Desenvolvimento de plataforma multiparamétrica substitutiva ao uso de animais de laboratório para avaliação do potencial alergênico de misturas da “vida real”
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Data
2018-12-07
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Universidade Federal de Goiás
Resumo
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.
Descrição
Palavras-chave
Alérgenos , Dermatite alérgica de contato , Hipersensibilidade tardia , Rotas de resultados adversos , Segurança de produtos ao consumidor , Técnicas in vitro , Testes de toxicidade , Toxicologia , Allergens , Dermatitis, allergic contact , Hypersensitivity, delayed , Adverse outcome pathways , Consumer product safety , In vitro techniques , Toxicity tests , Toxicology
Citação
ÁVILA, R. I. Desenvolvimento de plataforma multiparamétrica substitutiva ao uso de animais de laboratório para avaliação do potencial alergênico de misturas da “vida real”. 2018. 119 f. Tese (Doutorado em Ciências da Saúde) - Universidade Federal de Goiás, Goiânia, 2018.