The photoinduced inflammatory response is the result of a series of photochemical reactions taking place after the absorption of non-ionizing radiation by skin chromophores, each of them possessing a different absorption spectrum. At room temperature, most of the molecules are present in their ground state and as a result of absorption of radiation of a given energy (or of a specific wavelength) they undergo an electronic transition to an excited state. The nature of this excited state can be a singlet (if all the electrons have their spins paired) or a triplet (when there is an unpaired electron). Depending on the chromophore and on its environment, the lifetimes of these states can range from picoseconds or nanoseconds (for singlets) to microseconds (for triplets), and these lifetimes can be long enough to permit reactions to occur from these excited states resulting in chemical changes that generate different photoproducts known as free radicals or ROS. It is now well established that both species are continuously produced in vivo. Oxygen radicals can induce a number of disruptive cellular processes, including lipid peroxidation, DNA cleavage, altered enzyme activity, polysaccharide polymerization, and cell death. Because the production of radicals is a physiological process, the cells have developed several mechanisms to minimize the effects of these oxyradicals. The organism possesses relatively small molecules (a-tocopherol, ß-carotene, ascorbic acid, etc.) as well as more complex enzymatic systems (superoxide dismutase, catalase, thioredoxin reductase, glutathione peroxidase and reductase) for antioxidant purposes. Most of these UV light-induced cutaneous pathologies will be discussed later in this review.
There is a dose-response relation between the UV-induced erythema and the wavelength of irradiation. Solar radiation of shorter wavelength (UVB region, 290-320 nm) results in both epidermal and dermal changes. However, most UVB is absorbed by chromophores localized mainly in the epidermis, such as nucleic acids, amino acids, urocanic acid and melanin. Many of the chromophores act as protective agents against UV radiation. Melanin is the main chromophore in the epidermis absorbing photons of wavelengths ranging from 350 to 1200 nm.
The degree of the skin response to ultraviolet radiation depends on the localization and distribution of the chromophore as well as on skin thickness. Figures 1-5 summarize the photochemical reactions occurring when skin chromophores absorb UV radiation.
Of great photobiological interest (19,20) are the chemical changes of amino acids absorbing ultraviolet radiation. Tyrosine and tryptophan are the most common aromatic amino acids absorbing in the UVB region. At physiological pH, tryptophan (Trp) has an absorption maximum at 290 nm. Upon photon absorption, one of the reactions from the singlet excited state (Trp(S1)) involves photoionization and the generation of the tryptophan radical cation (Trp.+). The photoejected electron can react with molecular oxygen (3O2) forming the superoxide radical anion (O2.-). This reactive oxygen species commonly dismutates to form hydrogen peroxide, H2O2,and, in the presence of catalytic amounts of copper or iron metalloions (Haber-Weiss/Fenton reactions), can generate the hydroxy radical .OH that can produce damaging effects on biological systems. Trp(S1) can also undergo intersystem crossing to its triplet state (Trp(T1)) and react with 3O2 generating organic peroxyl radicals (.TrpOO.). Trp(T1) can also generate 1O2 through a type II reaction (i.e., energy transfer, see Figure 1).
It is commonly accepted that even if tyrosine (Tyr) absorbs radiation in the UV region it ultimately transfers the absorbed energy to tryptophan residues in proteins (20). In vitro studies have demonstrated that the 254 run photolysis of Tyr yields dopamine as one of the main photoproducts (21). After a complex series of reactions, polymerization to melanin occurs (Figure 2).
The nitrogen bases are the main chromophores in nucleic acids capable of absorbing ultraviolet radiation in the 250-270 nm range. Photochemical alterations in nucleic acids have a major impact at the cellular level, leading to cell death, mutagenesis and photocarcinogenesis.
A variety of photoproducts have been identified (22-25), with the cyclobutyldimers and photohydrates of the pyrimidine bases being formed at higher levels (Figure 3). The formation and yields of these products vary both with the nature of the base and the sequence in the nucleic acid strand. Cyclobutyldimers between adjacent thymines are produced at higher yields than any other pyrimidine combination and so are the cytosine photohydrates. Another product involving covalent coupling of two pyrimidine bases is the pyrimidine-(6-4)-pyrimidone photoadduct, formed in greater yields in thymine-cytosine sequences. Early observations have demonstrated that purine nucleic acid components and particularly guanine, as the free base or the related nucleoside and nucleotides, are more readily photooxidized by a variety of photosensitizers than are their pyrimidine analogs. Figure 5 shows the guanine radical intermediates from type I photoreactions and from the .OH reaction giving rise to identical decomposition products. Guanine can also be attacked by singlet oxygen in a type II photoreaction. These radical intermediates can be employed as a diagnostic tool for the assessment of photooxidative damage to DNA.
The cell possesses specific enzymes capable of repairing some of these photoproducts (i.e., endonucleases) and loss of activity of these enzymatic systems (i.e., xeroderma pigmentosum) increases the likelihood that damage will persist in the genetic material after UV exposure.
Urocanic acid (UA), a deaminated histidine, is the main chromophore absorbing UV radiation in the stratum corneum. It is produced by the action of the enzyme histidase on the amino acid histidine. The absence of urocanase in the epidermis prevents the transformation of UA into the imidazolone propionic acid. Several investigators (26-28) have suggested that UA is a natural sun block since its absorption spectrum covers the region from 240-300 nm (maximum at 275 nm) overlapping the main erythematogenic region (i.e., 290-310 nm). The trans-isomer of UA is naturally present in the skin and after absorption of photons undergoes photoisomerization to the cis-isomer (Figure 4) which mimics several of the immunomodulatory effects of UV radiation.
The majority of the reactive species generated by the action of UV radiation on skin chromophores are radicals. Free radicals are, by definition, chemically active species which possess an unpaired electron in their orbitals. Biological systems are usually exposed to different types of radicals generated either endogenously or as a result of some exogenous injury. Radicals can undergo addition or electron-transfer reactions with different cellular components and have thus been implicated in the ethiopathogenesis of several diseases.
Free radicals can be neutral or charged species. An example of a neutral radical is the thyoil radical (RS.) that can be produced by direct H-atom abstraction from a thiol group by another organic radical. This radical can be generated by the activation of thioredoxin reductase, an enzymatic antioxidant system (29). This epidermal membrane-associated free radical scavenging system that catalyzes the reduction of oxygen radicals to peroxide, although widely distributed in a variety of organisms and tissues, and located preferentially on the outer membrane surface in human epidermis, may be the first line of defense against free radical production induced by UV. The thioredoxin reductase/thioredoxin system has been implicated in a number of other antioxidant reactions (29).
Pyridinyl radicals are radical cations (i.e., positively charged radicals) which are involved in the formation of the chemical structure of two important coenzymes, NAD+ and NADP+ (30). The best known negatively charged radical is the oxyradical O2.-. The superoxide radical anion can be generated by different compounds and physical agents, such as ionizing radiation, ultraviolet radiation, hyperbaric oxygen and photosensitizing agents (31,32).
Although most of the oxygen consumed during cellular respiration is reduced to water by cytochrome C oxidase (33), a small amount of oxygen undergoes sequential stepwise univalent reduction and this is enough to produce a sufficient quantity of the superoxide radical and, ultimately, different ROS. These reactive species affect a variety of biological processes by damaging cellular membranes, enzymes, lipids, nucleic acids, and mitochondria by affecting the electron-transport chain. Among many processes that contribute to the generation of ROS are 1) the autooxidation of metabolites such as hydroquinones, thiols, hemoglobin (34), and catecholamines (33); 2) the enzymatic oxidation mediated by xanthine oxidase (35), aldehyde oxidase and orto-hydrodehydrogenase (31), and 3) the autooxidation of reduced species of the electron-transport chain generated from organelles such as mitochondria, microsomes or nuclei (36-38).
Oxidative stress may be regarded as an imbalance between the production of free radicals and their defense mechanisms. Such inefficiency of defense mechanisms may be due either to their relative deficit or inaccessibility or to their exhaustion following excess free radical production. Oxidative stress can be prevented or repaired by interventions that antagonize metabolic pathways of free radical generation, or that emulate or amplify physiological defense mechanisms. This toxic effect generated by hyperoxia or the inappropriate metabolism of oxygen is a well-defined cause of toxicity in biological systems. Most of the ROS generated after exposure of the skin to solar radiation (39-41) have a relatively short lifetime. Cellular injury caused by ROS involves generation of superoxide anion and hydrogen peroxide, but the most reactive mediator of such damage, however, appears to be the hydroxyl radical. Hydrogen peroxide is produced directly by a variety of oxidases and is also produced by dismutation of the superoxide anion (42-44).
This molecule must be considered dangerous because its small size and lack of charge allow it to diffuse across biological membranes and then, by reduction of H2O2 mediated by catalytic amounts of metal cations such as iron or copper in a reaction called iron-catalyzed Haber-Weiss reaction or sometimes Fenton reaction, produces OH- and .OH (32,43,45). The relevant reactions are:
Another reactive oxygen species formed from molecular oxygen is 1O2. In biological systems, 1O2 is generated by absorption of incident light of specific wavelengths by excitable endogenous or exogenous molecules known as photosensitizers. This type of photosensitization is known as type II (46,47). A large number of sensitizers occur naturally in organisms (riboflavin, 4-thiouridine and 2-thiouracil, bilirubin, etc.), but many others need to be added exogenously to the system. The energy of the triplet excited state for the sensitizers is then transferred to an adjacent triplet (unexcited) oxygen molecule, raising molecular oxygen to the singlet oxygen. There are two excited states of 1O2 of 23 and 37 kcal, the latter having the shorter lifetime. Even if the energy of the photons in the ultraviolet and visible regions is enough to generate singlet oxygen, O2 does not absorb at these wavelengths; therefore, 1O2 can only be generated, as established before, by an energy-transfer mechanism involving O2. This mechanism constitutes the underlying principle of photodynamic therapy (PDT). In PDT, an exogenous chromophore (i.e., usually a photosensitizing dye) is taken up by the cell and irradiated with light of a specific wavelength usually corresponding to the wavelength of maximum absorption of the dye (48,49). The excited sensitizer transfers the absorbed energy to O2, generating 1O2 and returning to its ground electronic state. 1O2 then reacts with different molecules through a series of complex biochemical reactions in different cellular components (DNA, membrane lipids, proteins, etc.), triggering the destruction and necrosis of neoplastic tissue.
Enzyme generation of 1O2 has been detected by IR spectroscopy and luminescence emission at 1268 nm from enzyme systems such as chloroperoxidase-H2O2-Cl-, lactoperoxidase-H2O2-Br-, and cyclooxygenase (50-55). This was the first direct experimental confirmation of 1O2 being produced as a chemical reactant generated by dark enzymatic systems, although this possibility has been critically discussed in the literature (56,57). As noted by Kanofsky (51), this biochemical production of 1O2 "should not be uncritically extrapolated to living systems".
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