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Comet Assay Modifications for Its Application in Food Safety

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Comet Assay Modifications for Its Application in Food Safety Facultad de Farmacia y Nutrición Comet assay modifications for its application in food safety Modificaciones del ensayo del cometa para su aplicación en seguridad alimentaria José Manuel Enciso Gadea Facultad de Farmacia y Nutrición El presente trabajo de investigación titulado: Comet assay modifications for its application in food safety Modificaciones del ensayo del cometa para su aplicación en seguridad alimentaria que presenta D. José Manuel Enciso Gadea para aspirar al grado de Doctor por la Universidad de Navarra en el Programa de Doctorado en Alimentación, Fisiología y Salud, ha sido realizado en el Departamento de Farmacología y Toxicología de la Facultad de Farmacia y Nutrición, bajo la dirección de la Dra. Adela López de Cerain Salsamendi y la co-dirección de la Dra. Amaya Azqueta Oscoz y la Dra. Ariane Vettorazzi Armental. Pamplona, 8 de febrero de 2018 Dra. Amaya Azqueta Oscoz Dra. Ariane Vettorazzi Armental Dra. Adela López de Cerain Salsamendi This work received financial support from: The “Asociación de Amigos” of the University of Navarra (Predoctoral grant). Ministry of Economy, Industry and Competitiveness (Spain). BIOGENSA Project: “Aplicación de una nueva estrategia de evaluación de genotoxicidad en ingredientes funcionales y en frituras de restauración colectiva” (AGL2015-70640-R) University of Navarra (PIUNA). Project: “Efecto cancerígeno de la ocratoxina A: influencia del sexo en el mecanismo de acción” [PIUNA 2012]. Acknowledgements / Agradecimientos Con estas breves palabras deseo expresar mi más sincero agradecimiento a todas aquellas personas que, de una manera u otra, me han ayudado a que esta tesis doctoral sea una realidad. En primer lugar, a la Universidad de Navarra, por haberme formado tanto en el plano intelectual como en el humano, y la Asociación de Amigos de la misma, por haberme dotado con los medios económicos para la consecución de este proyecto. A mi directora, la Dra. Adela López de Cerain Salsamendi, por haber confiado en ese chico que quería realizar la tesis sí o sí, aunque no empezara con una beca. Sé que tampoco era una situación ideal para ti, y, aun así, me diste tu voto de confianza, de lo cual siempre estaré agradecido. A mis codirectoras, Amaya y Ariane. Gracias a ti, Amaya, he perdido la cuenta de las horas y los días (incluso me atrevería a decir que son meses, y sabes que no estoy exagerando) que me he pasado contando cometas en el microscopio de fluorescencia, pero creo que ha valido la pena. Gracias por todo lo que me has enseñado y por todo lo que has trabajado para que este proyecto saliera adelante, siempre con una sonrisa; sin duda eres una gran investigadora. Gracias a ti, Ariane, por enseñarme la importancia del trabajo ordenado, por echarme la bronca cuando tocaba, y por ayudarme a finalizar este proyecto; sin duda llegarás allá donde te lo propongas. A todos los miembros del Departamento de Farmacología y Toxicología, en especial a mi Laboratorio de Toxicología: a Ana Gloria, con la que comencé mi andadura en CIFA cuando ni siquiera había acabado la carrera; a Celia, siempre dispuesta a ayudarme en mis momentos de crisis con una sonrisa; a Ismael, Maite, Javi, Violeta y Julen. A los que ya no están: Cecilia (nadie como tú para aprender las técnicas de cultivo celular), Leire, Esperanza y Maika; a las ya doctoras Laura y Tamara, por las risas y no tan risas en nuestro cuartito, los juevinchos, los días interminables en el animalario amenizados con la radio, … sois las mejores compañeras que podría haber tenido. A todos los alumnos/as de trabajo fin de grado y Máster, así como de intercambio que han pasado por el laboratorio, y a todo el personal de CIFA, en especial a Ángel, Amaya, las dos Elenas y Leire. Quizás nuestro edificio no sea el más bonito del campus, pero en él me he sentido como en casa. Por último, a los nuevos doctorandos, Damián, Julen, Estíbaliz y Bea, con los que he hecho muy buenas migas en poco tiempo, y a los que deseo lo mejor en sus respectivas tesis; pronto seremos colegas, ya lo veréis. To my Norwegian friends from the Norwegian Institute of Public Health, in particular to Kristine, Christine, Gunnar and Anka, whom kindly welcomed me, taught me everything they know about the comet assay, and made me feel the weather was warmer. It was a pleasure for me to work with you all. Thanks to Enrique, Elena, Rike, Marie, for all the good moments outside the lab enjoying Oslo. A todos y cada uno de mis compañeros y amigos, más o menos cercanos, en especial a aquellos con los que inicié mi andadura en la Universidad de Navarra hace ya más de 11 años, en la Licenciatura de Biología, a los que se incorporaron después en el Máster de I+D+i de Medicamentos, y a los pocos valientes que, como yo, decidieron continuar su formación con la elaboración de una tesis doctoral. A mis grandes amigos del Colegio Mayor Larraona, donde seguramente pasé los años más divertidos, del que guardo recuerdos inolvidables, y al que considero mi segunda casa. Gracias Manu, por abrirme las puertas del Mayor de par en par, y a todo el personal del mismo, por todos los buenos momentos allí vividos. Este trabajo también va por vosotros. A mi familia, en especial a mi padre José Manuel y a mi madre Merche, por dármelo todo sin esperar nada a cambio, y por ser los principales artífices de que haya llegado hasta aquí. No hay palabras para describir el esfuerzo, el cariño y los valores que nos habéis dado tanto a mí como a mi hermano. A él, por hacerme reír, por cabrearme otras veces, y por interesarse por lo que hago sin tener ni idea. A mi chica, Carlota, por darme todo su apoyo y energía en los días difíciles, cuando me preguntaba que estaría pensando el día en el que decidí realizar la tesis doctoral y me entraban ganas de mandar todo al garete, y sobre todo por escucharme, aunque te pareciera que te estaba hablando en chino. Sin duda has hecho que estos cuatro años sean mucho más llevaderos tesoro, y los que nos quedan. Summary Information on genotoxicity is of a key importance for the toxicological characterisation of different compounds. In this vein, and due to its various advantages, the comet assay is currently included in the genotoxicity testing strategy used in the food safety area. However, improvement points of particular interest have been identified. Thereby, the main objective of the present work was to evaluate some critical points of the comet assay, such as the time of lysis, in vitro, and the methodology used in the freezing/thawing procedures of tissue samples, their stability and the application of the Fpg-modified assay, in vivo. In addition, the in vivo comet assay was applied to frozen kidney samples obtained in a previous repeated-dose toxicity study of the food contaminant ochratoxin A. Finally, the knowledge derived from these objectives resulted in the development of standard operating procedures for both the in vitro and in vivo comet assays, which could be applied in good laboratory practice studies. Keywords: comet assay, genotoxicity, food safety, ochratoxin A Table of contents List of abbreviations ...................................................................................................................... 1 Chapter 1. General introduction ................................................................................................... 5 Chapter 2. Aim, objectives and outline ....................................................................................... 41 Chapter 3 (Paper 1). Does the duration of lysis affect the sensitivity of the in vitro alkaline comet assay? ............................................................................................................................... 47 Chapter 4 (Paper 2). Standardisation of the in vitro comet assay: influence of lysis time and lysis solution composition on the detection of DNA damage induced by X-rays ....................... 59 Chapter 5 (Paper 3). Applying the comet assay to fresh vs frozen animal solid tissues: a technical approach ...................................................................................................................... 69 Chapter 6 (Paper 4). Is oxidative stress involved in the sex-dependent response to OTA renal toxicity? ....................................................................................................................................... 91 Chapter 7. General overview .................................................................................................... 121 Chapter 8. Conclusions ............................................................................................................. 135 Abbreviations List of abbreviations 8-OHdG 8-hydroxy-2’-deoxyguanosine 8-oxoGua 8-oxo-7,8-dihydroguanine 8-oxodG 8-oxo-7,8-dihydro-2’-deoxyguanosine AAF 2-acetylaminofluorene Alk A 3-methyladenine glycosylase ALS alkali-labile sites AP apurinic/apyrimidinic COLIPA European Cosmetics Industry Association COM Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment CPN cisplatin CV coefficient of variation DAPI 4’,6-diamidino-2-phenylindole DMSO dimethylsulfoxide EC Enzyme Commission ECVAG European Comet Assay Validation Group ECVAM European Centre for the Validation of Alternative Methods EFPIA European Federation of Pharmaceutical Industries and Associations EFSA European Food Safety Authority EMA European Medicines Agency EMS ethyl methanesulfonate Endo III Endonuclease III ESCODD European Standards Committee on Oxidative DNA Damage EURL-ECVAM European Union Reference Laboratory for
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