Combinación de metodologías ómicas y mea-ómicas para estudiar el efecto de la suplementación de selenio y su interacción con la microbiota intestinal

El selenio (Se) es un elemento esencial para la salud humana con importantes funciones para el organismo. Está presente de forma natural en el medio ambiente tanto en formas orgánicas como inorgánicas. En el organismo, se incorpora en forma de selenocisteína (Sec) durante la biosíntesis de la cadena polipeptídica dando lugar a las selenoproteínas. Los efectos biológicos del selenio están en gran parte mediados por las selenoproteínas, ya que se ha reconocido que es un factor clave en el sistema inmune, el metabolismo de hormonas tiroideas y el desarrollo neurológico entre otros. La principal fuente de Se en humanos es la dieta, siendo las nueces de Brasil, pescados y carnes alimentos ricos en este elemento. Una deficiencia de Se puede ocasionar diversas condiciones fisiopatológicas como enfermedades cardíacas, enfermedades neurológicas, cáncer, infertilidad y procesos inflamatorios. Por ello, a veces es necesaria la suplementación de Se de forma directa mediante el consumo de complementos alimenticios. Desde el punto de vista de la reproducción humana, el Se es importante para el desarrollo normal testicular y la espermatogénesis. A nivel neurológico, el Se protege el cerebro del estrés oxidativo a través de las selenoproteínas, que a su vez, poseen numerosas funciones en el sistema nervioso central (CNS) como la producción de neurotransmisores o la señalización neuronal. El Se es un conocido antagonista frente a una gran cantidad de metales pesados tóxicos como el mercurio, el arsénico o el cadmio. Por otro lado, el Se, interacciona con la microbiota intestinal, la cual actúa como barrera en la absorción, interviene en la metabolización de especies de Se e incluso determinados géneros incorporan Se a sus selenoproteínas. La microbiota intestinal constituye el conjunto de microrganismos (bacterias, virus, hongos y arqueas) presentes en el cuerpo humano. La microbiota intestinal está involucrada en funciones inmunológicas, fisiopatológicas y metabólicas del huésped e interactúa con otros órganos y tejidos. Desde hace unos años está cobrando especial interés la interacción intestino-cerebro, en lo que se conoce como el eje microbiota intestinal-cerebro (gut-brain axis), demostrándose que numerosos problemas neurológicos como el Alzheimer o la depresión están relacionados con la alteración en la microbiota intestinal. Simultáneamente, la microbiota intestinal está relacionada con la producción de hormonas sexuales y disfunciones testiculares, teniendo un gran impacto en la reproducción masculina a través del eje microbiota intestinal-hipotálamo- hipofisiario-gonadal. Durante el desarrollo de esta Tesis Doctoral se han llevado a cabo diferentes estrategias analíticas para el estudio de la importancia del Se, enfocado principalmente al papel de las selenoproteínas, frente a metales tóxicos como el cadmio (Cd) y la modulación de la microbiota intestinal a través de los ejes microbiota intestinal-cerebro e hipotálamo-hipofisiario-gonadal. La evaluación del efecto protector del Se frente al Cd se ha realizado mediante un ensayo de exposición en la línea celular de hígado HepG2 por ser uno de los órganos metabólicamente más activos y sobre los que ya se han realizado estudios previos in vivo con ratones Mus musculus. Para ello, las células fueron expuestas durante 24h a diferentes concentraciones de Cd en forma de CdCl2 y de forma conjunta con Se en forma de selenometionina SeMet. Se determinó la viabilidad celular y la concentración de Cd y Se tanto en las células como los medios de cultivo. El contenido de selenoproteínas y selenometabolitos en las células HepG2 se determinó mediante la técnica de column switching y dilución isotópica de especies no específicas. Para conocer el impacto de la suplementación de Se y la microbiota intestinal en el selenoproteoma de los tejidos de cerebro y testículos se ha llevado a cabo un ensayo de exposición a ratones Mus musculus. Los ratones fueron sometidos a un pre-tratamiento con antibióticos para obtener una microbiota deprimida y a un tratamiento posterior con dieta suplementada en Se. La microbiota intestinal ha sido caracterizada mediante un análisis metataxonómico. En ambos tejidos, cerebro y testículos, se ha optimizado un método de extracción de selenoproteínas para su posterior análisis. Los daños testiculares tras la depresión de la microbiota y el efecto de la suplementación de Se se determinaron mediante un análisis histopatológico de los tejidos. Dada la importancia del eje microbiota intestinal-cerebro, se ha completado el estudio de las muestras de cerebro con un análisis metabolómico no dirigido empleando dos plataformas analíticas que han permitido abarcar un amplio rango de metabolitos. Además, se han analizado los niveles de expresión de todo el selenoproteoma de ratón mediante un análisis transcriptómico. Los análisis de correlación entre la abundancia de la microbiota intestinal, selenoproteínas y los metabolitos han mostrado asociaciones con bacterias clave para la reproducción y el neurodesarrollo. El conjunto de técnicas analíticas aplicadas en esta Tesis Doctoral ha aportado nueva información acerca de la respuesta biológica del organismo frente a la suplementación de Se, a través de los cambios en las selenoproteínas en modelos in vitro e in vivo. Asimismo, los estudios de la microbiota han permitido profundizar en los mecanismos de interacción intestino-cerebro e intestino-testículo.Selenium (Se) is an essential element for human health and crucial for body functions. It is naturally present in the environment in both organic and inorganic forms. In the body, it is incorporated in the form of selenocysteine (Sec) during the biosynthesis of the polypeptide chain, deriving to selenoproteins. The biological effects of selenium are largely mediated by selenoproteins, as it has been recognized to be a key factor for the immune system, thyroid hormone metabolism, and neurological development, among others. The main source of Se in humans is the diet, being Brazil nuts, fish, and meat foods rich in this element. A deficiency of Se can cause various pathophysiological conditions such as heart disease, neurological disease, cancer, infertility, and inflammatory processes. For this reason, sometimes it is necessary a Se supplementation directly through the consumption of food supplements. From the point of view of human reproduction, Se is important for normal testicular development and spermatogenesis. At the neurological level, Se protects the brain from oxidative stress through selenoproteins, which in turn have numerous functions in the central nervous system (CNS) such as the production of neurotransmitters or neuronal signalling. Se is a well-known antagonist against many toxic heavy metals such as mercury, arsenic, or cadmium. On the other hand, Se interact with the gut microbiota, which acts as a barrier to absorption, interferes in the metabolization of Se species, and certain genera incorporate Se into their selenoproteins. . The intestinal microbiota constitutes the set of microorganisms (bacteria, viruses, fungi, and archaea) present in the human body. The intestinal microbiota is involved in immunological, pathophysiological, and metabolic functions of the host and interacts with other organs and tissues. Recently, the interaction between the intestine and the brain, known as the gut-brain axis, has been gaining special interest, demonstrating that numerous neurological problems such as Alzheimer's or depression are related with alteration of the gut microbiota. Simultaneously, the gut microbiota is related to the production of sexual hormones and testicular dysfunctions, having a great impact on male reproduction through the gut microbiota-hypothalamic-pituitary-gonadal axis. During the development of this PhD Thesis, different analytical strategies have been carried out to study the importance of Se, mainly focused on the role of selenoproteins, against toxic metals such as cadmium (Cd) and the modulation of the microbiota through the gut microbiota-brain and hypothalamic-pituitary-gonadal axes. The evaluation of the protective role of Se against Cd has been carried out by an exposure assay in the liver cell line HepG2, as it is one of the most metabolically active organs and on which previous in vivo studies employing Mus musculus mice have already been carried out. To this end, the cells were exposed for 24h to different concentrations of Cd in the form of CdCl2 and in combination with Se in the form of selenomethionine SeMet. Cell viability and the concentration of Cd and Se were determined both in the cells and in the culture media. The content of selenoproteins and selenometabolites in HepG2 cells was analyzed using the column switching technique and isotopic dilution of non-specific species. To determine the impact of Se supplementation and the intestinal microbiota on the selenoproteome of brain and testis tissues, an animal experiment using Mus musculus mice was carried out. Mice were pre-treated with antibiotics to obtain a depressed microbiota and subsequently treated with a Se-supplemented diet. The intestinal microbiota has been characterized by metataxonomic analysis. In both tissues, brain, and testes, a selenoprotein extraction method has been optimized for subsequent analysis. Testicular damage after microbiota depression and the effect of Se supplementation were determined by histopathological analysis of the tissues. Given the importance of the gut-brain axis, the study of brain samples has been completed with an untargeted metabolomic analysis using two analytical platforms covering a wide range of metabolites. In addition, the expression levels of the entire mouse selenoproteome have been analyzed by transcriptomic analysis. Correlation analysis between gut microbiota abundance, selenoproteins, and metabolites have shown associations with essential bacteria for reproduction and neurodevelopment. The analytical techniques applied in this Doctoral Thesis has provided new information about the biological response of the organism to Se supplementation, through changes in selenoproteins both in vitro and in vivo models. Likewise, studies of the microbiota have made it possible to delve into the mechanisms of interaction between the gut-brain and intestine-testis

Antagonistic Interaction of Selenium and Cadmium in Human Hepatic Cells Through Selenoproteins

Cadmium (Cd) is a highly toxic heavy metal for humans and animals, which is associated with acute hepatotoxicity. Selenium (Se) confers protection against Cd-induced toxicity in cells, diminishing the levels of ROS and increasing the activity of antioxidant selenoproteins such as glutathione peroxidase (GPx). The aim of this study was to evaluate the antagonistic effect of selenomethionine (SeMet) against Cd toxicity in HepG2 cells, through the modulation of selenoproteins. To this end, the cells were cultured in the presence of 100 µM SeMet and 5 µM, 15 µM, and 25 µM CdCl2 and a combination of both species for 24 h. At the end of the experiment, cell viability was determined by MTT assay. The total metal content of Cd and Se was analyzed by triple-quadrupole inductively coupled plasma–mass spectrometry (ICP-QqQ-MS). To quantify the concentration of three selenoproteins [GPx, selenoprotein P (SELENOP), and selenoalbumin (SeAlb)] and selenometabolites, an analytical methodology based on column switching and a species-unspecific isotopic dilution approach using two-dimensional size exclusion and affinity chromatography coupled to ICP-QqQ-MS was applied. The co-exposure of SeMet and Cd in HepG2 cells enhanced the cell viability and diminished the Cd accumulation in cells. Se supplementation increased the levels of selenometabolites, GPx, SELENOP, and SeAlb; however, the presence of Cd resulted in a significant diminution of selenometabolites and SELENOP. These results suggested that SeMet may affect the accumulation of Cd in cells, as well as the suppression of selenoprotein synthesis induced by Cd.This work was supported by the projects PG2018-096608-B-C21 from the Spanish Ministry of Economy and Competitiveness (MINECO). SR-A thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2016-076364). The authors are grateful to FEDER (European Community) for financial support, grant UNHU13-1E-1611

Metabolic impairments caused by a “chemical cocktail” of DDE and selenium in mice using direct infusion triple quadrupole time of flight and gas chromatography mass spectrometry

Among organic contaminants, pesticides are one of the most important groups of chemicals due to their persistent character and toxicity. However, the biological systems are exposed to a complex environment in which the contaminants can interact in a synergistic/antagonistic fashion, and for this reason, the study of “chemical cocktails” is of great interest to fully understand the final biological effect. In this way, selenium is known for its antagonistic action against several toxicants. In this paper, metabolic impairments caused by the joint exposure of p,p′-dichloro diphenyl trichloroethane (DDE) and selenium (Se) have been issued for the first time. A metabolomic workflow was applied to mice fed DDE and DDE with Se diet, on the basis of the complementary use of two organic mass spectrometric techniques, combining direct infusion mass spectrometry (DI-ESI-QqQ-TOF MS) and gas chromatography–mass spectrometry (GC–MS). The results show a good classification between the studied groups caused by about 70 altered metabolites in the liver, kidney, or brain, including the pathways of energy metabolism, degradation of phospholipidic membrane, β-oxidation, and oxidative stress, which confirm the potential of combined metabolomic platforms in environmental studies.This work has been supported by projects CTM2015-67902-C2-1-P and PG2018-096608 B-C21 from the Spanish Ministry of Economy and Competitiveness and P12-FQM-0442 and B10-1657 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). G.R.-M. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2013-064501). S.R.-A. thanks the Spanish Ministry of Economy and Competitiveness for a PhD scholarship (BES-2016-076364). Finally, the authors are grateful to FEDER (European Community) for financial support, Grant Nos. UNHU13-1E-1611 and UNHU15-CE-3140

The role of selenium in shaping mice brain metabolome and selenoproteome through the gut-brain axis by combining metabolomics, metallomics, gene expression, and amplicon sequencing

Selenium (Se) is a trace element crucial for human health. Recently, the impact of Se supplementation on gut microbiota has been pointed out as well as its influence on the expression of certain selenoproteins and gut metabolites. This study aims to elucidate the link between Se supplementation, brain selenoproteins and brain metabolome as well as the possible connection with the gut-brain axis. To this end, an in vivo study with 40 BALB/c mice was carried out. The study included conventional ( n = 20) and mice model with microbiota depleted by antibiotics ( n = 20) under a regular or Se supplemented diet. Brain selenoproteome was determined by a transcriptomic/gene expression profile, while brain metabolome and gut microbiota profiles were accomplished by untargeted metabolomics and amplicon sequencing, respectively. The total content of Se in brain was also determined. The selenoproteins genes Dio and Gpx isoenzymes, SelenoH, SelenoI, SelenoT, SelenoV, and SelenoW and 31 metabolites were significantly altered in the brain after Se supplementation in conventional mice, while 11 selenoproteins and 26 metabolites were altered in microbiota depleted mice. The main altered brain metabolites were related to glyoxylate and dicarboxylate metabolism, amino acid metabolism, and gut microbiota that have been previously related with the gut-brain axis ( e.g., members of Lachnospiraceae and Ruminococcaceae families ). Moreover, specific associations were determined between brain selenoproteome and metabolome, which correlated with the same bacteria, suggesting an intertwined mechanism. Our results demonstrated the effect of Se on brain metabolome through specific selenoproteins gene expression and gut microbiota.This work was supported by the projects: PG2018-096608-B- C21 and PID2021-123073NB-C21 from the Spanish Ministry of Science and Innovation (MICIN) . Generación del Conocimiento . MCIN/ AEI /10.13039/50110 0 011033/ FEDER “Una manera de hacer Europa”, UHU-1256905 and UHU-202009 from the FEDER Andalusian Operative Program 2014-2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). S.R.A. thanks the Spanish Ministry of Science and Innovation for a PhD scholarship ( BES-2016-076364 ). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611 . The authors would like to acknowledge the support from The Ramón Areces Foundation (ref. CIVP19A5918 ). Funding for open access charge: Universidad de Huelva / CBUA

Targeted and untargeted metabolomic analysis of Procambarus clarkii exposed to a “chemical cocktail” of heavy metals and diclofenac

La contaminación del agua plantea un problema importante, pero se dispone de información limitada sobre los efectos conjuntos de xenobióticos de diferentes grupos químicos para evaluar la respuesta biológica real. Procambarus clarkii (P. clarkii) ha demostrado ser un buen bioindicador para evaluar la calidad de los ecosistemas acuáticos. En este trabajo, se ha estudiado la bioacumulación de cadmio (Cd), arsénico (As) y diclofenaco (DCF) en diferentes tejidos durante 21 días después de la exposición a un “cóctel químico” de As, Cd y DCF, y hasta 28 días considerando un período de purificación. Además, se llevó a cabo un análisis metabolómico dirigido y no dirigido para profundizar en las alteraciones metabólicas provocadas así como en la metabolización del DCF.Water pollution poses an important problem, but limited information is available about the joined effects of xenobiotics of different chemical groups to evaluate the real biological response. Procambarus clarkii (P. clarkii) has been demonstrated to be a good bioindicator for assessing the quality of aquatic ecosystems. In this work, we studied the bioaccumulation of cadmium (Cd), arsenic (As) and diclofenac (DCF) in different tissues of P. clarkii during 21 days after the exposure to a “chemical cocktail” of As, Cd and DCF, and until 28 days considering a depuration period. In addition, a combined untargeted and targeted metabolomic analysis was carried out to delve the metabolic impairments caused as well as the metabolization of DCF. Our results indicate that As and Cd were mainly accumulated in the hepatopancreas followed by gills and finally abdominal muscle. As and Cd show a general trend to increase the concentration throughout the exposure experience, while a decrease in the concentration of these elements is observed after 7 days of the depuration process. This is also the case in the abdominal muscle for Cd, but not for As and DCF, which increased the concentration in this tissue in the depuration phase. The hepatopancreas showed the greatest number of metabolic pathways affected. Thus, we observed a crucial bioaccumulation of xenobiotics and impairments of metabolites in different tissues. This is the first study combining the exposure to metals and pharmaceutically active compounds in P. clarkii by untargeted metabolomics including the biotransformation of DCF.This work was supported by the coordinated projects PGC 2018- 096608-B-C21 and PGC 2018-096608-B-C22 from the Spanish Ministry of Science and innovation (MCIN). (Generacion ´ del Conocimiento. MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa”). Authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611. Rodríguez-Moro, G. thanks to Plan Andaluz de Investigacion, ´ Desarrollo e Innovacion ´ (PAIDI 2020) and Fondo Social Europeo for a postdoctoral grant (DOC_01115). Au thors are grateful to Servicio General de Investigacion ´ de Microanalisis, ´ CENTRO DE INVESTIGACION ´ TECNOLOG´IA E INNOVACION ´ (CITIUS). Universidad de Sevilla, for the use of some of the chromatographic equipment. Funding for open access charge: Universidad de Huelva / CBUA

Metabolic Impairments Caused by Pesticides in Mammals and Their Interactions with Other Pollutants

The biological systems are exposed to a complex environment in which the contaminants can interact in a synergistic/antagonistic fashion and for this reason, the study of “chemical cocktails” is of great interest to fully understand the final biological effect. To evaluate the final biological response of a pollutant, it is essential to have an adequate analytical methodology that allows the correct monitoring of environmental systems in order to establish their quality, and, when appropriate, the application of corrective measures. Undoubtedly, massive methods “the omics” are among the most efficient current tools. To this end, transcriptomics, proteomics, metabolomics and chemical speciation can provide very useful information, mainly when they are combined. However, the combination of proteomics with metabolomics has some drawbacks as the temporal space is different (i.e. metabolomics gives information about what happens right now, but it can be related with numerous post-translational modifications happened previously). In this sense, it seems that the combination of genomics with metabolomics is easier. Thus, when metabolomics data are interpreted in combination with genomic, transcriptomic and proteomic results, in the so-called systems biology approach, a holistic knowledge of the organism/process under investigation can be achieved

Policy-making related actors' understandings about nature-society relationship : beyond modern ontologies? the case of Cuenca, Ecuador

Unidad de excelencia María de Maeztu MdM-2015-0552Over the last five decades the discursive debate on sustainability has reached prominence as the socio-ecological impacts of the human presence on Earth have grown rapidly. Nature discourses are interwoven with those of sustainability. Within this discursive field, a diverse set of competing discourses have emerged. Among the most radical ones, the discourse of Buen Vivir has recently gained relevance in Latin America. This position aims to depart from modern western ideologies, mainly those of nature-society dualism and Eurocentric universalism. In this study, the social perspectives about nature-society of subnational policy makers and other social actors involved in territorial planning in the city of Cuenca, Ecuador are examined. Four main social discourses are identified, which instead of breaking away from the society-nature divide, embrace it. Therefore, the case of Cuenca suggests that Ecuadorian citizens (including policy-makers) are still captured by the same discourses on nature-society belonging to the discursive field of modernity and its more contemporary corollaries: development and sustainable development. Hence, relational ontologies promoted by the discourse of Buen Vivir still do not resonate among Ecuadorian policy-related actors

Promoción de la salud y entornos saludables

A forestar forestalAplicación de un programa educativo participativo en salud  bucal a una comunidad de adultos mayoresBiblioteca móvil y su implementación en el hospital Padre HurtadoConsumo de riesgo de alcohol en Chile: una propuesta innovadora de intervenciónDiseño de un programa interactivo de promoción de la salud vocal para NB1Encuentro formativo en promoción de salud y gestión de entornos saludables para TenoExperiencia docente: programa intersectorial de promoción/prevención en preescolares de comunas vulnerables, Región MetropolitanaFiltrado glomerular, método preventivo aparición de fibrosis sistémica nefrogénica por gadolinio en examen de RMImplementación de consejerías en vida sana en APS, Región de los RíosMedicina preventiva en feria libre de la población San Gregorio: Cecof San Gregorio, Contagiando SaludMetodología innovadora en la enseñanza de una ectoparasitosisPrevención de accidentes por monóxido de carbono en edificios, Providencia 2002-2009Programa de promoción y prevención en salud bucal para preescolaresPromoviendo hábitos saludables en los vecinos de Reñaca Alto, Viña del Mar, 2009Rol de la capacitación en la implementación de acciones para la prevención de la obesidadSatisfacción usuaria en el Cesfam Natales a un año de su funcionamientoTres estrategias publicitarias y de comunicación aplicadas al consumo de alcohol de bajo riesgoTropa de la salud: uso de los medios como forma de promover la salu

Long-Term Real-World Effectiveness and Safety of Ustekinumab in Crohn’s Disease Patients: The SUSTAIN Study

Background Large real-world-evidence studies are required to confirm the durability of response, effectiveness, and safety of ustekinumab in Crohn’s disease (CD) patients in real-world clinical practice. Methods A retrospective, multicentre study was conducted in Spain in patients with active CD who had received ≥1 intravenous dose of ustekinumab for ≥6 months. Primary outcome was ustekinumab retention rate; secondary outcomes were to identify predictive factors for drug retention, short-term remission (week 16), loss of response and predictive factors for short-term efficacy and loss of response, and ustekinumab safety. Results A total of 463 patients were included. Mean baseline Harvey-Bradshaw Index was 8.4. A total of 447 (96.5%) patients had received prior biologic therapy, 141 (30.5%) of whom had received ≥3 agents. In addition, 35.2% received concomitant immunosuppressants, and 47.1% had ≥1 abdominal surgery. At week 16, 56% had remission, 70% had response, and 26.1% required dose escalation or intensification; of these, 24.8% did not subsequently reduce dose. After a median follow-up of 15 months, 356 (77%) patients continued treatment. The incidence rate of ustekinumab discontinuation was 18% per patient-year of follow-up. Previous intestinal surgery and concomitant steroid treatment were associated with higher risk of ustekinumab discontinuation, while a maintenance schedule every 12 weeks had a lower risk; neither concomitant immunosuppressants nor the number of previous biologics were associated with ustekinumab discontinuation risk. Fifty adverse events were reported in 39 (8.4%) patients; 4 of them were severe (2 infections, 1 malignancy, and 1 fever). Conclusions Ustekinumab is effective and safe as short- and long-term treatment in a refractory cohort of CD patients in real-world clinical practice