Efecto del arsénico inorgánico presente en los alimentos sobre el epitelio intestinal

El arsénico es un metaloide tóxico ampliamente distribuido en el medioambiente. Su forma inorgánica [As(III) y As(V)] es la más tóxica encontrada en agua y alimentos. La exposición a arsénico inorgánico, descrita en numerosas zonas del planeta y que en la actualidad se estima que afecta a 200 millones de personas, aumenta la incidencia de determinados tipos de cáncer y otras patologías no cancerígenas. Teniendo en cuenta que la principal vía de exposición es la oral, el tracto gastrointestinal se considera la puerta de entrada de este tóxico al organismo; sin embargo, los estudios para determinar los efectos tóxicos del arsénico inorgánico a nivel intestinal son escasos. Esta tesis doctoral tiene como objetivo evaluar la toxicidad del arsénico inorgánico sobre el epitelio intestinal en diferentes tipos de exposición in vitro e in vivo, así como la búsqueda de componentes de la dieta que puedan contrarrestar este efecto tóxico. Para tal fin se han empleado modelos celulares de colon (Caco-2, células procedentes de un adenocarcinoma y NCM460, línea celular no transformada) y ratones BALB/c. Como posibles estrategias de reducción de la toxicidad intestinal de este metaloide se han ensayado cepas de bacterias lácticas y suplementos dietéticos. Los estudios in vitro ponen de manifiesto que las exposiciones de corta duración a arsénico inorgánico (2-24 h) generan un aumento de la liberación de la citoquina pro-inflamatoria IL-8 y de la generación de especies reactivas de oxígeno y/o nitrógeno en células Caco-2 y NCM460. Esta respuesta pro­inflamatoria puede ser la responsable de las modificaciones estructurales que cursan con cambios en los patrones de distribución de la zonula occludens 1 y de expresión de la claudina 1, proteínas de las uniones intercelulares que interviene en el mantenimiento de la estructura del epitelio. Paralelamente a estos efectos se evidencia una pérdida de la función barrera de las monocapas intestinales. En las exposiciones subcrónicas in vitro (7-21 días), también se pone de manifiesto una respuesta pro-inflamatoria que se mantiene durante todo el tratamiento. Esta respuesta viene acompañada de cambios en el programa de proliferación celular y diferenciación, lo que conduce a un detrimento del proceso de reparación celular. Además, la exposición subcrónica afecta igualmente a la estructura epitelial, causando la pérdida de microvellosidades, y a la función de barrera del epitelio intestinal. Estos datos evidencian que la exposición aguda y subcrónica a arsénico inorgánico puede alterar la homeostasis intestinal, afectando la capa de la mucosa, que realiza las funciones más importantes de la pared intestinal. Adicionalmente se ha evidenciado in vitro que exposiciones más prolongadas (6 meses) favorecen la adquisición de características tumorogénicas en células NCM460, en parte debido al mantenimiento de la respuesta pro-inflamatoria. En las células expuestas crónicamente se observa un aumento de la expresión de CD133, disminución de la expresión de CDX1 y CDX2, mayor secreción de la metaloproteínasa de matrix MMP-2, modificaciones en el perfil de acetilación de la histona H3, aumento de la hiperproliferación y la formación de esferas flotantes. En general, estos datos sugieren que la exposición de células epiteliales de colon humano a As(III) provoca la adquisición de características de células transformadas. Los resultados obtenidos en la exposición subcrónica in vivo confirman los datos obtenidos in vitro. Esta exposición genera estrés oxidativo y una respuesta pro-inflamatoria, evidenciada por una mayor expresión génica y proteica de las citoquinas IL-1β, IL-2 e IL-6 en el intestino grueso de animales tratados con As(III). Asimismo, se observa un efecto sobre la morfología del epitelio, con evidencias de hiperplasia en las criptas. Los tratamientos con arsénico también reducen la expresión de la mucina 2 y posiblemente la formación de mucus. Estos efectos tóxicos pueden ser los causantes del aumento de permeabilidad observado en los animales tratados con arsénico. Este es el primer estudio que evidencia la pérdida de la función barrera del epitelio intestinal in vivo debido a una exposición a arsénico inorgánico. Finalmente, los ensayos in vitro para evaluar el papel protector de componentes de la dieta ponen de manifiesto que determinadas cepas de Lactobacillus y ciertos suplementos dietéticos modulan la toxicidad que el arsénico inorgánico ejerce sobre el epitelio intestinal. La recuperación de la función barrera y/o de la capacidad de regeneración celular tras los tratamientos indica que estas estrategias, cuya seguridad alimentaria está probada, podrían emplearse para aminorar los efectos que ejerce el arsénico inorgánico a nivel intestinal, y posiblemente a nivel sistémico, en poblaciones afectadas.Arsenic is a toxic metalloid widely distributed in the environment. Its inorganic form [As(III) and As(V)] is the most toxic form found in water and food. Human exposure to inorganic arsenic, described in numerous areas of the planet and that currently affects approximately 200 million people, increases the incidence of certain types of cancer and other non-cancer pathologies. Taking into account that the main route of the arsenic exposure is oral, the gastrointestinal tract is considered the entry of this contaminant into the organism; however, studies to determine the toxic effects of inorganic arsenic at the intestinal level are scarce. This thesis aims to evaluate the toxicity of inorganic arsenic in the intestinal epithelium in different types of exposure in vitro and in vivo, as well as the search for dietary components that can counteract this toxic effect. For this purpose, human cellular models of colon (Caco-2, cells from an adenocarcinoma and NCM460, a non-transformed cell line) and BALB/c mice were used. Strains of lactic acid bacteria and dietary supplements have been tested as possible strategies to reduce the toxic effect of the metalloid. In vitro studies show that acute exposures to inorganic arsenic (2-24 h) generates an increase in the release of the pro-inflammatory cytokine IL-8 and in the generation of reactive oxygen species and/or nitrogen in Caco-2 and NCM460 cells. This pro-inflammatory response may be responsible for the structural modifications that occur with changes in the distribution patterns of zonula occludens 1 and in the expression of claudin 1, proteins of the intercellular junctions involved in the maintenance of the structure of the epithelium. In addition to these effects, a loss of the barrier function of the intestinal monolayers is evidenced. In the subchronic exposures in vitro (7-21 days), a pro-inflammatory response is also observed, and it is maintained throughout the treatment. This response is accompanied by modifications in the cell proliferation and differentiation program, which leads to an impairment of the cell repair process. Subchronic exposure also affects the epithelial structure, causing loss of microvilli, and the barrier function of the intestinal epithelium. These data show that acute and subchronic exposure to inorganic arsenic can alter intestinal homeostasis, affecting the mucosal layer, which performs the most important functions of the intestinal wall. Additionally, it has been demonstrated in vitro that chronic exposures (6 months) stimulate the acquisition of tumorigenic characteristics in NCM460 cells, possibly due to the maintenance of the pro-inflammatory response. In cells chronically exposed, an increase in CD133 expression, a downregulation of CDX1 and CDX2 expression, an increase of the matrix metalloproteinase MMP-2 secretion, modifications in the histone H3 acetylation profile, an increase in the hyperproliferation and the formation of free-floating spheres were observed. In general, these data suggest that chronic exposure of human colon epithelial cells to As(III) causes the acquisition of transformed cell characteristics. The results obtained in subchronic exposure in vivo confirm the data obtained in vitro. This exposure generates oxidative stress and a pro-inflammatory response, evidenced by a higher gene and protein expression of the cytokines IL­1β, IL-2 and IL-6, in the large intestine of animals treated with As(III). In addition, a modification on the epithelium morphology is observed, with hyperplasia of the crypts. Arsenic treatments also reduce the expression of mucin 2 and possibly the formation of mucus. These toxic effects may be the cause of the increased permeability observed in animals treated with arsenic. This is the first study that demonstrates the loss of the intestinal epithelial barrier function in vivo due to exposure to inorganic arsenic. Finally, in vitro assays to evaluate the protective role of dietary components show that certain strains of Lactobacillus and certain dietary supplements modulate the toxicity exerted by the inorganic arsenic on the intestinal epithelium. The recovery of the barrier function and/or the capacity of cell regeneration after treatments indicate that these strategies, whose food safety has been proven, could be used to reduce the toxic effects exerted by inorganic arsenic at intestinal level and possibly at systemic level in affected populations

A Time-Course Comparison of Skeletal Muscle Metabolomic Alterations in Walker-256 Tumour-Bearing Rats at Different Stages of Life

Cancer cachexia is a severe wasting condition that needs further study to find ways to minimise the effects of damage and poor prognosis. Skeletal muscle is the most impacted tissue in cancer cachexia; thus, elucidation of its metabolic alterations could provide a direct clue for biomarker research and be applied to detect this syndrome earlier. In addition, concerning the significant changes in the host metabolism across life, this study aimed to compare the metabolic muscle changes in cachectic tumour-bearing hosts at different ages. We performed 1H-NMR metabolomics in the gastrocnemius muscle in weanling and young adult Walker-256 tumour-bearing rats at different stages of tumour evolution (initial, intermediate, and advanced). Among the 49 metabolites identified, 24 were significantly affected throughout tumour evolution and 21 were significantly affected regarding animal age. The altered metabolites were mainly related to increased amino acid levels and changed energetic metabolism in the skeletal muscle, suggesting an expressive catabolic process and diverted energy production, especially in advanced tumour stages in both groups. Moreover, these changes were more severe in weanling hosts throughout tumour evolution, suggesting the distinct impact of cancer cachexia regarding the host’s age, highlighting the need to adopting the right animal age when studying cancer cachexia

Potential use of agroindustrial byproducts in food: a study of nutrient availability

O Brasil está entre os dez países que mais desperdiçam alimentos, com cerca de 35% da produção agrícola indo para o lixo. O processamento agroindustrial de alimentos é uma das atividades que mais geram resíduos, com aproximadamente 50% de matéria-prima sendo descartada. A falta de informações sobre a qualidade nutricional desses subprodutos agroindustriais não possibilita seu potencial aproveitamento na fabricação de produtos alimentícios. Neste contexto, o objetivo do trabalho foi realizar a caracterização química de subprodutos resultantes do processamento industrial de frutas e vegetais e do beneficiamento de cereais. Os elementos químicos Br, Ca, Co, Cr, Cs, Fe, K, La, Na, Rb, Sc e Zn foram determinados através da análise por ativação neutrônica instrumental, a composição centesimal, através de métodos preconizados pela AOAC, os fatores antinutricionais, através das determinações de ácido fítico e taninos e a disponibilidade dos nutrientes in vitro para os elementos Ca, Fe, K e Zn e pelo sistema de células Caco-2 para o Fe. A maioria das amostras contém alto teor de fibras e proteína e baixo teor de lipídeos e valor calórico. O farelo de arroz, a casca da semente de cupuaçu, a semente de cupuaçu e o bagaço de framboesa apresentaram as maiores concentrações de ácido fítico, entre 19,9 e 10,7 mg g-1. Já a casca de uva apresentou a maior quantidade de taninos (23,8 mg/g de catequina). As amostras apresentaram boa disponibilidade in vitro de Ca e Zn. Porém, para Fe e K, os valores ficaram abaixo de 10% disponível para a maioria das amostras. Na análise de biodisponibilidade através do sistema de células Caco-2, a amostra que apresentou maior quantidade de ferritina foi a casca de pepino (56,8 ng ferritina/\'mü\'g proteína). Observou-se que os subprodutos, geralmente, apresentam quantidade maior ou igual de nutriente que a parte usualmente consumida do alimento, além de apresentar disponibilidade de nutrientes compatível com outros alimentos de origem vegetal. Os dados sugerem que os subprodutos agroindustriais são potenciais ingredientes para a indústria alimentícia, podendo agregar valor nutricional em novos produtos. Estudos futuros e mais específicos para cada subproduto devem ser considerados, como análise sensorial e maneiras de aumentar a qualidade nutricional dos subprodutosBrazil is amongst the ten countries that mostly waste food, with about 35% of agricultural production going to the trash. The agro-food processing is one of the activities which generate high amount of residues, with approximately 50% of raw material being discarded. The lack of information on the nutritional quality of agroindustrial byproducts does not enable its potential use in the manufacture of food products. In this context, the aim of this study was the chemical characterization of the by-products of industrial processing of fruits and vegetables and grain processing. The chemical elements Br, Ca, Co, Cr, Cs, Fe, K, La, Na, Rb, Sc e Zn were determined by instrumental neutron activation analysis. The proximate composition was evaluated by methods recommended by AOAC. The antinutritional factors, through the determination of phytic acid and tannins. The availability of nutrients in vitro for Ca, Fe, K and Zn and through the Caco-2 cells for Fe. Most samples contain high fiber and protein and low lipid content and calorific value. Rice bran, the peel of cupuaçu seed, the cupuaçu seed and the raspberry bagasse had the highest concentrations of phytic acid, between 19.9 and 10.7 mg g-1. The grape peel showed the highest amount of tannins (23.8 mg / g of catechin). The samples showed good in vitro availability for Ca and Zn, but the values for K and Fe were below 10% available for most samples. In the analysis of bioavailability through the Caco-2 cells system, the sample that showed the highest amount of ferritin was the peel of cucumber (ferritin 56.8 ng/\'mü\'g protein). It could be observed that the by-products generally exhibit similar or larger amounts of the nutrient than the food usually consumed, and nutrients availability compatible with other plant origin food. The data suggest that the agroindustrial byproducts are potential ingredients for the food industry and can add nutritional value to new products. Future studies more specific to each by-product should be considered like sensory analysis and ways to increase the nutritional quality of by-product

Effect of Enzymatic Biotransformation on the Hypotensive Potential of Red Grape Pomace Extract

Hypertension is a widespread health risk, affecting over a billion people and causing 9 million deaths per year. The Renin–Angiotensin–Aldosterone System (RAAS) is a primary target for hypertension treatment, and it is primarily treated through drugs that inhibit the Angiotensin I-Converting Enzyme (ACE). In addition to pharmacological treatment, various plants are recommended in traditional medicine for blood pressure regulation. This study aimed to produce high-phenolic-content extracts with and without enzymatic assistance from red grape pomace and evaluate their antioxidant capacity and ACE inhibitory potential. The total phenolic content (TPC) was measured, and phenolic identification was performed using HPLC analysis. In addition, the antioxidant capacity and anti-hypertensive potential were determined via in vitro assays. There was no statistical difference in the TPC antioxidant capacity between the extraction methods. Otherwise, when considering the extraction yield, the enzymatic process recovered around 70% more phenolic compounds from the pomace, and the phenolic profile was changed. Enzymatic assistance also significantly increased the ACE inhibitory potential in the grape pomace extract. This study demonstrates the viability of upcycling grape pomace to obtain bioactive compounds and to reduce their environmental impact, and highlights the influence of the enzymatic extraction on the hypotensive potential of the extract

Walker-256 Tumour-Induced Cachexia Altered Liver Metabolomic Profile and Function in Weanling and Adult Rats

Cancer cachexia occurs in up to 85% of advanced cancer patients, affecting different tissues and organs, mainly the liver, which plays a central role in body metabolism control. However, liver responses to cancer cachexia progression are still poorly understood. Considering the possible different challenges provided by the rodent’s phase of life and the cachexia progression, we evaluated the liver metabolic alterations affected by Walker-256 tumour growth in weanling and young-adult rats. For this, we applied a metabolomics approach associated with protein and gene expression analyses. Higher amino acid levels and impaired glucose metabolism were important features in tumour-bearing animals’ liver tissue. The weanling hosts had more pronounced cachexia, with higher carcass spoliation, liver lipid metabolism and impaired CII and CIV mitochondrial complexes. The liver alterations in young adult tumour-bearing rats were related to energy status and nucleotide metabolites, such as uridine, NAD+, xanthosine, hypoxanthine and inosine. In conclusion, the Walker-256 tumour-induced cachexia impaired liver metabolism, being more severe in the weanling hosts. Further studies are needed to correlate these changes in the preclinical model, which can be correlated to the clinical features of cancer cachexia, allowing for a translational potential involving the liver function and its responses to potential treatments

Leucine-Rich Diet Improved Muscle Function in Cachectic Walker 256 Tumour-Bearing Wistar Rats

Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia