**3. Role of ultraviolet radiation**

The demonization of UVR has occurred due to several investigations conforming its deadly role in eliciting different skin cancers. Consequently, it has been declared as a 'Type I carcinogen' by International Agency for Research on Cancer (IARC). Particularly of concern include UV-A and UV-B radiations. Major contribution of these radiations results in formation of photolesions such as cyclo-butane pyrimidine dimers (CPDs), (6–4) photoproducts (6–4 PPs) together with Dewar isomers through direct or indirect mechanisms [5]. CPDs generally stop advancement of cell cycle by activating the repair machinery whilst 6–4 PPs can prevent apoptosis from occurring [6]. UV-B halts maximum cells in S phase of cell cycle and triggers both p95 and p53. This does not seem to take place for UV-A which halts the cell cycle at S phase only for a limited time. Thus, lesions arising considering UV-A have a higher potential of causing mutation due to lack of active p53 which is vital for stimulating XPC, GADD45 and p48 that are required in turn to induce the Nucleotide excision repair pathway [7, 8].

Moreover, UVR act to dissociate Helicase and DNA polymerase during replication in the S-phase of cell cycle. Consequently, although Helicase keeps on generating ssDNA regions, DNA polymerase stops on encountering a lesion. If a 6–4 PP formation occurs in these ssDNA regions it can both slow repair process while stimulating ATR-Chk-1 pathway. Given the benefits of activating this pathway, one problem still resides which is the elevation of mutation level. Thus, unchecked 6–4 PPs can promote existence of cells portraying higher mutation [9].

*Skin Aging: Implications of UV Radiation, Reactive Oxygen Species and Natural Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.100102*

Formation of these UV induced photoproducts is not equally distributed throughout the genome, but depends on several factors. Formation of CPDs and 6–4 PPs rely upon nucleosome structure, nucleosomal DNA rotational setting, binding of proteins to transcription factor binding site, chromatin states and particularly certain hypersensitive sites [10]. Telomeres can function as an excellent example of hypersensitive sites prone to UVR. They portray several repeats of TTAGGG sequences and end in the single stranded form at the 3′ end. This end folds and forms a T-loop in assistance with proteins at the ending of a chromosomes thus inhibiting fusion with other chromosomes [11]. Formation and further accumulation of CPDs in such distinct sequences can be high due to near about absence of damage repair [12].

Another factor involves the differential penetration of UVR through the skin layers. As skin embodies numerous cells, so CPD formation could alter with cellular functions. This analysis can be proved as occurrence of 8-oxo dGua and CPD were observed more in fibroblast as compared to keratinocytes under UV-A irradiation. Explanation for this could be as of disparity in endogenous antioxidant activity and distribution of molecules that act as photosensitizers [13, 14]. UVRs particularly UV-B are also held accountable for strand breaks and inception of modified bases such as cytosine photohydrate, thymine glycol 5,6 dihydro thymine and 8-oxoguanine among others [15].

### **4. Role of reactive oxygen species**

An eminent feature of UVR is rise of ROS and reactive nitrogen species. They can either constitute of free radicals or other molecules that are highly toxic or mutagenic to the cell. A free radical is an atom or molecule which contains unpaired electrons in its outer orbit. Due to this property some free radicals are highly unstable and react with other surrounding molecules. They snatch an electron via reacting with another molecule and convert them into a free radical. This free radical generated performs the same process thus initiating a string of events. Among the ROS present, the 'red alert' species include hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide radical and superoxide radical [16].

Main pit for ROS production is the mitochondria. Till now, ten sites have been claimed in mitochondria to form ROS. Multiple sites are either linked to Electron Transport Chain or the Kreb cycle. Complex I has two sites for superoxide formation 1) the Flavin in NADPH oxidizing site 2) the ubiquinone reducing site. Complex III has one site for superoxide production, the quinol oxidizing site. Certain enzymes also take part in ROS generation such as glycerol-3 phosphate dehydrogenase, 2-oxoglutarate dehydrogenase and xanthine oxidase [17]. ROS production inside the cell also results during endoplasmic reticulum (ER) stress. ER is of high priority as it controls protein folding, attachment of additional groups after translation and regulating protein trafficking, to name a few. ROS mediated protein-alterations converts them into displaying an unusual protein structure which can get deposited in the lumen of the organelle. Consequently, this could inhibit translation of mRNA into proteins to limit the damage [18].

ROS concentration is also modulated by pH. Conventionally, the pH in stratum corneum approximates around 4.5 [19]. This value increases and at subsequently deeper layers a pH of about 7.0 is established [20]. Maintenance of this pH gradient is crucial as for its participation in specific skin functions such as defense against

microbes, recovery from injuries and activities of various proteases. This balance is however impaired with aging especially in people eighty years old or above [21]. Imbalance in pH gradient has a detrimental effect on skin from both chronological and photoaging perspective as it influences the photosensitizers contributing to ROS levels. For example, many photosensitizers such as riboflavin, porphyrins, quinones among others comprising of a carbonyl group employ type II mechanism of photosensitization for ROS production wherein energy absorbed by them is transferred directly to a O2 molecule. From this reaction, rise singlet oxygen and superoxide anion [22]. Other sources for ROS generation include 3-hydroxypyridine, N-alkyl 3 hydroxy pyridine and their respective derivatives such as pyridoxine, pyridoxal, pyridoxamine including others. They catalyze reactions by following type I mechanism of photosensitization [23].

Significant contribution towards ROS generation is lent by NADPH oxidase. Irradiation with UV increases the Ca2+ ions concentration in the cell. This increase in the Ca2+ ions level triggers NADPH oxidase to generate ROS species apart from activating Protein kinase C which can phosphorylate Rho GPIα thus permitting Rac to migrate towards the plasma membrane where it could bind to Nox1, NoxO1 and NoxA1 complex. This binding results in stimulating Nox1. Interestingly, UV-A irradiation can produce ceramides in keratinocytes. The underlying mechanisms have not been figured out yet but it is hypothesized that ceramide could activate PI3K which stimulates Rac1 by guanine nucleotide exchange factor β-Pix [24, 25]. Furthermore, leukotriene B4 (LTB4) is also associated with lysophosphatidic acid stimulated ROS level elevation in keratinocytes. LTB4 is synthesized from arachidonic acid by action of several enzymes including 5-lipoxygenase (5-LO). It behaves as a ligand and binds to receptor BLT2 which is expressed ubiquitously in all cells. Moreover, elevation of expression of LTB4 and BLT-2 takes place in keratinocytes under UV-B. BLT-2 triggers ROS levels by activating Nox-1. This event is ensued by stimulation of p38/JNK pathway in keratinocytes [26].

Lipid peroxidation involves lipid molecules reacting with free radicals to produce lipid peroxyl radicals along with hydroperoxides. Other significant outcomes include hexanal, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). 4HNE exhibit electrophilic properties and thus react with nucleophiles such as lysine's amino group, imidazole group of histidine and sulfhydryl group of cysteine. Certain metal ions like iron are of significance as lipid hydroperoxides on interacting with transition metals can form lipid peroxyl radicals or lipid alkoxyl radicals [27]. Un-chelated iron produce hydroxyl radical from hydrogen peroxide which can help initiate the lipid peroxidation process [28]. Further irradiation with UV-A has pointed towards elevation of intracellular peroxide levels. Fatty acids, specifically arachidonic acid serve as prime target of these radicals [29, 30]. The iron driven fenton reaction generates hydroxyl radicals along with many lipid peroxides which through the JNK-2 pathway elevate various matrix metalloproteinases (MMPs) [31]. 4 HNE is responsible for elastin modification which could potentially lead to actinic elastosis. 4HNE and acrolein are deposited on elastin fibers which decreases elastin fiber digestion by the elastase released by leukocytes [32].
