5.1. Anti-Oxidative Properties of the Cornified Envelope
To cope with these many sources of ROS the skin has developed sophisticated and in part very skin-specific anti-oxidative mechanisms. Most of the anti-oxidants show in fact a higher concentration in the epidermis than in the dermis [
113]. This correlates well with the fact that the ROS load is higher in the epidermis than in the dermis. The epidermis is built up in a very gradual way and displays an increasing calcium concentration from the
stratum basale to the
stratum granulosum where a peak is reached. Also the cornified envelope gradually increases in its density. The formation of the cornified envelope starts in the
stratum spinosum and is fully assembled in the
stratum corneum. An epidermal concentration gradient is also found in the case of anti-oxidants, especially the low-molecular-weight ones. Vitamin C, vitamin E, glutathione, ubiquinol, and uric acid are detectable in the
startum corneum, but their concentration increases steeply towards deeper cell-layers of the
stratum corneum [
105,
114]. These comparably low concentrations of non-enzymatic and lipophilic anti-oxidants in the outer layers of the
stratum corneum are possible, because the cornified envelope itself has anti-oxidative capabilities. These anti-oxidative capabilities of the cornified envelope rely on the SPRR proteins. Members of this protein family are not only rich in prolins but have an over-proportional enrichment incysteines. Therefore these proteins can quench ROS by forming intramolecular disulfide bonds. Interestingly these anti-oxidative properties were mainly found for the SPRR2 subfamily. This fact can be explained by different accessibilities of the cysteine residues of these cornified envelope proteins [
113]. According to Harman’s idea [
2] ROS levels increase in the aging process. Indeed, we have found that the CE is dramatically altered in the aging process. Based on our own work [
72] we argued that the loss of loricrin is compensated by increased levels of SPRRs. The biggest changes were found for the SPRR2 subfamily. In the light of the anti-oxidative capacities of the cornified envelope this increase in SPRRs during the aging process represents a valid tool to cope with the increasing ROS levels during aging. Below the
stratum corneum another, upside-down gradient of anti-oxidative substances and enzymes is found. In this gradient the highest concentrations of enzymes and anti-oxidants are found in the
stratum granulosum constantly declining towards the
stratum basale [
115]. In this way the suprabasal cells have lower ROS levels and are protected against UVB-induced apoptosis [
116]. The importance of the CE as an anti-oxidant/UV barrier is also stressed by the fact that UV can completely deplete the
stratum corneum of anti-oxidants/vitamins [
117]. Therefore only the remaining CE proteins (mainly SPRR2 subfamily) can exert their anti-oxidative properties and protect the epidermal cells.
5.2. The Non-Enzymatic Anti-Oxidants Vitamin C, Vitamin E, Beta-Carotene and CoQ10
The strong anti-oxidant L-ascorbate/vitamin C cannot be synthesized by primates and therefore has to be taken up with food [
118]. The water soluble vitamin C itself is an electron donor and is used as a cofactor for enzymatic reactions such as the crosslinking of collagen. Vitamin C is very prominent and the most abundant of all anti-oxidants [
106]. In addition this anti-oxidant can react with a potential dangerous free radical and can donate its electron. In this way vitamin C itself is oxidized and forms so called “semidehydroascorbic acid”. The big advantage of the resulting radical is that it is stable and comparably unreactive. This radical can either be reduced back or can react further to dehydroascorbic acid [
119]. It was shown that vitamin C has a strong effect on photoaged skin, most probably by quenching ROS that originate from UV-irradiation [
120]. It was found that the amount of ascorbate decreases in both intrinsic skin aging as well as extrinsic aged/photoaged skin [
121].
The second vitamin with anti-oxidative capacities is α-tocopherol/vitamin E. Vitamin E is more than one compound, but the most important one in humans is α-tocopherol. Similar to vitamin C α-tocopherol has a very important photoprotective and anti-photoaging role in the skin [
122]. In contrast to the water soluble vitamin C, vitamin E is lipophilic and is found in animal membranes. It can be nutritionally supplied by plant oils. The anti-oxidant α-tocopherol is highly important because it can stop ongoing lipid peroxidation, by the reduction of the lipid peroxyl radical to hydroperoxide. In the course of this detoxification process this anti-oxidant loses a proton and is itself transformed into a radical. However, the α-tocopheroxyl radical is not very reactive and is further on detoxified by ascorbate, glutathione, or enzymes [
123]. Recent literature also indicates that α-tocopherol exerts its photoprotective and anti-aging functions not only via its anti-oxidative role but also due to its role as activator/mediator of different signaling pathways. It has, for example, been shown that the protein kinase C pathway is affected by vitamin E [
124]. In the aging process the levels of α-tocopherol are unaffected in the dermis, whereas a clear decrease of this anti-oxidant was observed in the epidermis [
121]. Cutaneous application of vitamin E ameliorates photoaging, decreases lipid peroxidation and furthermore also reduces photocarcinogenesis, MMP-1 transcription levels and thymine dimer formation [
106].
Beta-Carotene is produced by plants and bacteria and also has to be taken up by food. This substance is a provitamin for retinol. In addition it has been shown that this precursor of vitamin A has a huge effect on skin aging and photoaging by either scavenging radicals or inhibiting lipoxygenases that are capable of producing ROS as discussed above [
125,
126]. Beta-carotene, a typical skin carotene, is anti-oxidative because the peroxyl radical is directly added to its backbone forming an epoxide that is decomposed afterwards [
127].
The next anti-oxidant discussed here is somehow ambiguous. Uric acid on the one hand is the final product of the degradation of purines and is created by an enzyme that itself is capable of producing ROS as discussed above. On the other hand it is an anti-oxidant. Similar to ascorbate, uric acid is a reductant for ROS and can scavenge radicals such as hydroxyl radicals, singlet oxygen, and oxo-heme oxidants. By absorbing one electron, uric acid itself is transformed into a radical, although not very reactive [
128]. It was also shown that uric acid is the main anti-oxidant in serum [
129]. Therefore the contribution to the anti-oxidative capacity of the skin is comparably low as the skin has a low blood supply. Moreover, it was demonstrated that the extracellular urate is a potent anti-oxidant but acts as a pro-oxidant within the cell [
130].
The last enzyme-free anti-oxidant discussed in this review is CoQ10. CoQ10 is known because of its contribution to the mitochondrial ETC. Ubiquinone is reduced to ubisemiquinone and ubiquinol at complex I and II and oxidized back to ubiquinone at complex III [
131]. Besides this important contribution to the ETC, ubiqinone has also been described as an anti-oxidant. The lipid soluble CoQ10H2 is a chain breaker in lipid peroxidation and protects lipids from lipid peroxidation [
132]. In comparison to vitamins C and E, ubiquinone seems to be ineffective in photoprotection [
132].
5.3. The Importance of Superoxide Dismutases, Catalases, Glutathione Peroxidases, Ferritin, and Peroxiredoxins in Quenching ROS
Among the most prominent enzymes that can handle reactive oxygen species are the superoxide dismutases. These enzymes “dismutate” superoxide to hydrogen peroxide [
133]. In mammals three isoforms can be distinguished that differ in their localization. The enzyme SOD1 is found in the cytosol and nucleus and has Cu/Zn as cofactor, SOD2 is found in mitochondria to dismutate superoxide originating from the mitochondrial ETC and binds Mn
2+, and SOD3 is found in the extracellular space harboring the metal ions Cu/Zn in its active center [
134]. In the first half-reaction the electron from the superoxide radical is transferred to the metal ion in the active center thereby reducing it. The superoxide itself is oxidized to O
2. In the second half-reaction the reduced metal in the superoxide enzymes is reoxidized by transferring the electron to superoxide resulting in the formation of hydrogen peroxide [
135]. All three human superoxide dismutases have a huge impact on aging skin. Generally a deletion of superoxide dismutase is lethal as demonstrated in mice, but with SOD mimetics life can be prolonged for several weeks.
Sod1−/− mice show a clear skin atrophy that is also observed in aged individuals [
136,
137]. In case of SOD2 deletions the phenotypes are even more dramatic. UV irradiation leads to the above discussed mtDNA deletions and results in a burst of radicals from the defective mitochondrial ETC. Not surprisingly, UV irradiation results in a dose dependent increase in SOD2 mRNA levels in wildtype mice [
138]. Although SOD2 overexpression had no obvious life prolonging effects [
139], distinct skin aging phenoytpes were observed in
Sod2−/− mice. These phenotypes comprise a thinning of the epidermis, a clear atrophy of the dermal connective tissue, a reduced amount of procollagen I, and an atrophy of the subcutaneous fat tissue [
140,
141,
142]. The SOD3 enzyme is expressed in the dermis as well as in the epidermis. By harboring a heparin-binding domain this enzyme is in close contact with the extracellular matrix and cell surfaces. In contrast to SOD1 and SOD2 very high doses of UV are needed to induce the expression of SOD3. Therefore the role of SOD3 in the skin is unclear although it has been shown that SOD3 is involved in skin inflammation and its expression is reduced in psoriasis [
134,
143].
A very prominent enzyme that detoxifies hydrogen peroxide is the peroxisomal localized catalase [
144]. This enzyme consists of four identical polypeptide chains, each harboring a heme group [
145]. In a first step hydrogen peroxide reacts with the heme group leading to an oxoferryl porphyrin cation radical and a water molecule. The so called compound I is very active and reacts immediately with a second hydrogen peroxide molecule producing water and molecular oxygen and regenerating the original prosthetic heme group [
146]. The catalase enzyme is very prominently expressed in the skin, especially in the
stratum corneum. The amount of catalase exceeds the amount of superoxide dismutases. Inside the
stratum corneum a gradient of activity, with a decreasing activity towards the surface of the skin, was detected [
147,
148]. In the aging process the activity of this enzyme is altered with a widening gap between the dermis and epidermis. Thus, catalase activity decreases in the dermis and increases in the epidermis of aged and photoaged skin. Because the ROS load of the cells, especially in the epidermis, increases with aging, increasing catalase activity is reasonable, whereas the reduction of catalase in the dermis remains mysterious [
148,
149]. A remarkable experiment showed that by targeting the peroxisomal catalase to mitochondria a statistical significant increase in medium and maximum lifespan was found in mice [
150].
A main contributor to the anti-oxidative potential of the cell is the tripeptide glutathione GSH, harboring a special gamma peptide linkage. This peptide is synthesized in a two-step process. The first step is performed by the gamma glutamylcysteine synthetase, the second step by the glutathione synthetase. The GSH acts as an anti-oxidant because of its thiol group. In the course of the process GSH is oxidized by reactive oxygen radicals and forms a dimer with another activated GSH via formation of a disulfidic bond (GSSG). GSH can be recovered in a reducing step by the glutathione reductase consuming NADPH [
151]. GSH not only detoxifies ROS, but can also regenerate oxidized α-tocopherol and retinol [
106]. In aged mice it was shown that both, the absolute amount of GSSG as well as the GSSG:GSH ratio strongly increases in the dermis in comparison to young skin [
152]. In photoaged skin the concentrations of glutathione are reduced, but this effect could be compensated by an increased activity of the glutathione reductase [
121]. It is estimated that in aged skin the concentration of anti-oxidants is strongly decreased, in line with this the levels of α-tocopherol, ascorbate and GSH have been shown to be reduced by 70% [
121]. The function and (inter)action of all anti-oxidants is deeply interwoven to keep the redox state in the skin tissue in balance. For example, vitamin C can reduce oxidized α-tocopherol and is itself oxidized; glutathione in turn can rescue vitamin E and the resulting GSSG is converted into GSH again by the glutathione reductase enzyme [
106].
Beside its role as an anti-oxidant, GSH is also a cofactor for enzymatic reactions. The glutathione peroxidase is an enzyme that fulfills two tasks: reduce hydrogen peroxide to water and stop lipid peroxidation. In humans, eight glutathione peroxidases are known, five of them containing selenium as a co-factor. In a first step, the peroxide, either lipid or hydrogen, oxidizes the enzyme bound Se, thereby forming SeOH. In a next step the enzyme reacts with a thiol group in GSH resulting in the formation of a selenylsulfide bond between the enzyme and the glutathione. A reaction with a second GSH regenerates the enzyme and GSSG is formed [
153,
154]. Alterations in the enzyme activities in aging skin and photoaged skin have not yet been characterized, however a targeted disruption of the glutathione peroxidase 4 in mice displayed severe skin phenotypes, like hyperplasia of the epidermis, dermal inflammation, increased rates of lipid peroxidation, and higher levels of the cyclooxygenase-2 [
155].
As already mentioned, free iron ions are a constant threat for the cell because the Fenton reaction is capable of starting a vicious cycle of ROS production in the cell, ultimately leading to its death. Therefore the cell has to conceal the iron ions very carefully. This iron storage is achieved by the protein ferritin. The protein consists of 24 subunits forming a sphere that surrounds the iron. The iron is only stored in its Fe(III) form as ferrihydrite and upon its release it has to be reduced to the Fe(II) form. The 24 subunits can be divided into two subtypes: the heavy (H)-type and the light (L)-type. The L-type is involved in the core-formation, the H-type in the Fe(II) oxidation [
156]. Ferritin is primarily stored in the cytosol, although mitochondrial and nuclear forms are also known. The iron release is also dependent on lysosomal ferritin degradation [
157]. Ferritin seems to be an important tool in the regulation of the redox homeostasis especially after UV irradiation. The highest concentrations of ferritin in the skin are found in the
stratum basale [
158]. The levels of ferritin in the epidermis are around three to seven fold higher than in the dermis. After UV irradiation, especially UVA, the levels of ferritin in the dermis as well as epidermis, strongly increase, indicating a potential anti-oxidative mechanism to stop ROS production in cells after disturbing the redox homeostasis [
158,
159]. However, the combination of iron storage, ferritin, and UV irradiation also has detrimental potential. It was demonstrated that UVA irradiation of primary dermal fibroblasts induces an immediate degradation of ferritin in the lysosomes, followed by a release of iron ions into the cytosol accompanied by a burst of ROS [
160]. The acceleration of skin aging in females after the menopause was also attributed to iron and ferritin. In females there are two ways to get rid of excessive iron: menstruation and desquamation. After the menopause the excessive iron ions are stored in the skin via ferritin and this could contribute to an increase in ROS levels that accelerates the aging process in the skin [
161].
The last class of enzymes that have anti-oxidative capacities and are discussed in this review are the peroxiredoxins. In mammals six isoforms were identified, whereas 2-Cys enzymes (PRDX1-5) and 1-Cys enzymes (PRDX6) can be distinguished [
162]. In the following, only the 2-Cys enzymes will be discussed. A peroxide substrate reacts with a conserved cysteine in the active center of these enzymes leading to the formation of a sulfenic acid residue. This is followed by a reaction of the sulfenic acid with a second cysteine (therefore the term 2-Cys enzyme), thereby forming an intra-molecular disulfide bond [
151]. The enzyme is regenerated by a flavoprotein disulfide reductase such as the thioredoxin reductase [
162]. More details of the function of this enzyme can be found in the Chapter “Oxidative stress in fungi” in the same issue. High levels of PRXD1-3 were found especially in the epidermis but also in the dermis of rats. Similar to the calcium distribution, PRDX1 and PRDX2 were found with increasing concentrations towards the
stratum granulosum. PRDX3 showed the opposite distribution. The highest concentration was found in the
stratum basale, the lowest concentration in the
stratum granulosum [
163]. The peroxiredoxins seem to be very important in the detoxification of ROS originating from UV irradiation. UVB-irradiation induced the expression of PRXD2, UVA the expression of PRDX1 [
163,
164]. Overexpression of PRDX6 leads to significantly reduced levels of oxidized lipids in mice and results in a reduced rate of UVB and UVA induced apoptosis, whereas loss of PRDX6 leads to an increased skin tumor rate [
165,
166]. There is also growing evidence that an increase of activity of peroxiredoxins has great potential in increasing lifespan [
167].
5.4. The Anti-Oxidant Treatment Paradox
An increase in ROS levels over time is a common feature of all human tissues and especially of the skin. Therefore many attempts were made to quench these ROS by topical treatment of the skin or supplementation with anti-oxidants, in the hope to improve or even rejuvenate aged skin. But the results are very controversial and are heavily disputed in the literature. Just recently it was demonstrated that a mixture of alpha hydroxy acids, vitamins B
3, C, and E applied on facial skin dramatically improves the quality of the epidermis and dermis including the smoothening of wrinkles and the refinement of skin texture without side effects [
168]. A similar effect was found on treating aged and photodamaged skin with a special combination of several anti-oxidants consisting of resveratrol, baicalin, and vitamin E. These antioxidants were partially sufficient to rejuvenate aged skin [
169]. Resveratrol was shown to stimulate the Nrf2 pathway in skin leading to an increase in the GSH content and improvement of skin quality [
170]. Also CoQ10 conjugated with nanoparticles (to improve the skin permeability) seems to have a beneficial effect on skin quality [
171,
172]. Some additional substances that seem to have a positive effect on aged skin, especially the epidermis, are summarized in Lorencini
et al. [
173]. Though many studies promote the use of antioxidants for preventing skin aging, others warn of potential side effects. Treatment of various model organisms with vitamin C gave a broad variety of results ranging from a prolonged lifespan to “no effect” [
174]. Surprisingly, no beneficial, statistically significant effect on lifespan elongation was found for food supplementation with vitamin E [
175,
176]. In the small rodent
Microtus agrestis supplementation with vitamin C and E led to a remarked reduction of lipid peroxidation, as expected, but significantly reduced the lifespan of this organism [
176,
177]. Also CoQ10 can lead to both, an increased (mice, nematodes) or decreased (
S. cerevisiae) lifespan [
176,
178,
179,
180]. Recent literature also warns of the excessive use of vitamins. Oral administration of beta-carotene, vitamins E and A in humans seems to lead to a higher mortality rate [
181] or increased risk of diseases [
182]. Quite surprisingly it was shown that increased ROS levels and increased oxidative damage can even lead to an increase in lifespan [
183,
184]. This controversy can be explained by the fact that ROS are not exclusively detrimental for cells, but can even be beneficial. There is growing evidence that ROS, especially hydrogen peroxide, have an important role in cells as a second messenger [
96,
185]. Therefore it is not desirable to quench away all the ROS, because this influences the ROS homeostasis of the cell with such detrimental effects as promoting tumor formation [
186]. A beneficial effect on skin aging by a treatment with antioxidants can onlybe achieved if the original ROS level of healthy cells is preserved.