Desenvolvimento e aplicação de modelo de pele humana reconstruída in vitro para estudos de citotoxicidade e genotoxicidade

Abstract

Alternative methods are being developed to reduce, refine, and replace (3Rs) animals used in experiments and have been applied to test the safety of cosmetics and medicines. It is important to consider the difficulties that Brazil faces with customs and perishability issues that hinder and even prevent the use of commercially available reconstructed human skin models. The aim of this work was to develop a reconstructed human skin model (PHR-UFAM) and test its applicability for cytotoxicity and genotoxicity assays. For this, skins were generated using permanent human keratinocytes and fibroblasts (embedded in collagen) cells. The quality control was performed by morphological and immunohistochemical analyses, viability by MTT test with negative control, and barrier function test. The applicability for genotoxicity assays was evaluated by micronucleus test, neutral and alkaline comet, and modified comet for detection of DNA methylation sites with positive controls of each test defined by OECD and literature. The best condition tested allowed the development of a reconstructed human skin (PHR-UFAM) with epidermis presenting expression of immunohistochemical markers: epithelial (cytokeratins) and dermal (vimentin). The model followed quality parameters (histology, viability, and barrier function) and presented morphological similarity with models recognized and validated by OECD. The applicability of skin model for genotoxicity assays demonstrated the effectiveness of the positive controls: Mitomycin C that was able to increase significantly the micronucleus frequency when tested at concentrations of 1,5 e 3 μg/mL (3.03% and 3.51%, respectively); Ethylmethanesulfonate caused damage to the DNA when tested at concentrations of 5 e 10 μM by comet assay in both alkaline (damage index values of 59.3 and 103.7, respectively) and neutral (damage index of 40.7 and 125, respectively) versions; and 5-azacytidine in the modified comet assay for methylation detection, tested at concentrations of 0.5, 1 e 1.5 μM showed reduced damage indexes (107.5, 83.1, and 89.3, respectively). The results were similar to skin models validated by OECD and literature. Thus, with this work it was possible to develop a reconstructed human skin model composed entirely of permanent cells. However, further validation studies are required to prove its effectiveness for cytotoxicity and genotoxicity tests of substances with therapeutic or cosmetic potential.