L’allergie de contact est une pathologie de la peau particulièrement répandue dans les pays industrialisés. Aucune thérapie ne permet actuellement de la soigner et seule l’éviction de l’allergène permet de la prévenir. Historiquement, l’évaluation du potentiel sensibilisant des molécules mises sur le marché a toujours été réalisée au moyen de tests sur l’animal. Cependant, le champ d’action de ces tests est aujourd’hui limité en raison de la nouvelle législation européenne sur les cosmétiques. Dans ce contexte, le développement de méthodes alternatives ne reposant pas sur l’utilisation d’animaux devient capital. L’allergie de contact repose sur une étape chimique clé : la formation d’un complexe antigénique allergène-protéine capable d’activer le système immunitaire cutané. Le but de ce travail de thèse a été d’étudier le comportement in situ d’allergènes au sein d’épidermes reconstruits de type SkinEthic®. A l’aide d’une technique d’analyse non invasive, la spectroscopie RMN HRMAS, il a été possible de suivre le devenir de différents allergènes, de leur éventuelle activation par voie métabolique, jusqu’à leur fixation sur les protéines épidermiques. / Contact dermatitis is a skin pathology particularly prevalent in industrialized countries. No therapy currently exists and only complete avoidance of the particular allergen can prevent an allergic reaction. Historically, the assessment of skin sensitisation potential of molecules placed on the market was always carried out by animal testing. However, the scope of this testing method is now limited by the new European cosmetics legislation. In this way, the development of alternative methods, not based on animal experimentation, become an important issue. Contact dermatitis results of a chemical key step: the formation of an antigenic complex allergen-protein complexe able to activate the cutaneous immune system. The aim of this PhD work was to study the in situ behaviour of allergens in reconstructed human epidermis (SkinEthic® model). By using an appropriate non-invasive analysis technique, HR-MAS NMR spectroscopy, it has been possible to study the mode of action of different allergens, from their possible activation through the metabolic pathway to the binding with epidermal proteins.

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Malondialdehyde (MDA) is a mutagenic and carcinogenic product of lipid peroxidation which has also been found at elevated levels in smokers. MDA reacts with nucleic acid bases to form pyrimidopurinone DNA adducts, of which 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimidol[1,2-α]purin-10(3H)-one (M1dG) is the most abundant and has been linked to smoking. Mutations in the TP53 tumour suppressor gene are associated with half of all cancers. This research applied a multidisciplinary approach to investigate the toxic effects of MDA on the human lung fibroblasts MRC5, which have an intact p53 response, and their SV40 transformed counterpart, MRC5 SV2, which have a sequestered p53 response. Both cell lines were treated with MDA (0-1000 µM) for 24 and 48 h and subjected to a variety of analyses to examine cell proliferation, cell viability, cellular and nuclear morphology, apoptosis, p53 protein expression, DNA topography and M1dG adduct detection. For the first time, mutation sequencing of the 5’ untranslated region (UTR) of the TP53 gene in response to MDA treatment was carried out. The main findings were that both cell lines showed reduced proliferation and viability with increasing concentrations of MDA, the cell surface and nuclear morphology were altered, and levels of apoptosis and p53 protein expression appeared to increase. A LC MS-MS method for detection of M1dG adducts was developed and adducts were detected in CT-DNA treated with MDA in a dose-dependent manner. DNA appeared to become more fragmented with increasing MDA concentration, and the number of mutations in the 5’ UTR region of the TP53 gene also increased. The majority of mutations observed were insertions, compared to lung cancer mutation data where the majority were G to T transversions. This was unexpected, suggesting that tobacco smoke compounds have a different role in mutagenesis than endogenous lipid peroxidation. Thus, MDA has been found to have a clear effect on human lung fibroblasts at both the cellular and DNA level.