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Development of a Novel in Vitro Liver 3D Model to Assess Potential (Geno)Toxicity of Nanomaterials Jefferson De Oliveira Mallia

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Development of a Novel in Vitro Liver 3D Model to Assess Potential (Geno)Toxicity of Nanomaterials Jefferson De Oliveira Mallia Development of a novel in vitro liver 3D model to assess potential (geno)toxicity of nanomaterials Jefferson de Oliveira Mallia To cite this version: Jefferson de Oliveira Mallia. Development of a novel in vitro liver 3D model to assess potential (geno)toxicity of nanomaterials. Biotechnology. Université Grenoble Alpes [2020-..]; University of Swansea (Swansea (GB)), 2020. English. NNT : 2020GRALS027. tel-03227420 HAL Id: tel-03227420 https://tel.archives-ouvertes.fr/tel-03227420 Submitted on 17 May 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE GRENOBLE ALPES préparée dans le cadre d’une cotutelle entre l’Université Grenoble Alpes et Swansea University Spécialité : Biotechnologie, instrumentation, signal et imagerie pour la biologie, la médecine et l'environnement Arrêté ministériel : le 6 janvier 2005 – 25 mai 2016 Présentée par Jefferson de Oliveira Mallia Thèse dirigée par M. Sylvain Bohic et Mme. Shareen H. Doak codirigée par M. Gareth Jenkins et M. Jean-Luc Ravanat préparée au sein des Laboratoires: European Synchrotron Radiation Facility (ESRF), Laboratoire des Lésions des Acides Nucléiques (CEA), et Institute of Life Science dans les Écoles Doctorales Ingénierie pour la santé la Cognition et l'Environnement (ISCE) Développement d'un nouveau modèle 3D in vitro pour évaluer le potentiel (géno)toxicité des nanomatériaux Thèse soutenue publiquement le 19 Mai 2020, devant le jury composé de : Mme. Cathy, Thornton Professeur, Personal Chair in Biomedical Sciences Swansea University, Président du Jury M. Nicolas, Foray Directeur de recherche, INSERM UMR 1052 à Lyon, Rapporteur M. Francis L., Martin Professeur, Chair in Biosciences of University of Central Lancashire, Rapporteur M. Francois, Esteve Professeur, Université Grenoble Alpes, Examinateur M. Martin, Clift Associate Professor, Swansea University, Examinateur Development of a novel in vitro liver 3D model to assess potential (geno)toxicity of nanomaterials Jefferson de Oliveira Mallia Swansea University Medical School Communauté Université Grenoble Alpes École Doctorale Ingénierie pour la Santé, la Cognition et l'Environnement Submitted to Swansea University and Communauté Université Grenoble Alpes in the fulfilment of the requirement for the Joint Research Degree of Doctor of Philosophy 2019 Summary The liver is the largest internal organ and plays an important part in maintaining the homeostatic balance in the body. It is also one of the primary sites of nanoparticle accumulation. HepG2 spheroids were used as an in vitro model to assess iron oxide (magnetite, Fe3O4 and maghemite, γ-Fe2O3) nanoparticle (NP) toxicity. The development of HepG2 spheroids by the hanging drop method was illustrated through cell viability and tissue morphology assessment of spheroids generated from 1000, 5000, and 10,000 cells per drop. The spheroids generated from 5000 cells per drop had the most optimal configuration. Liver function was assessed by albumin, urea and aspartate transaminase which were expressed in physiologically relevant concentrations by the HepG2 spheroid. Synchrotron X-ray fluorescence (SXRF) was used to assess NP distribution and metal homeostasis. The SXRF studies showed a dose-dependent accumulation of NPs in HepG2 spheroid transverse tissue sections. This accumulation also affected iron homeostasis by significantly increasing intracellular copper levels. The elemental distribution of calcium and selenium also increased upon challenge with NPs, which may be involved in the removal of reactive oxygen species (ROS). Glucose metabolism shifted towards anaerobic respiration in presence NPs, exhibiting the Warburg effect. Transmission electron microscopy also shows intracellular localisation of the iron oxide NPs. Cytokinesis block micronucleus (CBMN) and single-cell gel electrophoresis (comet) assays were used to assess genotoxicity. Both NPs induced a significant increase in micronuclei frequency. Pan-centromeric staining further indicated that the primary mode of action of DNA damage caused by iron oxide NPs was clastogenic. Comet scores showed no significant increases in tail intensities percentages. In conclusion, the HepG2 spheroid model is representative of an in vivo liver due to its ability to mimic glucose metabolism, liver functionality and metal homeostasis responses. Iron oxide NPs cause the HepG2 spheroids to respire anaerobically and disrupt iron homeostasis which can potentially lead to the generation of ROS resulting in the DNA damage. i | Résumé Le foie est le plus grand organe interne et joue un rôle important dans le maintien de l'équilibre homéostatique dans le corps. C'est également l'un des principaux sites d'accumulation de nanoparticules. Les sphéroïdes HepG2 ont été utilisés comme modèle in vitro pour évaluer la toxicité des nanoparticules (NP) d'oxyde de fer (magnétite, Fe3O4 et maghémite, γ-Fe2O3). Le développement des sphéroïdes HepG2 par la méthode des gouttes suspendues a été quantifié par l’évaluation de la viabilité cellulaire et de la morphologie des tissus des sphéroïdes générés à partir de 1000, 5000 et 10 000 cellules par goutte. Les sphéroïdes générés à partir de 5000 cellules par goutte avaient la configuration la plus optimale. La fonction hépatique a été évaluée par l'albumine, l'urée et l'aspartate transaminase, qui ont été exprimées à des concentrations physiologiquement pertinentes par le sphéroïde HepG2. La fluorescence des rayons X synchrotron (SXRF) a été utilisée pour évaluer la distribution des NP et l'homéostasie des métaux. Les études SXRF montrent une accumulation dépendante de la dose de NP dans les coupes transversales de tissu sphéroïde HepG2. Cette accumulation a également affecté l'homéostasie du fer en augmentant de manière significative les niveaux de cuivre intracellulaire. Les concentrations du calcium et du sélénium ont également augmenté en présence des NP, ces deux éléments pouvant être impliquées dans l'élimination des espèces réactives de l'oxygène (ROS). Le métabolisme du glucose s'est déplacé vers la respiration anaérobie en présence de NP, montrant l'effet Warburg. La microscopie électronique à transmission montre également la localisation intracellulaire des NP d'oxyde de fer. L'analyse des micronoyaux par blocage de la cytokinèse (CBMN) et l'électrophorèse en gel unicellulaire (comet) ont été utilisées pour évaluer la génotoxicité. Les deux NP ont induit une augmentation significative de la fréquence des micronoyaux. La coloration pan-centromérique a en outre indiqué que le principal mode d'action des dommages à l'ADN causés par les NP d'oxyde de fer était le clastogène. Les résultats comet n'ont montré aucune augmentation significative des pourcentages d'intensité de la queue. En conclusion, le modèle sphéroïde HepG2 est représentatif d'un foie in vivo en raison de sa capacité à imiter le métabolisme du glucose, la fonctionnalité du foie et les réponses à l'homéostasie des métaux. Les NP d'oxyde de fer amènent un métabolisme des sphéroïdes HepG2 anaérobie et perturbent l'homéostasie du fer, ce qui peut potentiellement générer la génération de ROS entraînant des lésions de l'ADN. ii | Declarations and Statements DECLARATION This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed ...................................................................... (candidate) Date .......................................20/12/2020 ................................. STATEMENT 1 This thesis is the result of my own investigations, except where otherwise stated. Where correction services have been used, the extent and nature of the correction is clearly marked in a footnote(s). Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed ..................................................................... (candidate) Date .............................................................20/12/2020 ........... STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ..................................................................... (candidate) Date ........................................................................20/12/2020 iii | Table of Contents Summary ................................................................................................................... i Résumé ..................................................................................................................... ii Declarations and Statements .................................................................................. iii Table of Contents .................................................................................................... iv Acknowledgements ................................................................................................. ix List of Figures .........................................................................................................
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