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Assessment of the viability of human skin ex vivo model (hOSEC) and its application efficacy studies of sunscreens in long-term photoaging


The worldwide incidence of cancer growing every year and the ultraviolet (UV) radiation is the major risk factor. In Brazil, approximately 30% of cancer cases correspond to skin cancer. Among prevention strategies, the use of broad-spectrum sunscreens is highly recommended by health organizations and the scientific community. The regulatory agencies require that photoprotective formulations have their efficacy and safety proved by in vitro or in vivo tests increasingly stringent. The effectiveness is evidenced mainly by testing in human in which the SPF and PPD photoprotection values are determined in development phase. These tests consist in a single application of the products and they evaluate the photoprotective potential against acute exposure of skin to UV radiation. However, the assessment of the efficacy and even the safety of these products should be performed in a model of chronic exposure employing multiple applications of product, mimicking its use by the consumer. However, in vivo tests (humans and animals) existing could not be used for this type of evaluation. Thus, due to the lack of in vivo chronic models, this project aims the implementation and application of alternative model human organotypic skin explant culture (hOSEC) to assess the effectiveness of sunscreens against the effects of chronic exposure of the skin to UV radiation, associated with multiple applications of sunscreens. The hOSEC models will be divided into groups: 1) not exposed to UV radiation and without treatment with sunscreens (viability of the skin control); 2) exposed to UV radiation daily without photoprotectors (negative control); 3) exposed to UV radiation and treated daily with placebo formulations (vehicle control) and 4) exposed to UV radiation and treated daily with sunscreen SPF-50 (positive control). All groups will be cultivated for 30 days and analyzed at 0, 7, 15 and 30 days. Initially, the group 1 will be assessed regarding long-term viability: i) structural and proliferative by histological analysis, immunohistochemical analysis of cytokeratins 5, 6 and 10 and of the Ki-67 antibody and by quantification of CPDs and, ii) biochemical by measuring of the activity of antioxidant enzymes, by the quantification of non-enzymatic antioxidants and evaluation ofthe organization of collagen and elastin fibers. After confirmation of the viability of hOSEC model (group 1), the skins of group 2 will be exposed to different UV radiation doses and they will be analyzed by cell viability assay (MTT) and by lipid peroxidation assay to define the daily dose applied on the skin. The other groups protected or not by placebo formulations and sunscreens, will be evaluated regarding to the effects induced by chronic exposure to UV radiation. This will occurs by determining the oxidative damages (TBARS, hydroperoxide and oxidized proteins). It will also quantified antioxidant enzymes by Western Blotting technique and by spectrophotometric methods and the non-enzymatic antioxidants. Collagens I and III of the dermis will be determined by immunofluorescence. The enzymes metalloproteinases 1 and 9 will be quantified by Western Blotting techniques and traditional zymography. In addition, the determination of epidermis/dermis thickness and the organization of collagen and elastic fibers will be performed using histological techniques. The present project will contribute to increase knowledge related to structural and functional stability of hOSEC model in cultivation and consequently increase their application in vitro studies with high reliability. Furthermore, biological parameters that best reflect the photoaging induced by chronic exposure of the skin to UV radiation may be used as markers for in vitro evaluation of the photoprotective efficacy of sunscreens and other commercial products. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MASSON-MEYERS, DANIELA S.; ANDRADE, THIAGO A. M.; CAETANO, GUILHERME F.; GUIMARAES, FRANCIELLE R.; LEITE, MARCEL N.; LEITE, SAULO N.; FRADE, MARCO ANDREY C.. Experimental models and methods for cutaneous wound healing assessment. International Journal of Experimental Pathology, v. 101, n. 1-2, . (14/23662-1, 16/16437-7)
PEGORIN, GIOVANA S.; LEITE, MARCEL N.; ANTONIASSI, MARCIO; CHAGAS, ANA LAURA D.; SANTANA, LUISIANE A.; GARMS, BRUNA C.; MARCELINO, MONICA Y.; HERCULANO, RONDINELLI D.; CIPRIANI FRADE, MARCO ANDREY. Physico-chemical characterization and tissue healing changes by Hancornia speciosa Gomes latex biomembrane. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS, v. 109, n. 7, . (17/19603-8, 09/09355-0, 16/16437-7)
LEITE, MARCEL NANI; ROSA VIEGAS, JULIANA SANTOS; GARCIA PRACA, FABIOLA SILVA; DE PAULA, NATALIA APARECIDA; ZAMBELLI RAMALHO, LEANDRA NAIRA; LOPES BADRA BENTLEY, MARIA VITORIA; CIPRIANI FRADE, MARCO ANDREY. Ex vivo model of human skin (hOSEC) for assessing the dermatokinetics of the anti-melanoma drug Dacarbazine. European Journal of Pharmaceutical Sciences, v. 160, . (16/16437-7, 14/50928-2, 19/04448-2)

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