Desenvolvimento de plataforma multiparamétrica substitutiva ao uso de animais de laboratório para avaliação do potencial alergênico de misturas da “vida real”

• Ano de defesa: 2018
• Autor(a) principal: Marcelino, Renato Ivan de Ávila
• Orientador(a): Valadares, Marize Campos
• Banca de defesa: Valadares, Marize Campos, Cordeiro, Lorena Rigo Gaspar, Fonseca, Simone Gonçalves da, Souza, Pedro Paulo Chaves de, Cunha, Luiz Carlos da
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Goiás
• Programa de Pós-Graduação: Programa de Pós-graduação em Ciências da Saúde (FM)
• Departamento: Faculdade de Medicina - FM (RG)
• País: Brasil
• Palavras-chave em Português: Alérgenos; Dermatite alérgica de contato; Hipersensibilidade tardia; Rotas de resultados adversos; Segurança de produtos ao consumidor; Técnicas in vitro; Testes de toxicidade; Toxicologia
• Palavras-chave em Inglês: Allergens; Dermatitis, allergic contact; Hypersensitivity, delayed; Adverse outcome pathways; Consumer product safety; In vitro techniques; Toxicity tests; Toxicology
• Área do conhecimento CNPq: MEDICINA::ANATOMIA PATOLOGICA E PATOLOGIA CLINICA
• Link de acesso: http://repositorio.bc.ufg.br/tede/handle/tede/9307

Animal experimentation has been a great ally of man in the progress of Sciences. However, the predictive capacity of in vivo models to identify toxic chemicals to humans has been widely debated. The advance of the 21st Century scientific knowledge has promoted the search for predictive models that consider the human physiology as well as the pathophysiology of the physically and chemically induced-adverse reactions. In this context, the present study focused on the evaluation of dermal toxicity of ingredients of hair dyes and agrochemicals and their finished products, referred to herein as "real-life" mixtures (the real condition of which the consumer is exposed to). An integrated strategy of in vitro tests was developed to evaluate the potential of these materials to induce allergic reactions when in contact with human skin. This strategy involved different human-relevant innovative techniques based on key-events of the adverse outcome pathway of skin sensitization (protein reactivity, activation of keratinocytes and/or dendritic cells). The first method implemented in house was the direct peptide reactivity assay (DPRA, OECD 442C/2015). The refinement process conducted was able to reduce the final reaction volume of the test, yielding in considerable reduction of cost and use of organic solvents. These modifications allowed the "minituarization of the technique", called micro-DPRA (mDPRA), which showed a similar performance to conventional DPRA in the identification of allergenic substances and finished products as surfactants and glyphosate-containing formulations. A second refinement led to the development of the photo-mDPRA, a technique capable of identifying contact photoallergens, which was not possible by the already validated phototoxicity technique (3T3 neutral red uptake phototoxicity test - OECD 432/2004). Similar to mDPRA, photo-mDPRA showed applicability to pure substances and mixtures based on glyphosate and surfactants. This work also established an in vitro multiparametric platform to evaluate the skin sensitization of cosmetic materials: cosmetic ingredients and hair dye products containing henna [Lawsonia inermis (Lythraceae)]. In addition to mDPRA and photo-mDPRA, seven different innovative methods were implemented. Four of them showed superior capacity to identify cosmetic ingredients with allergenic potential as compared to the traditional rodent models, which are: mDPRA, measurement of interleukin (IL)-18 in human HaCaT keratinocytes, U937 cell line activation test (U-SENSTM, OECD 442E/2018) and genomic allergen rapid detection (GARDTM skin). GARDTM skin had the highest predictive capacity in relation to human data (concordance = 100%), while mDPRA (concordance = 91.7%), measurement of IL-18 in HaCaT keratinocytes and U-SENSTM (both with concordance = 92.3%) showed similar predictions. Expanding the applicability of this strategy, it was used to evaluate ten natural henna-containing hair dye products sold in commercial establishments located in Goiânia, GO, Brazil. The first step was the label analysis followed by characterization and quantification of the potent contact allergen pphenylenediamine (PPD) and the henna biomarker lawsone using high performance liquid chromatography. In contrast to what was stated in the labels by the manufacturers, the chromatographic analyses showed that all products contained PPD, and one of them did not present detectable contents of lawsone. Therefore, revealing the adulteration and falsification of henna products. Furthermore, toxicological evaluation using the integrated in vitro testing approach established in this study showed that hennas generally triggered high peptide reactivity, increased IL-18 levels in HaCaT keratinocytes and CD86 expression in U937 cells in U-SENSTM assay, and induced changes in the 200 genes from the GARDTM skin predictive signature. Additionally, all hennas had the potential to trigger allergic contact dermatitis in humans. In parallel, hair dye ingredients, including PPD, promoted modulation of genes related to the oxidative stress response in human HaCaT keratinocytes, including nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase 1 (HO1), and Fos proto-oncogene (FOS). Therefore, the toxicological consequences and the associated risks of undeclared use of PPD in natural henna products are highlighted, which include sensitization of susceptible consumers and cases of allergic contact dermatitis in previously sensitized individuals. In addition, this study contributes to promotion of innovation and technological autonomy of Brazil within the area of Toxicology for the 21st Century through the establishment and development of an in vitro human-relevant approaches for skin sensitization assessment.