Antecedentes Bibliofraficos
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Universitat de València Facultat de Farmàcia Departament de Medicina Preventiva i Salut Pública, Ciències de l’Alimentació,Toxicologia i Medicina Legal. - Area de Toxicologia – Doctorat en Ciències de l’Alimentaciò Tesi Doctoral internacional IN VITRO EVALUATION OF TOXIC EFFECTS, BIOAVAILABILITY AND BIOACCESSIBILITY OF BEAUVERICIN, ENNIATINS AND FUSAPROLIFERIN. EVALUACIÓN IN VITRO DE LOS EFECTOS TÓXICOS, BIODISPONIBILIDAD Y BIOACCESIBILIDAD DE BEAUVERICINA, ENNIATINAS Y FUSAPROLIFERINA. Presentada per: Dirigida per: Alessandra Prosperini Dra. Guillermina Font Pérez Dra. María José Ruiz Leal Las Dras, Guillermina Font Pérez y María José Ruiz Leal, catedrática y profesora titular, respectivamente del Área de Toxicología del Departamento de Medicina Preventiva i Salut Pública, Ciències de l’Alimentaciò, Toxicologia i Medicina legal, de la Universitat de València, INFORMAN QUE: La licenciada en “Scienze dell’Alimentazione e Nutrizione Umana” ha realizado bajo nuestra dirección el trabajo que lleva por título: “In vitro evaluation of toxic effects, bioavailability and bioaccessibility of beauvericin, enniatins and fusaproliferin”. Y autorizamos la presentación para optar al título de Doctor Internacional. Burjassot 24 de Junio 2013. María José Ruiz Leal Guillermina Font Pérez Esta tesis Doctoral ha dado lugar a los siguientes trabajos: 1. Interaction effects of Fusarium enniatins (A, A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells. Toxicology in Vitro (DOI: 10.1016/j.tiv.2013.06.021). Índice de impacto: 2.775. 2. Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells. Toxicology Letters (under review). Índice de impacto: 3.230. 3. Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1. Toxicology Letters (under review). Índice de impacto: 3.230. 4. Study of the cytotoxic activity of beauvericin and fusaproliferin and bioavailability in vitro on Caco-2 cells. Food and Chemical Toxicology. Índice de impacto: 2.999. 5. Effect of polyphenols on enniatins-induced cytotoxic effects in mammalian cells. Toxicology Mechanisms and Methods. Índice de impacto: 1.033. 6. Bioaccessibility of enniatins A, A1, B, and B1 in different commercial breakfast cereals, cookies, and breads of Spain. Journal of Agricultural and Food Chemistry. Índice de impacto: 2.823. La presente Tesis Doctoral Europea es parte de un proyecto financiado por el Ministerio de Ciencia e Innovación AGL 2010/17024/ALI: “Evaluación del riesgo de las micotoxinas emergentes de Fusarium”. Para su realización, la estudiante de Doctorado Alessandra Prosperini: Ha disfrutado de una beca predoctoral orientada a la formación de personal investigador estranjero en organismos de investigación de la Comunitat Valenciana, dentro del programa Santiago Grisolía, concedida por la Conselleria de Educación (Comunitat Valenciana). Ha conseguido el título de “Master en Calidad y Seguridad Alimentaria” (2010/2011) para poder acceder a los estudios de Doctorado. Ha disfrutado de una subvención para favorecer la movilidad de estudiantes en estudios de Doctorado con Mención hacia la Excelencia Curso 2011-2012. Dirección General de Universidades. Ministerio de Educación. Además la presente Tesis Doctoral forma parte del Programa de Doctorado Internacional: “Chemistry, Toxicology and Healthiness of foods” dirigida por el Prof. Pietro Damiani y la Prof.ssa Lina Cossignani. Università degli Studi di Perugia (Italy). ÍNDICE Índice ii Índice INDEX Page INDEX OF TABLES VI INDEX OF FIGURES IX LIST OF ABBREVIATIONS XVII ANTECEDENTES BIBLIOGRÁFICOS 1. INTRODUCTION 3 1.1 Las micotoxinas: una cuestión importante de seguridad alimentaria. 3 1.2 Fusarium spp. and fusarotoxins 5 1.3 Minor Fusarium mycotoxins 8 1.3.1 Beauvericin 8 1.3.1.1 Biological properties 9 1.3.1.2 Biochemical activity 10 1.3.1.3 Toxicological activity 12 1.3.1.4 Occurrence in food 18 1.3.2 Enniatins 24 1.3.2.1 Biological properties 25 1.3.2.2 Biochemical activity 26 1.3.2.3 Toxicological activity 27 1.3.2.4 Occurrence in food 35 iii Índice Page 1.3.3 Fusaproliferin 36 1.3.3.1 Biological and toxicological properties 37 1.3.3.2 Occurrence in food 37 1.4 In vitro cytotoxicity of individual and combined mycotoxins 40 1.4.1 Cytotoxicity studies 40 1.4.2 In vitro effects of combinations of mycotoxins 41 1.5 Mycotoxin-induced oxidative stress 43 1.5.1 Reactive oxygen species and oxidative stress 43 1.5.2 Oxidative lipid damage: lipid peroxidation 45 1.6 Antioxidant defense system 46 1.6.1 Intracellular defense system 46 1.6.2 Antioxidants from diet as protective system 49 1.7 Impairment of cell proliferation: the cell cycle distribution 51 1.8 Mechanism of apoptosis/necrosis cell death 52 1.9 Mitochondrial membrane potential disruption 54 1.10 DNA damage 55 1.11 Bioavailability studies 56 1.12 Bioaccesibility studies 60 1.13 References 65 iv Índice Page 2. OBJETIVOS 91 3. RESULTADOS 93 Interaction effects of Fusarium enniatins (A, A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells. 95 Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells. 121 Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1. 157 Cytotoxic effect and oxidative stress induction by Fusaproliferin in Caco-2 cells 195 Effect of polyphenols on enniatins-induced cytotoxic effects in mammalian cells 207 Study of the cytotoxic activity of beauvericin and fusaproliferin and bioavailability in vitro on Caco-2 cells 239 Bioaccessibility of enniatins A, A1, B, and B1 in different commercial breakfast cereals, cookies, and breads of Spain 261 4. DISCUSIÓN GENERAL 287 CONCLUSIONES 307 Anexos 311 Anexo I - Metodología 313 Anexo II - Difusión de risultados 341 v Índice de tablas INDEX OF TABLES Page INTRODUCTION Tabla 1. Algunas micotoxinas más comunes relacionadas con los hongos micotoxigenicos (adaptada desde Paterson and Lima, 2010). 4 Table 2. Mycotoxigenic species and their produced mycotoxins, isolated from FHB of wheat in Europe. 6 Table 3. In vivo studies of beauvericin. 13 Table 4. In vitro cytotoxicity studies on beauvericin, as determined by different toxicity endpoint, times of exposure and cell types. 14 Table 5. Natural occurrence of BEA, ENs A, A1, B and B1 and other mycotoxins in food and feed. 19 Table 6. In vivo studies of enniatins. 28 Table 7. In vitro cytotoxicity studies on enniatins A, A1, B and B1, as determined by different toxicity endpoint, times of exposure and cell types. 29 Table 8. Occurrence of fusaproliferin and other mycotoxins in food and feed. 38 Table 9. Biological endpoints for detecting cytotoxicity. 40 RESULTS AND DISCUSSION Interaction effects of Fusarium enniatins (A, A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells. Table 1. Dose-effect relationship parameters and mean combination index values (as a function of fractional inhibition of proliferation) of ENs A, A1, B and B1 and their binary, ternary and quaternary combinations on Caco-2 cells, after 24h of exposure. 109 vi Índice de tablas Page Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells. Table 1. Medium inhibitory concentration (IC50) values of BEA on Caco-2 cells, after 24, 48 and 72 h of exposure by the MTT and NR assays. IC50 values were calculated from full-dose response curve. Results are mean ± SD of three experiments. 131 Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1 Table 1. The medium inhibitory concentration (IC50) of ENs A, A1, B and B1 in Caco-2 cells after 24, 48 and 72 h of exposure by the MTT and NR assays. IC50 values are given as percent of control cells (1% MeOH). Results are mean ± SD of three experiments. 173 Study of the cytotoxic activity of beauvericin and fusaproliferin and bioavailability in vitro on Caco-2 cells. Table 1. Transport profile of beauvericin and fusaproliferin in Caco-2 cell monolayers after different incubation time. The values are expressed in percentage (%). 255 Bioaccessibility of enniatins A, A1, B, and B1 in different commercial breakfast cereals, cookies, and breads of Spain Table 1. Nutrients nutritional properties of the analyzed samples referred to 100 g of product (as reported in nutritional labels). 268 Table 2. Bioaccessibility (%) of the enniatins A, A1, B and B1, spiked with 1.5 and 3.0 µM concentration in different breakfast cereals, cookies and breads digested by an in vitro simulated gastrointestinal model. 273 vii Índice de figuras INDEX OF FIGURES Page INTRODUCTION Figure 1. Chemical structure of beauvericin. 8 Figure 2. Mechanism of action of BEA. Dashed arrow indicate those mechanism that are still unclear. MMP = mitochondrial membrane potential. 11 Figure 3. Chemical structure of enniatins. 24 Figure 4. Chemical structure of fusaproliferin, deacetyl- fusaproliferin and acetyl-fusaproliferin. 36 Figure 5. Stepwise reduction of molecular oxygen in biological systems leads to ROS production. 44 Figure 6. The chemical reaction between TBA and MDA that yields to the MDA-TBA adduct. Schematic diagram of the dynamic, multi-compartmental model of the stomach and small intestine. 46 Figure 7. Metabolic pathway of glutathione. 48 Figure 8. Chemical structure of (a) resveratrol, (b) quercetin, (c) t-pterostilbene. 50 Figure 9. Graphical representation of in vitro trans-epithelial transport. 59 Figure 10. Schematic representation of the in vitro digestion model as reported by Gil-Izquierdo et al. (2002). 62 Figure 11. Schematic diagram of the dynamic, multi- compartimental model of the stomach and small intestine. 63 Figure 12. A schematic diagram representing the in vitro digestion experiment (Kabak et al., 2009; 2012). 64 viii Índice de figuras Page RESULTS AND DISCUSSION Interaction effects of Fusarium enniatins (A, A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells. Figure 1.