by glutathione peroxidase (GPx), which
is available in high levels in the cytoplasm of animal cells. Glutathione peroxidase
d

reduces H2O2 to H2O. Hydrogen peroxide can also be decomposed to H 2O + O2 by

e

the enzyme catalase, which is mainly located in the peroxisomes organelles.

Myeloperoxidase (MPO) is another enzyme that can convert H2O2. This lysosomal
pt

enzyme produces the cytotoxic hypochlorous acid (HOCl) from H2O2 (Folkes et al.,
1995). HOCl is also stored in the granules of neutrophils and is involved in the
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destruction of invading micro-organisms. H2O2 can react with free Fe(II) that is
released from the metal-binding sites on proteins under influence of UV radiation
Ac

(Bissett et al., 1991). This is called a Fenton reaction (Fig. 8A). This reaction yields
hydroxide-ions (OH-), but also the very reactive hydroxyl radical (HO·). Exposure of
H2O2 to UV radiation at shorter wavelength than 320 nm (UVB), induces a H2O2
photolysis reaction and the formation of hydroxyl radicals (Fig 8B). Other sources of
ROS are flavoprotein oxidases, xanthine oxidases and peroxisomal enzymes
(Redmond, 1999). They can be involved in fatty acid metabolism and cytochrome
P450 enzymes, predominantly producing superoxide anions. (Bissett et al., 1991;
Castro and Freeman, 2001)

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Although ROS can damage important (bio-)molecules and have a role in
carcinogenesis, they are also involved in physiological processes such as cell
signaling, proliferation, tumor suppression, support of the immune system in the
defence against pathogens and the oxygen homeostasis (Rhee, 2006a). The
influence of ROS particles is determined by the dosage and the persistence of such
particle, as well as the cell type involved. Epidermal cells are relatively resistant to
ROS influence and other types of UV-induced degradation mechanisms (Tyrrell and

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Pidoux, 1986). Low ROS levels can be mutagenic, medium levels can result in
replicative senescence, and high ROS levels usually lead to cell death by apoptosis

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or necrosis (McCord, 2000). The oxygen homeostasis is maintained by multiple
redox-reactions. If the oxygen homeostasis is disturbed, the cellular environment

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becomes oxidatively stressed, in other words: the oxidant effects (such as those of
ROS) overrule the antioxidant effects. Oxidative stress is a consequence of ROS
an
production, often stimulated by UV irradiation, heat exposure, or degranulation of
immune cells such as neutrophils during inflammation (Devasagayam et al., 2004).
The subsequent tissue degeneration will contribute to accelerated tissue aging.
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The generation of reactive nitrogen species (RNS) originates mainly from nitric oxide
d

(NO) reactions with other radical species. One main representative of RNS is
peroxynitrite, ONOO-, which particle can be formed from superoxide anion and NO.
e

Peroxynitrite can nitrate proteins, predominantly tyrosine residues, rendering those

pt

targets compromised with deteriorated function, comparable to carbonyl proteins.

ce

ROS- and RNS-particles are not only involved in adverse effects, but paradoxically
also function in signaling. Superoxide anions and hydroxyl radicals are very short-
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lived and only attack biomolecules in the very near proximity (half-lives of ~ 1 µs and
1 ns, respectively). In contrast, H2O2 is stable in „clean‟ solution and is much longer
lived in cellular environment (half-life ~ 1 ms) and may form a major signaling particle
next to nitric oxide (NO) as a major RNS signaling particle. The role of NO in the skin
is reviewed in Cals-Grierson and Ormerod, 2004. ROS-signaling can regulate stress
response pathways, and is involved in normal growth processes and development.
(Labunskyy and Gladyshev, 2013). Redox signaling by H2O2 can occur by H2O2-
induced oxidation of Cys-thiol groups (R-SH) into disulfide bridges (R1-S-S-R2) within
proteins. This disulfide bond formation can be reduced back into the thiol status by

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either thioredoxin or glutaredoxin systems. Extensive overviews of redox signaling
are described in (Labunskyy and Gladyshev, 2013; Ray et al., 2012)

3.2 Antioxidants
Human cells possess multiple protection mechanisms against damage by ROS and
RNS, either by preventing damage or by damage repair. Antioxidants disarm ROS by
functioning as reducing agents (Sies, 1997). The function of antioxidants is to

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maintain the oxygen homeostasis, not to completely remove all oxidants because
they can have important functions (Rhee, 2006). The most important antioxidant

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enzymes that eliminate free radicals, such as ROS, is glutathione peroxidase (GPx),.
This antioxidant enzyme can remove singlet oxygen, hydrogen peroxide and organic

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peroxides by permitting a reaction with the thiol-group of glutathione (Tyrrell and
Pidoux, 1986). Other antioxidant enzymes are superoxide dismutase (SOD) and
an
catalase that eliminate superoxide radicals and hydrogen peroxide, respectively.

Non-enzymatic antioxidants include ubiquinol, vitamin C (ascorbic acid) and vitamin

M
E (α-tocopherol) (Sies, 1997; Pinnell, 2003). Also, the dark type of melanin;
eumelanin, can eliminate UV-induced radicals, besides their ability to absorb UV
d

directly (Pavel ,1993). In contrast, the lighter type of melanin, pheomelanin, can
e

produce ROS upon exposure to UV radiation (Pavel, 1993). DNA damage that
occurs despite prevention, can be repaired by several enzymatic mechanisms. The
pt

most important one is the nucleotide-excision repair (NER) mechanism, in which

multiple enzymes are involved that almost completely repair the UV-induced DNA
ce

damage. The damaged DNA is recognized, removed and replaced to recover the
original DNA chain. Xeroderma pigmentosum (XP) patients, with a genetic defect in
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the NER-mechanism, cannot be exposed to UV radiation without developing serious

skin disorders (Bonn, 1998).

3.3. Lipid damage

ROS can modify lipids, DNA and proteins and have been implicated in skin aging.
Lipids are important structural components of cellular membranes in the form of
phospholipids. Lipid (LH) peroxidation of unsaturated fatty acid chains by ROS yields
lipid hydroperoxides as primary products. An important route is lipid photooxidation
via UVA/VIS-photosensitized reactions type I and type II (section 2.3). Peroxidized

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Page 18 of 53
lipids (LOOH) tend to accumulate in cellular membranes proportionally with the
cumulative UVA/VIS radiation dose received by the skin. Substantial concentrations
of LOOH perturb the integrity of the membranes, and thus of the cells involved
(Girotti, 2001). Oxidized phospholipids (oxPL) tend to stick out of the membranes of
senescent and apoptotic cells and form a ligand for scavenging receptor CD36 on
phagocytic cells. The recognition of oxPL by CD36 has led to the development of the
Lipid Whisker Model (Greenberg, Li et al., 2008). This surveillance system for the

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integrity of host tissues can account for the removal of oxPL through complete
removal of apoptotic and senescent cells by phagocytic cells, such as macrophages.

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Lipid peroxides can be further decomposed to a number of reactive aldehyde species

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such as malondialdehyde (MDA), 4-hydroxynonenal, hexanal and other saturated
and α,β-unsaturated aldehydes and ketones (Marnett, 1999). These reactive side
an
products of lipid peroxidation can be mutagenic and carcinogenic because of their
reactivity towards DNA bases, creating MDA-DNA adducts, that cannot be repaired
(Marnett, 1999). The lipid peroxidation products such as aldehydes can also
M
introduce modifications in amino acids like histidine and lysine (section 3.6) (Uchida,
2003) that may be recognized by the immune system as non-self and may initiate
d

autoimmunity. Furthermore, the UV-induced oxidation of the polyunsaturated fatty

e

acid arachidonic acid (AA) in cell membranes is catalyzed by the enzyme

cyclooxygenase (COX), which can result in the formation of oxidation products such
pt

as immunomodulating prostaglandins (Needleman et al., 1986; Nicolaou, 2013).

Membrane lipid damage further leads to altered membrane fluidity, cell membrane
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leakage or dysfunction of membrane-bound cellular receptors (Cai and Harrison,

2000).
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3.4. DNA and RNA damage by ROS

ROS can damage DNA indirectly by lipid oxidation products, and directly by
interaction with the DNA bases. Several types of DNA damage exist, but the most
studied type of DNA damage by ROS is the oxidation of guanine into 8-oxoguanine
(8-oxoG), mediated by singlet oxygen (Kasai, Crain et al., 1986). Its nucleotide
analog, 8-oxo-deoxyguanosine (8-oxo-dG), generates GC to TA conversion during
replication by pairing an adenine instead of a cytosine molecule, and conversion of a
guanine to a thymine molecule. This leads to abnormal cell function, but these

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transversions are also found in oncogenes and tumor suppressor genes such as p53,
indicating a role in carcinogenesis (Shibutani et al., 1991).

RNA is more sensitive for ROS damage for several reasons; it is only a single strand
molecule leaving vulnerable spots more exposed to ROS. It is also not protected by
histones, nor by cellular compartments (as with nuclear DNA). RNA is more randomly
present throughout the cell, not confined to the nucleus (Li et al., 2006). 8-oxo-G

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lesions are found in RNA strands in a similar way as the formation of 8-oxo-dG.
These oxidation lesions lead to lowered translation efficiency resulting in abnormal

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proteins and aberrant enzyme function (Dukan et al., 2000).

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3.5. Mitochondrial DNA damage by ROS
Another target for ROS is mitochondrial DNA (mtDNA). MtDNA is susceptible for
an
oxidative stress induced damage and has a higher mutation frequency than genomic
DNA due to the lack of a repair mechanism and histones (Yakes and Van, 1997).
Most abundant ROS in mitochondria are superoxide anions, which are produced as
M
„side‟-products from the oxidative phosphorylation. Every O2 would be normally
reduced to water and gains 4 electrons, but O2 molecules can also escape with one
d

electron, as superoxide anions, O2- . These reactive particles are neutralized by a

canonical antioxidant enzyme, superoxide dismutase, which converts O2- into the
e

generally less reactive hydrogen peroxide (H2O2) (section 1.2).

pt

Singlet oxygen, repeatedly generated by the interaction of UVA and a photosensitizer,

can cause large scale deletion in mtDNA; the so called common deletion (Berneburg
ce

et al., 1999). This damage in mtDNA leads to defects in the electron transport chain
and oxidative phosphorylation process, ultimately resulting in reduced ATP
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production. Impaired oxidative phosphorylation also increases the generation of

singlet oxygen, which in turn leads to more mtDNA mutations (Wallace et al., 1998).
The common deletion occurs most frequently in mtDNA of fibroblasts upon repeated
UVA exposure in a dose-dependent manner. MtDNA deletions in fibroblasts lead to
altered gene expression patterns through a retrograde pathway between
mitochondria and the nucleus (Butow and Avadhani, 2004). Functional and structural
changes in fibroblasts affect dermal structures, such as collagen, and contributes to
skin photoaging.

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3.6. Protein damage by ROS
ROS-induced oxidative damage to the structural dermal proteins collagen and elastin
can result in changes in protein conformation and unfolding, leading to modifications
in mechanical properties of the skin (section 4). Collagen degradation and abnormal
elastin accumulation are visible in photoaged skin (Krieg et al., 1988).
Proteins undergo modifications and subsequent conformational changes when
certain amino acids are converted to their oxidized forms during oxidative stress

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(Stadtman, 1995). The amino acids can be oxidized by different ROS generated by
different pathways, as described in section 2.3. Proteins in the skin can be oxidized

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by singlet oxygen (1O2), generated by UVA-induced excitation of various
photosensitizers (type II photooxidation, section 2.3). An extensive compilation of 1O2

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–yields from biologically relevant compounds is reviewed in Redmond and Gamlin,
1999. Singlet oxygen damages primarily the side chains of the amino acids (Grune,
an
et al., 2003) tryptophan, histidine, tyrosine, lysine, methionine and cysteine. The
oxidation reaction can also generate further reactive species such as endo- and
hydroperoxides. These reactive species can oxidize other biological targets and
M
induce protein cross-linking. Especially histidine and lysine residues are targets for
oxidative modification and can turn into 2-oxohistidine and aminadipic semialdehyde
d

(a carbonyl moiety), respectively (Uchida, 2003). Oxidation of these side chains can
e

lead to a change in the property and function of the concerned protein or enzyme.
Moreover, histidine and lysine residues can react with reactive aldehydes from lipid
pt

peroxidation such as acrolein and 4-hydroxynonenal (section 3.3). If moderately

labeled with these aldehydes, the modified protein, can be degraded by the
ce

proteasome. If heavily labeled, the damaged proteins tend to aggregate, forming an

insult for cell viability (Grune and Davies, 2003; Grune et al., 2003; Jung et al., 2007).
Ac

Oxidized proteins are poor substrates for ubiquitination, which does not seem to play
a major role in recognition and targeting of oxidized proteins (Grune et al., 2003). The
resulting carbonyl proteins may reflect the total fraction of proteins with impaired
function. Carbonyl proteins may be regarded as a measure for the degree of
oxidative damage and can be detected in a variety of diseases, as well as in aged
tissue (Berlett and Stadtman, 1997; Levine, 2002).

At least one spontaneous protein degradation pathway is identified, not initiated via
ROS. Aspartyl and asparaginyl residues in protein chains can form a succinimidyl

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rings within the protein chain, if there is no steric hindrance. Via its methylation and
subsequent demethylation by methyltransferases, the original protein structure can
be restored. However, individual genetic makeup determines the efficacy in the
restoration process, and failure to repair or to degrade altered proteins can result in
accumulation of potentially toxic species in tissues (Clarke, 2003). This phenomenon
may contribute to the rate of tissue degeneration and aging.

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To summarize, UV-induced ROS can alter genetic material such as genomic DNA,
RNA and mitochondrial DNA and other cell constituents, such as lipid membranes,

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enzymic and non-enzymic, structural proteins. Depending on the extent of UV-
induced molecular changes, it can impair signal transduction pathways related to

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differentiation, growth and senescence. These defects can lead to abnormal cell
function, including abnormal protein production, enhanced connective tissue
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degradation and carcinogenesis. However, complete abolishment of oxidative stress
is not favorable and not desired, because ROS-induced signaling is regarded as a
vital tool in cell homeostasis (section 5).
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4. UV-induced damage to skin connective tissue
As a result of chronic UV exposure, most visible damage occurs in the connective
tissue, also referred to as the dermal extracellular matrix (ECM). The most important
and abundant structures of the dermal ECM are collagen, elastin and
glycosaminoglycanes (GAGs). Their main functions are to maintain the strength,
elasticity and hydration of the skin (Oxlund and Andreassen, 1980). In both intrinsic
and extrinsic aging, qualitative and quantitative changes in these structures are

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observed. This leads to loss of tensile strength and recoil capacity, wrinkle formation,
dryness, impaired wound healing and increased fragility (Poljsak et al., 2012).

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Although some aging mechanisms overlap, intrinsically aged skin and photoaged
skin show different changes in the ECM. Globally, intrinsically aged skin shows

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atrophy of dermal structures, whereas photoaged skin is characterized by the
accumulation of aberrant elastin fibers and glycosaminoglycans, together with
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damaged and diminished collagen (Scharffetter-Kochanek et al., 2000). The
pathways by which UV radiation damages these structures is reviewed in this chapter.
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4.1 Collagen damage
Collagen is the main component of the dermal ECM in mammals and is the major
d

determinant for skin appearance. It is produced and secreted by fibroblasts, together

e

with other dermal proteins of the extracellular matrix (ECM). Collagen in the dermal
ECM predominantly consists of collagen type I and III and is responsible for the
pt

tensile strength and stability of the dermal tissue and blood vessels (Oxlund and
ce

Andreassen, 1980).

Protein aging modifications are partially caused by direct UV interaction, but mainly
Ac

indirectly by ROS-induced damage (section 3.6). UVA- and UVB-induced ROS

stimulate the synthesis of matrix-degenerating metalloproteases (MMPs) (Birkedal-
Hansen, 1987). MMPs are a family of zinc-dependant endopeptidases. Four
members, collagenase (MMP1), 92 kDa gelatinase (MMP2), stromelysin (MMP3) and
72 kDa gelatinase (MMP9), are in particular responsible for the degradation of ECM.
Together, they can fully degrade collagen (Birkedal-Hansen, 1987). MMP1 is the only
MMP that can break down intact fibrillar collagen (Krieg et al., 1988), while the other
MMP types can further break down the already degraded collagen fragments. MMPs
mainly target connective tissue molecules and basement membrane proteins. MMPs

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Page 23 of 53
play a complex role in many physiological and pathological processes, including skin
aging.

Alternatively, ROS can affect ECM structures through the oxidation products of DNA,
lipid, and protein oxidation. These products can inactivate receptor type protein-
tyrosine phosphatase κ (RPTP-κ) with subsequent loss of its inhibiting function on
cell surface receptors. As a consequence, various cell receptors are free to bind to

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mediators, leading to activation of intracellular signal transduction pathways. This
applies to receptors for epidermal growth factor (EGF), TNF-α, and IL-1 on the

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membranes of fibroblasts and keratinocytes (Xu et al., 2006) (Fig. 9). These
receptors are so-called G-protein-coupled receptors, characterized by a GTPase

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enzyme coupled to the intracellular protein structures Ros, Rac and Cdc42. When a
ligand binds to this receptor, GDP is converted to GTP by GTPase (Coso et al.,
an
1995). Subsequently, various intracellular kinases (MAPK, ERK, etc.) will become
activated, of which certain members can also directly be activated by ROS (Fisher et
al., 1998) (Fig. 9). Ultimately, transcription factor complexes AP-1 and NF-κB can be
M
formed and regulate the transcription of MMPs. AP-1 and NF-κB levels have been
shown to be elevated within hours after low-dose UVB irradiation of the skin.
d

Activated NF-κB may contribute to the induction of heme oxygenase-1 (HO-1), which
e

elevates cellular levels of free iron that can promote further ROS generation via a so-
called Fenton reaction (Whitmarsh and Davis, 1996)(section 3.1).
pt

Besides activation of the MMPs, activated AP-1 and NF-κB can also have other
ce

consequences for cellular function. They control the transcription of the MMP tissue
inhibitor (TIMP), a family of four protease inhibitors that regulates MMP activity. The
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MMP/TIMP balance determines the rate of tissue degenera

tion (Hall et al., 2003) . Although MMP transcription is increased in UV-irradiated
fibroblasts, TIMP-1 expression is only slightly increased (Lahmann et al., 2001) and
TIMP-2 expression is not upregulated (Oh et al., 2004). This results in a
predominance of MMP activation over TIMP activity leading to degradation of
collagen and other fibrillar structures of the ECM, such as elastin and fibrillin
(Ashworth et al., 1999) (Fig. 9). Activated NFκB in fibroblasts also stimulates the
infiltration of inflammatory cells, such as neutrophils, by inducing the transcription of
proinflammatory cytokines IL-1, IL-6, VEGF and TNF-α. These mediators stimulate

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neutrophils to release neutrophil collagenase (MMP8) at the irradiated site that leads
to further matrix degradation (Sardy, 2009) and accelerated skin aging. These
proinflammatory cytokines also stimulate inflammatory cells to release NADPH
oxidase (Nox) enzymes. These intracellular enzymes generate superoxide anions
and are constantly produced at a low concentration by keratinocytes and
inflammatory cells to destroy bacteria through ROS, predominantly through
superoxide anions. In the course of UV-induced inflammation the production of Nox

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enzymes is increased, leading to enhanced ROS production and oxidative stress in
the concerned dermal tissue. Increased UV-induced transcription and subsequent

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activation of AP-1 also reduces the biosynthesis of collagen indirectly. AP-1-induced
downregulation of the transforming growth factor-β (TGF-β) type II receptor reduces

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the specific binding of the cytokine TGF-β. As a result, reduced phosphorylation of
the transcription factors Smad2 and Smad3 occurs, which impairs the whole Smad
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signaling pathway (Quan, He et al. 2004). This results in the reduced transcription of
COL3A1 and COL1A1 genes, that encode for precursors of collagen type III and I
respectively (Talwar et al., 1995), leading to a reduction in collagen type III and I in
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photoaged skin (Fig. 9).
d

4.2. Elastin damage

e

Typical for a photoaged skin, is the deposition of abnormal elastin. Histological

examination reveals course, deranged and highly branched elastic fibers that form
pt

aggregates of elastotic material (Naylor et al., 2011). This material is formed by a

combination of UV- or ROS-induced degradation of elastin, and biosynthesis of
ce

amorphous and dysfunctional elastin and fibrillin. Moreover, the increased

degradation of elastin is also caused by an elevated activity of the elastin-degrading
Ac

enzyme elastase. Macrophages, neutrophils and fibroblasts, that infiltrate after UV-
induced inflammatory effects, release various members of the elastase enzyme
family, among which are the MMPs (section 4.1) (Starcher and Conrad, 1995). Other
degraders of elastin are gelatinase B (MMP9) and stromelysin-1 (MMP3). They are
produced by activation of the intracellular MAPK pathway, either by ROS and/or
infiltrated inflammatory cells (Fig. 9). Repeated suberythemal exposures to UVB and
UVA in the skin showed increased deposition of abnormal elastin (Lavker et al.,
1995), which might be due to increased UV-induced ROS levels. ROS was shown to
enhance elastin mRNA levels in dermal fibroblasts (Kawaguchi et al., 1997). ROS

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and RNS generated during skin inflammation, can affect the assembly of elastic
fibers (Akhtar et al., 2010).

4.3 Glycosaminoglycane damage

GAG molecules are long polysaccharides consisting of glucosamine-, galactosamine-,
hexuronic -, iduronic acid and galactose units and sulfate groups at various positions.
They are important structures in the connective tissue, next to collagen and elastin.

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When complexed to a protein core, GAGs are transformed into proteoglycans. These
hydrophilic complexes gives support, strength and flexibility to the connective tissue

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and keep the tissue hydrated by their hygroscopic properties (Kjellen and Lindahl,
1991). GAGs do not directly absorb UVA, nor UVB and do not belong the

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chromophores of the skin. UV-induced damage to these structures in the framework
of photoaging occurs via indirect ways, such as ROS-induced oxidative damage to
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the disaccharide backbone of these compounds. A known member of the GAG family
for the skin is non-sulfated hyaluronic acid (HA) or hyaluronan (Fraser et al., 1997),
which is involved in migration of cells and differentiation processes for skin tissue
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repair (Tammi et al., 2005). Repetitive, chronic UVB exposure of mouse skin resulted
in loss of HA from the dermis (Dai et al., 2007).
e d
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ce
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5. Relation between exposure to ROS and aging.
The collected evidence for damage and degeneration by oxidative events during
aging raises the question: do we live shorter when massive oxidative degeneration to
tissue occurs? Results from animal experiments relating oxidative stress (the „free
radical theory‟) to aging and life span (Bakaev and Lyudmila, 2002; Chang et al.,
2012; Dai et al., 2009; Ishii et al., 1998; Ishii et al., 2011; Kim et al., 2008; Melov et al,
2000; Quick et al., 2008; Schriner, et al., 2005; Shibamura et al., 2009) are

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apparently conflicting with other studies that show the induction of low levels of
oxidative stress was even found to be beneficial with regard to lifespan and health.

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Low levels of oxidative stress even seem to induce resistance to degeneration, aging
and shortening of lifespan. This adaptive response, which is primarily related to the

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mitochondrion, is referred to as mitohormesis (Ristow and Schmeisser, 2014). ROS
are regarded as signaling particles in this adaptive response. The particular ROS
an
signaling agent may be H2O2. Lifespan extension was found in studies with yeast (S.
cerevisiae) and worms (C. elegans) that obtained H2O2-resistance elicited by caloric
restriction (Molin et al., 2011; Schulz et al., 2007). This effect was paradoxally
M
obtained by a controlled increase of mitochondrial ROS production, which apparently
triggers cellular metabolism to stress resistance. This effect is also referred as to
d

„redox regulation‟, from which phenomenon several pathways are reviewed (Ray et
e

al., 2012). It has also been recognized that aged cells show a reduced ability to
respond to cellular oxidative damage than in young organisms (Labunskyy and
pt

Gladyshev, 2013). Several in vivo studies with mice having the major antioxidant
ce

genes knocked out, do show the expected elevation in oxidative tissue damage, but
do not show a reduction of lifespan. The link between ROS, aging and lifespan may
therefore not be a simple link, but is likely more complex (Speakman and Selman,
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2011).

Despite the complexity, several in vivo studies are consistent with the „free radical
theory‟, as largely outlined in this review. Mitochondrial SDHC-mutations result in
premature death in C. elegans, Drosophila, cancer in mouse embryonic fibroblasts
and infertility in transgenic mice (Ishii et al., 2011). Aging was accelerated in
nematodes having a mutation in succinate dehydrogenase cytochrome b (Ishii et al.,
1998) and nematode lifespan extention was shown by antioxidants (Melov, 2000;
Bakaev and Lyudmila, 2002; Kim et al., 2008; Shibamura et al., 2009)

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Overexpressing mouse catalase, targeted to mitochondria, showed lifespan
extension (Schriner et al., 2005; Dai, 2009), as well as the administration of a special
mimetic SOD (Quick, 2008). From epidemiological studies a relation was found
between colorectal cancer and reduced activity of the catalase gene, indicating that
higher levels of H2O2 may mutate certain genes ending up in carcinogenesis (Chang
et al., 2012). Apparently, there is no indication of an adaptive response in this human
study.

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The conflicting results may be explained by differences in induced levels of ROS

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among studies, and the targeting of ROS to relevant cellular compartments. When
induction of ROS leads to moderate levels of ROS in mitochondria, vicious redox

us
cycles were proposed, in which ROS plays an intermediate role between intertwined
redox systems as NAD/NADH, NADP/NADPH and GSH/GSSG. However, when
an
ROS-levels become more elevated, it will lead to irreversible structural changes in
mitochondria and beyond (Ristow and Schmeisser, 2014; Dai et al., 2014). The
effects of mitohormesis in contrast to the free radical theory may yield confusing
M
outcomes from experiments aiming to determine aging or lifespan changes.
Experimentally induced ROS levels may be difficult to quantify, thereby implying that
d

experimental outcomes could easily be biased and yield unexpected results. Studies
e

with non-targeted antioxidant supplementation often show no effect on lifespan and

aging, which findings could mean a poor distribution/transport to mitochondria.
pt

Recent studies using mitochondria-targeted antioxidants look promising (Dai et al.,

2014), indicating that lowering ROS to standard levels counteracts on intrinsic aging
ce

and promotes healthspan.

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Page 28 of 53
6. Discussion and conclusions
In this review evidence is collected that multiple oxidation pathways lead to
degeneration and aging. Several studies from approximately 2006 have dampened
the relevance of the „free radical damage theory of aging‟, as also outlined in this
review. The correlation between elevated oxidative stress conditions and lifespan is
dependent on the extent of local ROS-levels induced. Tissue degeneration,
inherently linked to aging, becomes more significant through the lack of tissue

t
ip
regeneration. An example for the lack of tissue regeneration is the well-described
loss of telomeric ends leading to cellular senescence. Several pathways lead to

cr
cellular senescence, but the final decline of cells or tissue seems to be induced by
the effects of oxidative degeneration from endogenous or exogenous sources. Tissue

us
decline or aging may primarily be caused by oxidative processes. Senescence is a
status quo for individual cells, in contrast to oxidative degeneration, which is a
an
progressive event and also affecting whole tissue composition. It will finally prevail,
unless there is inexhaustible availability of antioxidants and massive tissue
regeneration, which will slow down the aging process.
M
Degeneration and aging phenomena proceed in a variable, individual degree,
d

initiated by oxidative events. The final outcome will differ for each person, depending
e

on genetic makeup and lifestyle. Regeneration or repair of cells/tissue is often not

sufficient for compensation and degeneration as well as aging phenomena prevail.
pt

The main structural components that confer an aged skin appearance are damaged
ce

protein and glycosaminoglycane structures that form the ECM. UV-induced damage
of DNA can lead to mutations and subsequent apoptosis or malignant
transformations of cells. UV-exposure can directly damage biomolecules, or indirectly
Ac

via the generation of radicals. Damage to these structures leads to cell death,
degraded proteins and inflammatory responses. Reactive intermediates, often
radicals, can further damage other biomolecules. These findings are generally more
clear from experiments in vitro than in vivo, due to complicated interpretation of
results obtained in vivo by potential involvement of adaptive responses
(mitohormesis). Many photochemical reactions in the skin, concerning endogenous
or exogenous components, are still unknown, and deserve further research to clarify
(photo)dermatoses and skin aging.

29
Page 29 of 53
Acknowledgement
The authors wish to thank Ms. A. van Lier for her initial literature search on this
subject and prof. dr. J.D. Bos for critical reading of the manuscript.

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Figure 1

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Ground state oxygen, O2

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Superoxide anion, .O2-

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Page 42 of 53
Figure 2

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Page 43 of 53
Figure 3

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Solar UV
intensity per wavelength

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Irradiance (W/m2 per nm)

UVB UVA

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0.00001

290 300 310 320 330 340 350 360 370 380 390 400
wavelength (nm)
Page 44 of 53
Figure 4

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Page 45 of 53
Figure 5

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Figure 6

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. . .. .

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sensitizer .O .O .
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UV / VIS -
Singlet oxygen, 1O2

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radiation

sensitizer*
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Page 47 of 53
Figure 7

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. . .. . . 1O + 2 Cl + 2 H2O

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2
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Superoxide anion, .O2-
2 HOCl

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hypochlorous acid
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O2
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H2O + O2

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Page 48 of 53
Figure 8

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Hydrogen peroxide, H2O2 Hydrogen peroxide, H2O2

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. . . .. . . . . . .. . .
A H. O . . .H
. . .O B H. O . . .H
. . .O

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UV radiation
< 320 nm

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H O . O .H H. O . . O. H
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Hydroxide-ion, Hydroxyl
OH - radical, .OH very reactive
very reactive

Fenton reaction H2O2 photolysis

Page 49 of 53
Figure 9

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Highlights

Highlights of ‘Oxidation events and skin aging’ by A. Kammeyer

Manuscript ref. ARR-D-14-00107-revised version.

 Skin aging is more influenced by extrinsic than intrinsic processes

 Due to extrinsic factors, skin aging differs from aging of other tissue

 Oxidative degeneration in the skin overrules anti-aging adaptive responses

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 Oxidative degeneration is the prime cause of skin aging, rather than
senescence

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 The rate of skin aging depends on individual genetic makeup and lifestyle

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Page 51 of 53
Figure captions.

Figure 1. The superoxide anion radical can be formed by the addition of an electron
to an oxygen molecule. The dots represent the electrons of one molecule O2 in the

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outer shell.

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Figure 2. Example of photoaged skin in the face and neck. Notable is the leathery,
coarsely wrinkled skin with reduced resilience. On the chest, intrinsically aged skin is

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visible, which has a smoother look. (taken from: Wlaschek, 2001)

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Figure 3. Solar UV intensity per wavelength in Albuquerque (380 N) at noon with
clear sky conditions. (taken from: Diffey, 2002).
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Figure 4. Pathways of solar UV radiation through the atmosphere and into our skin.
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Note the absorption of UVC by the ozone layer, high in the atmosphere.
p te

Figure 5. The UVB- or UVC-induced formation of cyclobutane-pyrimidine dimers

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from thymine (T) and cytosine (C) to form the dimer (TC-CPD) or pyrimidine-
pyrimidone (6-4) photoproducts (TC6-4PP), and Dewar isomers. (taken from:
Ravanat JL et al., UV damage to nucleic acid components, in: Sun Protection in
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Man, Ed: Giacomoni PU, Elsevier 2001)

Figure 6. Ground state oxygen is converted into singlet oxygen after reaction with a
sensitizer that is excited by UV radiation.

Page 52 of 53
Figure captions (cont.)

Figure 7. Superoxide anion can be converted into the less reactive hydrogen
peroxide by superoxide dismutase. Hydrogen peroxide can undergo subsequent

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reactions catalyzed by different enzymes to form other molecules like the reactive,

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cytotoxic hypochlorous acid (HOCl).

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Figure 8. The formation of very reactive hydroxyl radicals from hydrogen peroxide. A:
hydrogen peroxide can react with iron to form hydroxyl radicals and hydroxide-ions.
This is named the Fenton reaction. B: when hydrogen peroxide is exposed to UVB or

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shorter wavelength radiation, a photolysis reaction takes place where hydroxyl
radicals are formed.
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Figure 9. Schematic view of the influence of ROS on skin aging. ROS are produced
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by the mitochondria and extrinsic factors, like UV radiation or cigarette smoke. They
induce the MAPK pathway, which promotes the transcription of MMPs or the release
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of inflammatory cytokines that further activate MMP and ROS production.

Additionally, the production of procollagen type I and II is reduced. (taken from:
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Sardy, 2009).
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