In recent years a significant increase of gluten-related disorders (GRDs) has been observed. Two factors seem at the basis of this increase. The first set would be related to the diffusion of serological tests. The second set of data associated with the increased prevalence of CD and GRDs could be related to an increase in the global consumption of wheat in recent decades; Cereals that contain gluten are widely consumed and current wheat varieties have a higher content in gluten compared to the past due to changes directed by both technology and nutritional reasons. In the past, classification of GRDs was very simple, because CD and Dermatitis Herpetiformis (DH) were the only known diseases with a well-documented role of gluten in their pathogenesis. Increasing complexity in the nomenclature and clinical presentation of GRDs has led to the development of a consensus document by a panel of experts on new classification including: CD, Non-Coeliac Gluten Sensitivity (NCGS), Wheat Allergy (WA) DH, and gluten ataxia (GA) although there can show considerable overlap in the clinical presentation. Each gluten-related disorder exhibits a specific pathophysiological response to gluten ingestion. Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cereals, including rye and barley. The toxic protein fractions of gluten include gliadins and glutenins, with gliadins containing monomeric proteins and glutenins containing aggregated proteins. In wheat allergy (WA) and celiac disease (CD) the reaction to gluten is mediated by T-cell activation in the gastrointestinal mucosa. However, in WA it is the cross-linking of immunoglobulin (Ig)E by repeat sequences in gluten peptides (for example, serine-glutamine-glutamine -glutamine-(glutamine-) proline-proline-phenylalanine) that triggers the release of chemical mediators, such as histamine, from basophils and mast cells. Whereas in CD it is characterized as a chronic duodenal inflammation in which the increased secretion of inflammatory cytokines may in turn derange intestinal permeability and produce large amounts of reactive oxygen species (ROS), altering the redox state at the cellular level. Oxidative stress has been defined as the imbalance between the production of ROS and the antioxidant defenses of the cells in favor of the oxidants, leading to potential damage. ROS are produced during the cellular metabolic processes; if the production of ROS overwhelms a cell’s antioxidant (AO) capability, a condition known as oxidative stress occurs. The impairment of redox equilibrium proved to cause severe damage in proteins, lipids and DNA. In the last decade several studies showed that gluten exposure reflects in an intracellular oxidative imbalance, characterized by: increased levels of lipid peroxidation products (4-hydroxy-2(E)-nonenal (4-HNE)), increased oxidized (GSSG)/reduced (GSH) glutathione ratio and decreased number of protein-bound sulfhydryl groups. Finally, ROS can induce the formation of oxidative DNA lesion products (8-oxodG), which is considered as a mutagenicity marker. Oxidative damage can lead to single or double-strand breaks, point and frameshift mutations and chromosome abnormalities. There is considerable circumstantial evidence that oxidative DNA damage may play an important role not only in carcinogenesis, being used as a predictive marker of cancer development, but also in aging. Nowadays, studies concerning oxidative damage on DNA have not been developed with regard to CD. The aim of this study was to investigate a possible gliadin-induced genotoxic damage and its correlation with oxidative stress in vitro and in vivo. The in vitro models consist of Caco-2 cell line of heterogeneous human epithelial colorectal adenocarcinoma cells generally used for this type of study. Whereas the in vivo model has been used serum and duodenal biopsies of patients. For this study the Caco-2 cells have been exposed for maximum 24h to increasing concentrations (250µg/mL‒1000µg/mL) of digested gliadin (PT gliadin). Starting from a 500µg/mL dose (with a 12-hour contact time), an increase of reactive oxygen species (ROS) (DCFDA probe) was observed. The alkaline Comet assay showed DNA damage at a concentration of 1000 g/mL after 24h, characterized by a significant increase of DNA in the tail. Furthermore, the enzyme-modified Comet assay showed oxidative damage mainly with endonuclease-III, calculated as ∆ tail moment after 24h treatment at 1000µg/mL. Moreover, immunohistochemistry ɣ-H2AX detection (focal phosphorylation of histone H2AX at serine 139 to generate ɣ-H2AX in response to double-strand breaks) demonstrated an increase of the number of foci at 500µg/mL and 1000 µg/mL as genotoxic signature. The transglutaminase type 2 (TG2) activity was evaluated by western blot and ELISA method. The results show an increase of the enzyme expression in the chromatin and cytoskeleton at different doses (250, 500, and 1000µg/mL) compatible with apoptosis, as confirmed by annexin V (cytofluorometric staining method). Has been observed an increase of apoptotic cells starting from a 250 µg/ml dose. The oxidative stress has been evaluated through the analysis of biomarkers into the serum of patients: naïve celiac disease (nCD), celiac patient responders, undergoing to gluten free diet at least 12 months (CD-GDF), refractory celiac patient non responders, undergoing to gluten free diet at least 12 months (RCD) and healthy subjects (CTRL). The results demonstrated that Total Antioxidant Capacity (TAC) appeared to be significantly decreased in patients RCD respect to CD-GDF (p<0.05) and to CTRL (p<0.001). Plasmatic concentrations of TBARS, assessed as marker of lipid peroxidation and protein carbonyls (PC), assessed as marker of protein oxidation, were found significantly increased in RCD respect to CD-GDF, while the plasma levels of CD-GDF resulted similar to CTRL in both plasma biomarkers. Whereas the genotoxic damage was confirmed in vivo at the duodenal biopsy of celiac patients by means of H2AX e 8-OHG immunohistochemistry. In conclusion digested gliadin induces an increase of ROS production in Caco-2 cells with an alteration of the cellular redox state. Moreover, the high concentration of ROS induces DNA damage and the stimulation of the apoptotic process. This mechanism seems present also in vivo as demonstrated by the findings from CD patients.

EFFETTO DELLA GLIADINA SUL BILANCIO OSSIDATIVO E DANNO AL DNA NELLA LINEA CELLULARE CACO-2 E IN PAZIENTI CELIACI / E. Monguzzi ; tutor: D. Conte, L. Elli ; direttore della scuola: A. D'Arminio Monforte. - : . DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2017 Apr 26. ((29. ciclo, Anno Accademico 2016. [10.13130/monguzzi-erika_phd2017-04-26].

EFFETTO DELLA GLIADINA SUL BILANCIO OSSIDATIVO E DANNO AL DNA NELLA LINEA CELLULARE CACO-2 E IN PAZIENTI CELIACI

E. Monguzzi
2017-04-26

Abstract

In recent years a significant increase of gluten-related disorders (GRDs) has been observed. Two factors seem at the basis of this increase. The first set would be related to the diffusion of serological tests. The second set of data associated with the increased prevalence of CD and GRDs could be related to an increase in the global consumption of wheat in recent decades; Cereals that contain gluten are widely consumed and current wheat varieties have a higher content in gluten compared to the past due to changes directed by both technology and nutritional reasons. In the past, classification of GRDs was very simple, because CD and Dermatitis Herpetiformis (DH) were the only known diseases with a well-documented role of gluten in their pathogenesis. Increasing complexity in the nomenclature and clinical presentation of GRDs has led to the development of a consensus document by a panel of experts on new classification including: CD, Non-Coeliac Gluten Sensitivity (NCGS), Wheat Allergy (WA) DH, and gluten ataxia (GA) although there can show considerable overlap in the clinical presentation. Each gluten-related disorder exhibits a specific pathophysiological response to gluten ingestion. Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cereals, including rye and barley. The toxic protein fractions of gluten include gliadins and glutenins, with gliadins containing monomeric proteins and glutenins containing aggregated proteins. In wheat allergy (WA) and celiac disease (CD) the reaction to gluten is mediated by T-cell activation in the gastrointestinal mucosa. However, in WA it is the cross-linking of immunoglobulin (Ig)E by repeat sequences in gluten peptides (for example, serine-glutamine-glutamine -glutamine-(glutamine-) proline-proline-phenylalanine) that triggers the release of chemical mediators, such as histamine, from basophils and mast cells. Whereas in CD it is characterized as a chronic duodenal inflammation in which the increased secretion of inflammatory cytokines may in turn derange intestinal permeability and produce large amounts of reactive oxygen species (ROS), altering the redox state at the cellular level. Oxidative stress has been defined as the imbalance between the production of ROS and the antioxidant defenses of the cells in favor of the oxidants, leading to potential damage. ROS are produced during the cellular metabolic processes; if the production of ROS overwhelms a cell’s antioxidant (AO) capability, a condition known as oxidative stress occurs. The impairment of redox equilibrium proved to cause severe damage in proteins, lipids and DNA. In the last decade several studies showed that gluten exposure reflects in an intracellular oxidative imbalance, characterized by: increased levels of lipid peroxidation products (4-hydroxy-2(E)-nonenal (4-HNE)), increased oxidized (GSSG)/reduced (GSH) glutathione ratio and decreased number of protein-bound sulfhydryl groups. Finally, ROS can induce the formation of oxidative DNA lesion products (8-oxodG), which is considered as a mutagenicity marker. Oxidative damage can lead to single or double-strand breaks, point and frameshift mutations and chromosome abnormalities. There is considerable circumstantial evidence that oxidative DNA damage may play an important role not only in carcinogenesis, being used as a predictive marker of cancer development, but also in aging. Nowadays, studies concerning oxidative damage on DNA have not been developed with regard to CD. The aim of this study was to investigate a possible gliadin-induced genotoxic damage and its correlation with oxidative stress in vitro and in vivo. The in vitro models consist of Caco-2 cell line of heterogeneous human epithelial colorectal adenocarcinoma cells generally used for this type of study. Whereas the in vivo model has been used serum and duodenal biopsies of patients. For this study the Caco-2 cells have been exposed for maximum 24h to increasing concentrations (250µg/mL‒1000µg/mL) of digested gliadin (PT gliadin). Starting from a 500µg/mL dose (with a 12-hour contact time), an increase of reactive oxygen species (ROS) (DCFDA probe) was observed. The alkaline Comet assay showed DNA damage at a concentration of 1000 g/mL after 24h, characterized by a significant increase of DNA in the tail. Furthermore, the enzyme-modified Comet assay showed oxidative damage mainly with endonuclease-III, calculated as ∆ tail moment after 24h treatment at 1000µg/mL. Moreover, immunohistochemistry ɣ-H2AX detection (focal phosphorylation of histone H2AX at serine 139 to generate ɣ-H2AX in response to double-strand breaks) demonstrated an increase of the number of foci at 500µg/mL and 1000 µg/mL as genotoxic signature. The transglutaminase type 2 (TG2) activity was evaluated by western blot and ELISA method. The results show an increase of the enzyme expression in the chromatin and cytoskeleton at different doses (250, 500, and 1000µg/mL) compatible with apoptosis, as confirmed by annexin V (cytofluorometric staining method). Has been observed an increase of apoptotic cells starting from a 250 µg/ml dose. The oxidative stress has been evaluated through the analysis of biomarkers into the serum of patients: naïve celiac disease (nCD), celiac patient responders, undergoing to gluten free diet at least 12 months (CD-GDF), refractory celiac patient non responders, undergoing to gluten free diet at least 12 months (RCD) and healthy subjects (CTRL). The results demonstrated that Total Antioxidant Capacity (TAC) appeared to be significantly decreased in patients RCD respect to CD-GDF (p<0.05) and to CTRL (p<0.001). Plasmatic concentrations of TBARS, assessed as marker of lipid peroxidation and protein carbonyls (PC), assessed as marker of protein oxidation, were found significantly increased in RCD respect to CD-GDF, while the plasma levels of CD-GDF resulted similar to CTRL in both plasma biomarkers. Whereas the genotoxic damage was confirmed in vivo at the duodenal biopsy of celiac patients by means of H2AX e 8-OHG immunohistochemistry. In conclusion digested gliadin induces an increase of ROS production in Caco-2 cells with an alteration of the cellular redox state. Moreover, the high concentration of ROS induces DNA damage and the stimulation of the apoptotic process. This mechanism seems present also in vivo as demonstrated by the findings from CD patients.
CONTE, DARIO
D'ARMINIO MONFORTE, ANTONELLA
Settore MED/12 - Gastroenterologia
EFFETTO DELLA GLIADINA SUL BILANCIO OSSIDATIVO E DANNO AL DNA NELLA LINEA CELLULARE CACO-2 E IN PAZIENTI CELIACI / E. Monguzzi ; tutor: D. Conte, L. Elli ; direttore della scuola: A. D'Arminio Monforte. - : . DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2017 Apr 26. ((29. ciclo, Anno Accademico 2016. [10.13130/monguzzi-erika_phd2017-04-26].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/490822
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