STUDIO DEI MECCANISMI DI DANNO DA RAGGI UVA E UVB E DEGLI EFFETTI PROTETTIVI DA PARTE DI COMPOSTI POLIFENOLICI IN SISTEMI CELLULARI E MODELLI EX VIVO DI CUTE UMANA

Sun radiation consists of several spectrum regions, depending on the wavelengths, such as ultraviolet, visible and infrared portions. The ultraviolet rays (UV) are associated with both positive and negative effects on humans’ health. Ultraviolet light is used in the process of disinfection, it shows immunoregolatory activity and it stimulates vitamin D and serotonin production. It is well known that artificial sources of UVR are employed in the health field for sterilization procedures and for therapeutical purposes, such as treatment of dermatological pathologies like psoriasis, vitiligo and icterus neonatorum, as well as for aesthetic purposes (sunbed). In the last decade, besides these positive effects, UV side effects on skin have been demonstrated by different studies which correlated the intense and prolonged exposure to UV light with skin cancer development. Based on wavelength, UV spectrum can be divided into three segments: UVA (320-400 nm), UVB (280-320 nm) and UVC (100-280 nm). Radiation in the UVC range, considered to be the most cytotoxic and harmful, is largely absorbed by the stratospheric ozone layer, so UV regions mainly involved in photocancerogenesis development are considered to be ultraviolet radiation A (UVA) and ultraviolet radiation B (UVB). The amount and the UVA/UVB ratio that reach the Earth’s surface is influenced by a lot of environmental factors as latitude, seasons, weather/atmospheric conditions and the time of day. UVA is by far the most abundant solar UV radiation that reaches the Earth (about 90-95% of total UV radiation), penetrates the human epidermis down to the proliferative basal cells and even further to the derma and mediate biological events generating reactive oxygen species (ROS) in cells. ROS oxide other cellular constituents, particularly carbohydrates, lipids, proteins and DNA. Interactions between ROS and DNA induce formation of single strand breaks (SSBs), DNA-proteins crosslink and oxidized bases. The most abundant oxidized base is the guanine that produces the 8-oxoguanine, considered as promoter of carcinogenesis. UVB represents only 5% of total UV radiation that reaches the Earth. Besides it acts mainly at the epidermis basal layer of the skin, it is considered extremely damaging because of its high energy. UVB photons are directly absorbed by DNA and cause the formation of cyclobutane pyrimide dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), which in turn can lead to apoptotic events or carcinogenesis development. Also tryptophan and tyrosine are modified by UVB inducing the alteration of structure and function of proteins. Moreover, UVB is able to produce reactive oxygen species (ROS) and reactive nitric species (RNS). Cells have developed defense mechanisms and endogenous repair systems in order to reduce genotoxic damage UV-induced: complexes of antioxidant enzymes which take advantage of the presence of a metal to inactivate reactive oxygen species, and DNA repair systems like the base excision repair system (BER), used for oxidized bases, and the nucleotide excision repair (NER), used to repair photolesions like CPD and 6-4 PP. Recently, several molecules have been studied for their ability to give a contribution to these antioxidant defenses. For these reasons, there is a considerable interest in the concept of the use of phytochemicals and micronutrients present in the diet such as carotenoids, vitamin E and C, and polyphenols. This last group of compounds are well known for their several biological properties, such as their antioxidant activity, their ability to enhance the activity of DNA repair systems and also their skills in inflammatory and apoptotic events modulations. Several epidemiological studies have demonstrated their biological effects and their efficiency in the prevention and treatment of several chronic diseases like cardiovascular, respiratory, neurodegenerative and inflammatory diseases and cancer. So, in view of these reasons, the aim of my study was double: first, to confirm literature data regarding the different mechanisms used by UVA and UVB rays to cause damage in various models, then to investigate whether different plant-derivatives compounds, all belonging to the pholiphenols category, were able to prevent UVA- and UVB-induced damage. I reproduced the experiments on in vitro and ex vivo models. In vitro models consist of two human keratinocytes cell lines (HaCaT and NCTC 2544 cells), generally used for routine toxicological tests and photogenotoxic evaluations. Both cell lines are non tumorigenic, but showed a different degree of differentiation, higher in HaCaT cells compared to NCTC 2544. The ex vivo model consist of a three-dimensional model of organotypic human skin cultures useful to reproduce the physiological conditions. I tested the efficiency of three compounds: a water soluble extract of Bilberry only in the in vitro models (Vaccinium Myrtillus extract, rich in polyphenols and considered to be the plant with the highest content of anthocyanins), a Thymus Vulgaris leaf extract (Thyme specie) and the synthetic Thymol (its major component), used in comparable dosages, in both models. All experimental models were pretreated in serum free medium for 1 hour with each compound and then irradiated with UVA or UVB. Control samples were treated in the same way, but not UV exposed. In order to confirm mechanisms of UV damage and to compare results obtained in the experimental models, I performed several tests, each with a specific endpoint. I evaluated the intracellular redox status, quantifying the ROS formed and the lipid peroxidation (malondialdeide levels, MDA), immediately after the end of UVA and UVB exposure and only in the in vitro models; after, I performed some genotoxic tests (the alkaline comet test to detect single and double strand breaks in single cells, the immunostaining of the histone H2AX phosphorilated to detect only double strand breaks and the micronucleous test to detect alterations to mitotic apparatus or to chromosomes); finally I investigated effects of rays on apoptotic events, using the Annexin V test and evaluating the alteration of mitochondrial membrane potential UV-induced. After, using this same assays, I tested the protective effect of Vaccinium Myrtillus extract, thymol and Thymus Vulgaris L. extract. Vaccinium Myrtillus’ effect was evaluated only in the in vitro models, the protection of thymol and Thymus Vulgaris L. extract in both models (in vitro and ex vivo). Results obtained confirmed the different machanisms of action of UVA and UVB in all models. Particularly, confirming the different mechanisms of damage used by UVA and UVB rays. My results showed the more oxidant UVA effect and the more genotoxic and apoptotic UVB effect. Three natural compounds showed their different protective effect against UVA- and UVB- induced damage, in in vitro models. Moreover, thymol and Thymus Vulgaris L. extract reduced damage UV-related both in the NCTC 2544 and HaCaT cell lines but also in the ex vivo model. Vaccinium Myrtillus extract, thymol and Thymus Vulgaris L. extract reduced, to a different extent, UVA-caused damage, on account of their antioxidant properties. Compounds revealed their ability to reduce also UVB-induced damage. The efficiency against UVB damage was lower compared to the one against UVA rays. Probably the protection was related with the ability of pholyphenols, and because of my compounds, to repair DNA damage UVB-induced increasing the expression of genes involved in NER system (specifically involved in UVB-induced damage repair). Vaccinium Myrtillus, thymol and Thymus Vulgaris L. extract showed also their protection in the apoptotic assays, avoiding the increase of extremely damaged cells which could lead to mutagenesis development. Results obtained in my thesis activity should be considered as preliminary data useful to investigate, in detail, mechanisms used by the three natural compounds to prevent genotoxic damage UVA- and UVB-induced. Moreover, it should be very interesting to investigate the bioavailability and the permeability of tested compounds in order to use them as food supplements for a diet enriched in antioxidants (systemic effect) or in sunscreens (topical effect).