ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH
Tesi di Dottorato
Data di Pubblicazione:
2014
Citazione:
ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH / V. Galbiati ; tutor: C.L. Galli ; correlatore: A. Panerai. Università degli Studi di Milano, 2014 Dec 15. 27. ciclo, Anno Accademico 2014. [10.13130/galbiati-valentina_phd2014-12-15].
Abstract:
Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-α, IL-8 and IFN-γ (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001).
Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity.
The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the ‘universe’ of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity.
Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011).
In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equi
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
V. Galbiati
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