Subcellular distribution and Nrf2/Keap1-interacting properties of Glutathione S-transferase P in hepatocellular carcinoma.

The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2-related factor 2 (Nrf2). Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments. GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and ß-TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and ß-TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation. In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.

Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Randomised controlled trial combining vitamin E-functionalised chocolate with physical exercise to reduce the risk of protein-energy malnutrition in predementia aged people: study protocol for Choko-Age.

OBJECTIVE: Protein-energy malnutrition and the subsequent muscle wasting (sarcopenia) are common ageing complications. It is knowing to be also associated with dementia. Our programme will test the cytoprotective functions of vitamin E combined with the cortisol-lowering effect of chocolate polyphenols (PP), in combination with muscle anabolic effect of adequate dietary protein intake and physical exercise to prevent the age-dependent decline of muscle mass and its key underpinning mechanisms including mitochondrial function, and nutrient metabolism in muscle in the elderly. METHODS AND ANALYSIS: In 2020, a 6-month double-blind randomised controlled trial in 75 predementia older people was launched to prevent muscle mass loss, in respond to the 'Joint Programming Initiative A healthy diet for a healthy life'. In the run-in phase, participants will be stabilised on a protein-rich diet (0.9-1.0 g protein/kg ideal body weight/day) and physical exercise programme (high-intensity interval training specifically developed for these subjects). Subsequently, they will be randomised into three groups (1:1:1). The study arms will have a similar isocaloric diet and follow a similar physical exercise programme. Control group (n=25) will maintain the baseline diet; intervention groups will consume either 30 g/day of dark chocolate containing 500 mg total PP (corresponding to 60 mg epicatechin) and 100 mg vitamin E (as RRR-alpha-tocopherol) (n=25); or the high polyphenol chocolate without additional vitamin E (n=25). Muscle mass will be the primary endpoint. Other outcomes are neurocognitive status and previously identified biomolecular indices of frailty in predementia patients. Muscle biopsies will be collected to assess myocyte contraction and mitochondrial metabolism. Blood and plasma samples will be analysed for laboratory endpoints including nutrition metabolism and omics. ETHICS AND DISSEMINATION: All the ethical and regulatory approvals have been obtained by the ethical committees of the Azienda Ospedaliera Universitaria Integrata of Verona with respect to scientific content and compliance with applicable research and human subjects' regulation. Given the broader interest of the society toward undernutrition in the elderly, we identify four main target audiences for our research activity: national and local health systems, both internal and external to the project; targeted population (the elderly); general public; and academia. These activities include scientific workshops, public health awareness campaigns, project dedicated website and publication is scientific peer-review journals. TRIAL REGISTRATION NUMBER: NCT05343611.

Melatonin as a Repairing Agent in Cadmium- and Free Fatty Acid-Induced Lipotoxicity.

(1) Background: Cadmium (Cd) is a potentially toxic element with a long half-life in the human body (20-40 years). Cytotoxicity mechanisms of Cd include increased levels of oxidative stress and apoptotic signaling, and recent studies have suggested that these aspects of Cd toxicity contribute a role in the pathobiology of non-alcoholic fatty liver disease (NAFLD), a highly prevalent ailment associated with hepatic lipotoxicity and an increased generation of reactive oxygen species (ROS). In this study, Cd toxicity and its interplay with fatty acid (FA)-induced lipotoxicity have been studied in intestinal epithelium and liver cells; the cytoprotective function of melatonin (MLT) has been also evaluated. (2) Methods: human liver cells (HepaRG), primary murine hepatocytes and Caco-2 intestinal epithelial cells were exposed to CdCl2 before and after induction of lipotoxicity with oleic acid (OA) and/or palmitic acid (PA), and in some experiments, FA was combined with MLT (50 nM) treatment. (3) Results: CdCl2 toxicity was associated with ROS induction and reduced cell viability in both the hepatic and intestinal cells. Cd and FA synergized to induce lipid droplet formation and ROS production; the latter was higher for PA compared to OA in liver cells, resulting in a higher reduction in cell viability, especially in HepaRG and primary hepatocytes, whereas CACO-2 cells showed higher resistance to Cd/PA-induced lipotoxicity compared to liver cells. MLT showed significant protection against Cd toxicity either considered alone or combined with FFA-induced lipotoxicity in primary liver cells. (4) Conclusions: Cd and PA combine their pro-oxidant activity to induce lipotoxicity in cellular populations of the gut-liver axis. MLT can be used to lessen the synergistic effect of Cd-PA on cellular ROS formation.

Transcriptomics of natural and synthetic vitamin D in human hepatocyte lipotoxicity.

Vitamin D (VD) has been used to prevent nonalcoholic fatty liver disease (NAFLD), a condition of lipotoxicity associated with a defective metabolism and function of this vitamin. Different forms of VD are available and can be used for this scope, but their effects on liver cell lipotoxicity remain unexplored. In this study we compared a natural formulation rich in VD2 (Shiitake Mushroom extract or SM-VD2) with a synthetic formulation containing pure VD3 (SV-VD3) and the bioactive metabolite 1,25(OH)2-D3. These were investigated in chemoprevention mode in human HepaRG liver cells supplemented with oleic and palmitic acid to induce lipotoxicity. All the different forms of VD showed similar efficacy in reducing the levels of lipotoxicity and the changes that lipotoxicity induced on the cellular transcriptome. However, the three forms of VD generated different gene fingerprints suggesting diverse, even if functionally convergent, cytoprotective mechanisms. Main differences were (1) the number of differentially expressed genes (SV-VD3 > 1,25[OH]2-D3 > SM-VD2), (2) their identity that demonstrated significant gene homology between SM-VD2 and 1,25(OH)2-D3, and (3) the number and type of biological functions identified by ingenuity pathway analysis as relevant to liver metabolism and cytoprotection annotations. Immunoblot confirmed a different response of VDR and other VDR-related proteins to natural and synthetic VD formulations, including FXR, PXR, PPARγ/PGC-1α, and CYP3A4 and CYP24A1. In conclusion, different responses of the cellular transcriptome drive the cytoprotective effect of natural and synthetic formulations of VD in the free fatty acid-induced lipotoxicity of human hepatocytes.

Wheat germ oil vitamin E cytoprotective effect and its nutrigenomics signature in human hepatocyte lipotoxicity.

Wheat germ oil (WGO) is rich in α-tocopherol (vitamin E, VE), a vitamin that has long been suggested to exert hepatoprotective effects. In this study, this function of WGO-VE and its transcriptomics fingerprint were investigated in comparison with RRR-α-tocopherol and all-rac-α-tocopherol (nVE and sVE, respectively), in human liver cells treated with oleic acid (OA) to develop steatosis and lipotoxicity. Used in chemoprevention mode, all the VE formulations afforded significant reduction of the OA-induced steatosis and its consequent impact on lipotoxicity indicators, including ROS production and efflux (as H2O2), and apoptotic and necrotic cell death. A trend toward a better control of lipotoxicity was observed for WGO-VE and nVE compared to sVE. Gene microarray data demonstrated that these effects of VE formulations were associated with significantly different responses of the cellular transcriptome to compensate for the modifications of OA treatment, including the downregulation of cellular homeostasis genes and the induction of genes associated with defects of liver cell metabolism, fibrosis and inflammation, liver disease and cancer. Ingenuity Pathway Analysis data showed that WGO-VE modulated genes associated with liver carcinogenesis and steatosis, whereas nVE modulated genes involved in liver cell metabolism and viability biofunctions; sVE did not significantly modulate any gene dataset relevant to such biofunctions. In conclusion, WGO-VE prevents lipotoxicity in human liver cells modulating genes that differ from those affected by the natural or synthetic forms of pure VE. These differences can be captured by precision nutrition tools, reflecting the molecular complexity of this VE-rich extract and its potential in preventing specific cues of hepatocellular lipotoxicity.

How Aging and Oxidative Stress Influence the Cytopathic and Inflammatory Effects of SARS-CoV-2 Infection: The Role of Cellular Glutathione and Cysteine Metabolism.

SARS-CoV-2 infection can cause a severe respiratory distress syndrome with inflammatory and thrombotic complications, the severity of which increases with patients' age and presence of comorbidity. The reasons for an age-dependent increase in the risk of severe COVID-19 could be many. These include defects in the homeostatic processes that control the cellular redox and its pivotal role in sustaining the immuno-inflammatory response to the host and the protection against oxidative stress and tissue degeneration. Pathogens may take advantage of such age-dependent abnormalities. Alterations of the thiol redox balance in the lung tissue and lining fluids may influence the risk of infection, and the host capability to respond to pathogens and to avoid severe complications. SARS-CoV-2, likewise other viruses, such as HIV, influenza, and HSV, benefits in its replication cycle of pro-oxidant conditions that the same viral infection seems to induce in the host cell with mechanisms that remain poorly understood. We recently demonstrated that the pro-oxidant effects of SARS-CoV-2 infection are associated with changes in the cellular metabolism and transmembrane fluxes of Cys and GSH. These appear to be the consequence of an increased use of Cys in viral protein synthesis and to ER stress pathway activation that interfere with transcription factors, as Nrf2 and NFkB, important to coordinate the metabolism of GSH with other aspects of the stress response and with the pro-inflammatory effects of this virus in the host cell. This narrative review article describes these cellular and molecular aspects of SARS-CoV-2 infection, and the role that antivirals and cytoprotective agents such as N-acetyl cysteine may have to limit the cytopathic effects of this virus and to recover tissue homeostasis after infection.

A Dynamic Model for Estimating the Interaction of ROS-PUFA-Antioxidants in Rabbit.

Defining optimal nutrition in animals and humans remains a main scientific challenge. The objective of the work was to develop a dynamic model of reactive oxygen species (ROS)-polyunsaturated fatty acid (PUFA)-antioxidant homeostasis using the rabbit as a model. The problem entity was to evaluate the main metabolites generated from interactions between traits included in the conceptual model and identified by three main sub-models: (i) ROS generation, (ii) PUFA oxidation and (iii) antioxidant defence. A mathematical model (VENSIM software) that consisted of molecular stocks (INPUTs, OUTPUTs), exchange flows (intermediate OUTPUTs) and process rates was developed. The calibration was performed by using standard experimental data (Experiment 1), whereas the validation was carried out in Experiments 2 and 3 by using supra-nutritional dietary inputs (VIT E+ and PUFA+). The accuracy of the models was measured using 95% confidence intervals. Analytical OUTPUTs (ROS, PUFA, Vit E, Ascorbic acid, Iso-/NeuroProstanes, Aldehydes) were well described by the standard model. There was also good accuracy for the VIT E+ scenario, whereas some compensatory rates (Kc1-Kc4) were added to assess body compensation when high levels of dietary PUFA were administered (Experiment 3). In conclusion, the model can be very useful for predicting the effects of dietary treatments on the redox homeostasis of rabbits.