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Sample manipulation for storage and storage itself, interfere with the stability of labile lipids in human plasma, including vitamin E (α-tocopherol), polyunsaturated fatty acids (PUFAs), and their enzymatic and free radical-derived oxidation metabolites. This remains a main limit of lipidomics studies that often lack of sufficient standardization and validation at the pre-analytical level. In order to characterize the stability of these lipids in human plasma and to develop a standardized pre-analytical protocol for lipidomics methods, the oxidation metabolites of α-tocopherol, the free form of ω3 and ω6 PUFAs, and some arachidonic acid (AA)-derived eicosanoids were investigated in human plasma during storage at different freezing temperatures. The effect of a protection/defense cocktail of antioxidants and lipoxygenase inhibitors (PD solution) on these lipid parameters was also evaluated. The temperature of storage markedly affected the formation of α-tocopheryl quinone (α-TQ), the main lipoperoxyl radical-derived oxidation metabolite of vitamin E, with the lowest production rate observed in samples stored at −80 °C or in liquid nitrogen. A similar effect of the storage temperature was observed for the free form of the ω-3 species eicosapentaenoic and docosahexaenoic acid, and for the ω-6 AA. Freezing samples at −20 °C resulted in a time-dependent formation of the pro-inflammatory eicosanoid LTB4. The PD solution prevents non-specific alterations of these lipid parameters in samples that are processed for direct analysis and protects from the temperature-dependent modifications of free PUFAs. Combining PD solution and preservation at −80 °C or in liquid nitrogen, resulted in levels of α-TQ and PUFAs that remained stable over 1 month and up to 8 months of storage, respectively. This method paper provides indications for the optimal processing and storage of human plasma utilized in lipidomics studies.

Lipid disorders have been implicated in overweight and menopause. However, evidence on lipidomic analysis of fatty acids in erythrocytes of menopausal women is scarce. The aim of this study was to investigate the relationship between the body mass index within or beyond 5 years of menopause and erythrocyte fatty acid profile. This case-control study was conducted on out of 37 menopausal women total patients, 22 with body mass index ≥ 25 and 12 matched controls (body mass index 25 and non-physiological content of fatty acids, were richer in percentage of saturated fatty acids and poorer of monounsaturated fatty acids and polyunsaturated fatty acids than women with BMI25 than in women with BMI

Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in β-amyloid (Aβ) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aβ oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator–activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aβ aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aβ deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aβ oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein.

Vitamin E is the term to identify a group of eight plant-derived and fat-soluble molecules with antioxidant and gene regulation functions. The most relevant form for human nutrition and health is α-tocopherol, the functions of which have been demonstrated in humans and animal models of vitamin E deficiency to be essential for homeostatic processes primarily of the central nervous system and possibly of other tissues/systems, such as red blood cells, the immune system, and the liver. The molecular mechanisms behind vitamin E’s essential role remains elusive. It is apparent however that α-tocopherol affects structural and metabolic aspects of the cellular membrane and possibly of other subcellular structures, such as the transport of vesicles and lipid droplets. Its activity as an H atom donor influences at the same time the metabolism and molecular integrity of membrane phospholipids, affecting enzymatic and spontaneous (free radical-dependent) oxidations of polyunsaturated lipids to form eicosanoids and lipid by-products, many of which are potent mediators of immunovascular processes and/or toxic molecules. Nonalpha forms of vitamin E have tissue levels, distribution and functions alternative to α-tocopherol that are regulated at different levels of liver and peripheral tissue metabolism (discussed in the accompanying Chapter 24).

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor with a key role in drug metabolism and disposition. Its activity regulates a number of physiological processes in the liver and intestine, and it is now a validated target for human diseases associated with inflammation and dysregulation of the immune system. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified the naturally-occurring garcinoic acid as a selective and efficient PXR agonist. The properties of garcinoic acid as a specific PXR agonist was demonstrated using different approaches - screening on a panel of nuclear receptors, physical and thermodynamic evaluation of binding affinity, and co-crystallization study. Cytotoxicity assays, transcriptional and functional experiments were carried out in human liver cells, in mouse liver and gut tissue to prove compound activity and target engagement. Taken together, these data support the conclusion that garcinoic acid efficiently activates PXR and may prove to be an amenable lead toward the development of differentially acting PXR regulating compounds.

Vitamin E metabolism is a complex and highly regulated process intimately associated with lipoprotein metabolism. Hepatic steps of this process markedly differ between alpha-tocopherol (α-TOH) and other forms of this vitamin, including the cytochrome P450-dependent transformation to a series of metabolites that are now utilized as both nutritional and functional indicators of the vitamin status and metabolism. In the last three decades a number of protein interactions and gene modulation effects that characterize these differences have identified. These aspects are described in this chapter along with the recently identified role of enzymatic and free radical-derived metabolites of this vitamin as bioactive lipids. Compelling in vitro and in vivo data demonstrate that long-chain metabolites of α-TOH are more potent than their precursor in modulating peroxidation reactions and lipid metabolism, inflammatory genes, cell cycle regulation, and antiatherogenic pathways. The majority of these studies took advantage of the recently developed lipidomics technology allowing unbiased identifications and quantification of the entire spectrum of physiological metabolite in biological fluids, cell culture samples, and tissues. Other achievements also included the recognition of new metabolites, the identity of which are currently under investigation. These studies on the functional metabolome of vitamin E have led to speculation that, similarly to other fat-soluble vitamins, vitamin E may provide some of its biological roles by behaving as a provitamin. If confirmed this would represent a real breakthrough in human nutrition and a major step forward in defining vitamin E essentiality and adequacy, and their assessment strategy as well.

Specific microRNAs (miRs), including the "angio-miR-126" and the "inflamma-miR-146a-5p," have been proposed as biomarkers and even therapeutic targets of obesity-associated metabolic diseases. Physical activity, a key measure of prevention for obesity and its complications, is reported to influence the expression of these miRs. In this study, we investigate whether a physical activity program proven to improve metabolic parameters in obese patients can correct the circulating levels of these miRs. Plasma miR-126 and miR-146a-5p were measured in a cohort of obese patients (n = 31, 16F + 15M) before and after the 3-month physical activity program of the CURIAMO trial (registration number for clinical trials: ACTRN12611000255987) and in 37 lean controls (24F + 13M). miR-146a-5p, but not miR-126, was significantly increased in obese patients as compared with lean controls and decreased in approximately two-thirds of the participants post-intervention with a response that positively correlated with pre-intervention levels of this miR. Waist circumference, the inflammatory cytokine IL-8 and lipid parameters, principally total cholesterol, showed the strongest correlation with both the baseline levels and post-intervention correction of miR-146a-5p. Post-hoc analysis of experimental data supports the use of miR-146a-5p as a biomarker and predictor of the clinical response to physical activity in obese patients. Furthermore, miR-146a-5p expression was confirmed to increase together with that of the inflammatory genes TLR4, NF-κB, IL-6, and TNF-α in LPS-stimulated human mononuclear leukocytes. In conclusion, the inflamma-miR-146a-5p can serve as a personalized predictor of clinical outcome in obese patients entering physical activity weight-reduction programs. © 2018 IUBMB Life, 70(10):1012-1022, 2018.

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.

The role of vitamin E in both enzymatic and free radical-dependent metabolism of polyunsaturated fatty acids (PUFAs) has been well demonstrated. This study proposed a new LC-MS/MS method to quantify the main vitamin E forms, their metabolites and main PUFA species in human blood, since, at present, there are not procedures able to simultaneously determine these two classes of compounds. After the optimization of sample treatment and reverse-phase separation conditions, tandem mass spectrometry detection was evaluated experimenting both positive and negative electrospray ionisation modes. The procedure was also preliminarily adapted to assess five arachidonic acid-derived eicosanoids that could be under the influence of vitamin E function, such as LTB4 (leukotriene B4), 20-HETE (20-hydroxyeicosatetraenoic acid) and their ω-oxidation metabolites. After the validation study, the performance characteristics were confirmed analysing a certified reference material (SRM® 1950 - frozen human plasma by NIST). Finally, an application of the method in the analysis of lipid abnormalities of chronic kidney disease patients was shown.

Non-alcoholic fatty liver disease (NAFLD) represents the most common form of chronic liver disease worldwide (about 25% of the general population) and 3-5% of patients develop non-alcoholic steatohepatitis (NASH), characterized by hepatocytes damage, inflammation and fibrosis, which increase the risk of developing liver failure, cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD, particularly the mechanisms whereby a minority of patients develop a more severe phenotype, is still incompletely understood. In this review we examine the available literature on initial mechanisms of hepatocellular damage and inflammation, deriving from toxic effects of excess lipids. Accumulating data indicate that the total amount of triglycerides stored in the liver cells is not the main determinant of lipotoxicity and that specific lipid classes act as damaging agents. These lipotoxic species affect the cell behavior via multiple mechanisms, including activation of death receptors, endoplasmic reticulum stress, modification of mitochondrial function and oxidative stress. The gut microbiota, which provides signals through the intestine to the liver, is also reported to play a key role in lipotoxicity. Finally, we summarize the most recent lipidomic strategies utilized to explore the liver lipidome and its modifications in the course of NALFD. These include measures of lipid profiles in blood plasma and erythrocyte membranes that can surrogate to some extent lipid investigation in the liver.

Two recent randomized controlled trials demonstrated improved radiographic, histological and hepatometabolic cues of non-alcoholic steatohepatitis (NASH) in pediatric patients treated with the ω-3 fatty acid docosahexaenoic acid (DHA) in combination with vitamin D (VD) or with choline (CHO) and vitamin E (VE), the DHA-VD and DHA-CHO-VE trials, respectively). In the present study we verified the nutritional compliance to these DHA-based multivitamin treatments; lipidomics biomarkers of the reported outcome on NASH indicators were also investigated. Samples were obtained from 30 biopsy-proven pediatric NASH patients of the DHA-CHO-VE trial randomized in multivitamin treatment group and placebo group (n = 15 each), and from 12 patients of the treatment group of the DHA-VD trial. All patients underwent 6-month therapy plus 6 months of follow-up. Plasma samples and clinical data were obtained at baseline and at the end of the study (12 months). Selected biomarkers included the free form of DHA and other ω-3 fatty acid arachidonic acid (AA), indices of the vitamin E status, and some hepatic metabolites of these lipids. Radiographic and histological improvements of treated patients were associated with increased concentrations of DHA, α-linolenic acid and α-tocopherol (i.e. VE), and with decreased AA that was also investigated in complex lipids by untargetd lipidomics. As a result a significantly lowered AA/DHA ratio was observed to represent the main indicator of the response to the DHA-based therapy. Furthermore, baseline levels of AA/DHA showed strong association with NAS and US improvement. A stable correction of DHA AA metabolism interaction is associated with the curative effect of this therapy and may represent a key nutritional endpoint in the clinical management of pediatric NASH.

2,2'-diselenyldibenzoic acid (DSBA) is a chemical probe produced to explore the pharmacological properties of diphenyldiselenide-derived agents with seleno-hormetic activity undergoing preclinical development. The present study was designed to verify in vivo the drug’s properties and to determine mechanistically how these may mediate the protection of tissues against stress conditions, exemplified by ionizing radiation induced damage in mouse bone marrow. In murine bone marrow hematopoietic cells, the drug initiated the activation of the Nrf2 transcription factor resulting in enhanced expression of downstream stress response genes. This type of response was confirmed in human liver cells and included enhanced expression of glutathione S-transferases (GST), important in the metabolism and pharmacological function of seleno-compounds. In C57 BL/6 mice, DSBA prevented the suppression of bone marrow hematopoietic cells caused by ionizing radiation exposure. Such in vivo prevention effects were associated with Nrf2 pathway activation in both bone marrow cells and liver tissue. These findings demonstrated for the first time the pharmacological properties of DSBA in vivo, suggesting a practical application for this type of Se-hormetic molecules as a radioprotective and/or prevention agents in cancer treatments.

Enzymatic and non-enzymatic (free-radical-derived) metabolites of vitamin E are objects of increasing interest as both are indicators of the metabolism and biological functions of this fat-soluble vitamin. These include bioactive long-chain metabolites of alpha-tocopherol formed by the activity of cytochrome P450, which have recently been measured for the first time in human plasma using a new unbiased LC-MS/MS procedure. This chapter summarises the available knowledge on analysis strategies and techniques currently in use to measure the different types of metabolites of this vitamin so far identified and studied in biological samples.

After more than 90 years from its discovery and thousands of papers published, the physiological roles of vitamin E (tocopherols and tocotrienols) are still not fully clarified. In the last few decades, the enzymatic metabolism of this vitamin has represented a stimulating subject of research. Its elucidation has opened up new horizons to the interpretation of the biological function of that class of molecules. The identification of specific properties for some of the physiological metabolites and the definition of advanced analytical techniques to assess the human metabolome of this vitamin in vivo, have paved the way to a series of hypotheses on the functional implications that this metabolism may have far beyond its catabolic role. The present review collects the available information on the most relevant analytical strategies employed to assess the status and metabolism of vitamin E in humans as well as in other model systems. A particular focus is dedicated to the analytical methods used to measure vitamin E metabolites, and particularly long-chain metabolites, in biological fluids and tissues. Preliminary information on a new LC-APCI-MS/MS method to measure these metabolites in human serum is reported.

Glutamate-mediated excitotoxicity, neuroinflammation, and oxidative stress are common underlying events in neurodegeneration. This pathogenic "triad" characterizes the neurobiology of epilepsy, leading to seizure-induced cell death, increased susceptibility to neuronal synchronization and network alterations. Along with other maladaptive changes, these events pave the way to spontaneous recurrent seizures and progressive degeneration of the interested brain areas. In vivo models of epilepsy are available to explore such epileptogenic mechanisms, also assessing the efficacy of chemoprevention and therapy strategies at the pre-clinical level. The kainic acid model of pharmacological excitotoxicity and epileptogenesis is one of the most investigated mimicking the chronicization profile of temporal lobe epilepsy in humans. Its pathogenic cues include inflammatory and neuronal death pathway activation, mitochondrial disturbances and lipid peroxidation of several regions of the brain, the most vulnerable being the hippocampus. The importance of neuroinflammation and lipid peroxidation as underlying molecular events of brain damage was demonstrated in this model by the possibility to counteract the related maladaptive morphological and functional changes of this organ with vitamin E, the main fat-soluble cellular antioxidant and "conditional" co-factor of enzymatic pathways involved in polyunsaturated lipid metabolism and inflammatory signaling. The present review paper provides an overview of the literature supporting the potential for a timely intervention with vitamin E therapy in clinical management of seizures and epileptogenic processes associated with excitotoxicity, neuroinflammation and lipid peroxidation, i.e. the pathogenic "triad".

2,2’-diselenyldibenzoic acid (DSBA) is a mild thiol peroxidase agent presently in preclinical development. This study reports that the drug has novel seleno-hormetic properties in both murine bone marrow and human liver cells. According with previous in vitro findings, mechanistic aspects of such properties were confirmed to include the activation of Nrf2 transcription factor and an increased expression of downstream stress response genes in the liver and in hematopoietic stem and progenitor cells of the myeloid lineage. These genes include glutathione S-transferase that is reported to represent a major player in the metabolism and pharmacological function of seleno-organic compounds. As a practical application, DSBA administration prevented bone marrow toxicities following acute exposure to sub-lethal doses of ionizing radiation in C57 BL/6 mice.In conclusion, this study demonstrates for the first time the pharmacological properties of DSBAin vivo. The findings suggest applications for this selenohormetine in radioprotection and prevention protocols.

The glutathione (GSH) S-transferase family of detoxification and signalling proteins represents a major hub for the metabolism of Selenium-derived compounds. At the same time, these compounds can be used to modulate the expression and multiple activities of GSTs and other glutathione-dependent genes, that are important aspects in both the chemoprevention and therapy of drug-resistant cancers. In this context, the isoform GSTP-1 (GSTP) appears to play a fundamental role. Besides promoting GSH-dependent detoxification of cellular electrophiles, GSTP physically interacts with a number of small molecules and cellular proteins producing regulatory effects across the main signal transduction and transcription pathways (identified as the "regulatory interactome of GSTP"). An emerging molecular mechanism behind such regulatory function is the activity of GSTP as a redox chaperonine responsible for the selective glutathionylation of protein Cys residues in the different subcellular compartments. The redox-sensitive transcription factor Nrf2 was recently identified as one of the regulatory nodes of this interactome at the interface between inflammation, adaptive stress response, and cell death pathways. The influence of Nrf2 in the stress response to cellular electrophiles and its regulatory interaction with GSTP are discussed in this review suggesting the hypothesis that this interaction may represent the actual pharmacological target of Se compounds with thiol peroxidase activity. These points are critically evaluated with a view to further development of these compounds in cancer prevention and the chemotherapy of drug-resistant tumours.

Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood-brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1β, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.

Glutathione is considered the major non-protein low molecular weight modulator of redox processes and the most important thiol reducing agent of the cell. The biosynthesis of glutathione occurs in the cytosol from its constituent amino acids, but this tripeptide is also present in the most important cellular districts, such as mitochondria, nucleus, and endoplasmic reticulum, thus playing a central role in several metabolic pathways and cytoprotection mechanisms. Indeed, glutathione is involved in the modulation of various cellular processes and, not by chance, it is a ubiquitous determinant for redox signaling, xenobiotic detoxification, and regulation of cell cycle and death programs. The balance between its concentration and redox state is due to a complex series of interactions between biosynthesis, utilization, degradation, and transport. All these factors are of great importance to understand the significance of cellular redox balance and its relationship with physiological responses and pathological conditions. The purpose of this review is to give an overview on glutathione cellular compartmentalization. Information on its subcellular distribution provides a deeper understanding of glutathione-dependent processes and reflects the importance of compartmentalization in the regulation of specific cellular pathways. © 2018 BioFactors, 45(2):152-168, 2019.

In two consecutive randomized placebo-control trials, DHA has been used in combination with other micronutrient vitamins (choline (CHO), vitamin E as alpha-tocopherol and vitamin D) to treat obese nonalcoholic steatohepatitis (NASH) patients along with dietary restriction and physical activity. Regardless of micronutrient vitamins combination, the two DHA-based trials resulted in an improved liver echogenicity (primary end-point), NAFLD Activity Score (NAS) that included biopsy-derived fibrosis and steatosis indices, and metabolic parameters such as fasting blood glucose, LDL, triglycerides and AST. In these trials increased concentrations of plasma free DHA and, even more, a decreased AA/DHA ratio were the strongest predictors of the clinical outcome. A stable correction of DHA concentrations and metabolism may thus represent a major nutritional end-point and precious indicator in the clinical management of pediatric NASH. DHA therapy deserves further investigation as a first-lane intervention in these patients.

Deregulation of autophagy is proposed to play a key pathogenic role in hepatocellular carcinoma (HCC), the most common primary malignancy of the liver and the third leading cause of cancer death. Autophagy is an evolutionarily conserved catabolic process activated to degrade and recycle cell's components. Under stress conditions, such as oxidative stress and nutrient deprivation, autophagy is an essential survival pathway that operates in harmony with other stress response pathways. These include the redox-sensitive transcription complex Nrf2-Keap1 that controls groups of genes with roles in detoxification and antioxidant processes, intermediary metabolism, and cell cycle regulation. Recently, a functional association between a dysfunctional autophagy and Nrf2 pathway activation has been identified in HCC. This appears to occur through the physical interaction of the autophagy adaptor p62 with the Nrf2 inhibitor Keap1, thus leading to increased stabilization and transcriptional activity of Nrf2, a key event in reprogramming metabolic and stress response pathways of proliferating hepatocarcinoma cells. These emerging molecular mechanisms and the therapeutic perspective of targeting Nrf2-p62 interaction in HCC are discussed in this paper along with the prognostic value of autophagy in this type of cancer.

Selenium (Se) is an essential micronutrient that functions as "redox gatekeeper" and homeostasis factor of normal and cancer cells. Epidemiology and experimental studies, in the last years suggested that both inorganic and organic forms of Se may have favorable health effects. In this regard, a protective action of Se on cellular systems that may help preventing cancer cell differentiation has been demonstrated, while the hypothesis that Se compounds may cure cancer and its metastatic diffusion appears speculative and is still a matter of investigation. Indeed, the overall actions of Se compounds in carcinogenesis are controversial. The recognition that cancer is a stem cell disease instigated major paradigm shifts in our basic understanding of cancer and attracted a great deal of interest. Although current treatment approaches in cancer are grounded in the need to kill the majority of cancer cells, targeting cancer stem cells (CSCs) may hold great potential in improving cancer treatment. In this respect, Se compounds have been demonstrated modulating numerous signaling pathways involved in CSC biology and these findings are now stimulating further research on optimal Se concentrations, most effective and cancer-specific Se compounds, and inherent pathways involved in redox and metabolic regulation of CSCs. In this review, we summarize the current knowledge about the effects of Se compounds on CSCs, by focusing on redox-dependent pathways and main gene regulation checkpoints that affect self-renewal, differentiation, and migration responses in this subpopulation of cancer cells.

Selenium (Se) is an essential micronutrient that functions as "redox gatekeeper" and homeostasis factor of normal and cancer cells. Epidemiology and experimental studies, in the last years suggested that both inorganic and organic forms of Se may have favorable health effects. In this regard, a protective action of Se on cellular systems that may help preventing cancer cell differentiation has been demonstrated, while the hypothesis that Se compounds may cure cancer and its metastatic diffusion appears speculative and is still a matter of investigation. Indeed, the overall actions of Se compounds in carcinogenesis are controversial. The recognition that cancer is a stem cell disease instigated major paradigm shifts in our basic understanding of cancer and attracted a great deal of interest. Although current treatment approaches in cancer are grounded in the need to kill the majority of cancer cells, targeting cancer stem cells (CSCs) may hold great potential in improving cancer treatment. In this respect, Se compounds have been demonstrated modulating numerous signaling pathways involved in CSC biology and these findings are now stimulating further research on optimal Se concentrations, most effective and cancer-specific Se compounds, and inherent pathways involved in redox and metabolic regulation of CSCs. In this review, we summarize the current knowledge about the effects of Se compounds on CSCs, by focusing on redox-dependent pathways and main gene regulation checkpoints that affect self-renewal, differentiation, and migration responses in this subpopulation of cancer cells.

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

Recently we described the role of glutathione-S-transferase P-1 (GSTP) expression in regulating Nrf2 transcriptional activity by protein-protein interaction. A functional interplay between Nrf2 and GSTP to control glutathione (GSH) biosynthesis, detoxification function and signal transduction through protein Cys glutathionylation can be hypothesized. To explore such interplay the effect of GSTP gene manipulation on the cellular levels and redox of main thiols in murine embryonic fibroblasts (MEFs) was investigated. GSTP knockout MEFs showed increased levels of all the identified forms of thiols that included GSH, GSSG, GSSP, Cys, Cyss, CyssP, HCys, HCyss, HCyssP, γ-GluCys and CysGly. The exposure to the Se-organic thiol-peroxidase compound Ebselen, further and massively up-regulated GSH, Cys and HCys and their corresponding disulfides in GSTP-KO, but not in wild-type (WT) cells. GSSP (protein glutathionylation), and to a lower extent HCyssP, increased after treatment with Se-organic compounds only in WT cells (confirmed by both HPLC and immunoblot analysis). These findings confirms the role of GSTP as a feedback component of Nrf2 pathway (control of GSH levels) and identify with an unbiased approach the role of this gene in mediating protein glutathionylation signalling during the exposure to Se-organic thiol-peroxidases.

Several studies are increasingly underlying the biological role of vitamin E metabolites as bioactive compounds with anti-inflammatory, anti-proliferative and anti-atherogenic activity. A quantitative method for the simultaneous determination in human plasma and serum of vitamin E (α-tocopherol, α-T and γ-tocopherol, γ-T) and its cytochrome P-450 metabolites: 13'-hydroxychromanol (α-13'-OH), 13'-carboxychromanol (α-13'-COOH) and carboxyethyl hydroxychromanols (α-CEHC and γ-CEHC), was developed and validated. After enzymatic hydrolysis and deproteinization, the metabolites were extracted with a mixture of hexane/ methyl tertiary butyl ether (2/1, v/v). The separation was achieved by reversed phase chromatography and the analytes detected by a triple quadrupole mass analyser using electrospray ionization in positive mode (LC-MS/MS). α-T and γ-T were extracted separately without enzymatic hydrolysis. The analytes were quantified with the isotopic dilution method. After an extensive validation study (three levels in three different occasions for a total of 54 experiments), the procedure was successfully applied to the analysis of sera of healthy volunteers (before and after supplementation with α-T) and plasma of patients affected by chronic kidney disease. Finally, the structures of three unknown compounds found in blood and related to the long chain metabolites (α-13'-OH and α-13'-COOH) were further investigated using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS).

Hepatic lipid peroxidation is a key pathogenic event in the progression from NAFLD to the more severe inflammatory and fibrotic lesions of non-alcoholic steatohepatitis (NASH). This more advanced stage of the disease is a major risk factor for cirrhosis and hepatocellular carcinoma. Steatosis and inflammation of the liver are associated with an impaired metabolism and function of α-tocopherol, the main fat-soluble antioxidant of the cellular membranes and circulating lipoproteins. With this background, an increased vitamin E oxidation rate should occur in NAFLD. The lack of unbiased pre-analytical and analytical protocols leaves this assumption unsupported. A new LC-MS/MS analytical procedure was thus set up to measure the vitamin E oxidation metabolite α-TQ in human plasma. The application of this procedure in a pilot observation study demonstrated increase α-TQ levels in NAFLD patients in association with increased 4-hydroxynonenal (4-HNE) protein adducts and decreased polyunsaturated fatty acid (PUFA) levels. In conclusion these findings point to a role of α-TQ as a causal biomarker of lipid peroxidation and impaired liver metabolism of vitamin E in NAFLD.

Cytochrome P450-derived long-chain metabolites are gaining increasing interest as bioactive intermediates of vitamin E. In this study we first report on the HPLC-ECD and GC-MS analysis in human serum of the earliest metabolite of this vitamin, namely α-(13'-hydroxy)-6-hydroxychroman (α-13'-OH). The two chromatographic procedure are sensitive enough (LOQ of 10nM) to measure α-13'-OH after hexane extraction of 1 ml of sample obtained from healthy volunteers supplemented for 1-week with 1000 IU/d (671 mg/d) RRR-α-tocopherol. The observed concentrations ranged between 15 and 50 nM, with minor differences between fasting and 4-hr post-meal state. Baseline (non-supplemented state) levels of 7.2 ± 1.6 nM were observed extracting higher volumes of serum. Biological effects of α-13'-OH investigated for the first time in RAW264.7 murine macrophages involved transcriptional control of inflammatory cytokines, and transcriptional and functional regulation of COX2 and iNOS enzymes in response to lipopolysaccharides. In conclusion, here we present the first quantitative evaluation of serum α-13'-OH also providing early evidence of the anti-inflammatory potential of this metabolite that is worth of further investigation in the area of functional and nutraceutical implications of vitamin E metabolism.

The enzymatic metabolism of vitamin E in liver cells generates long chain metabolites (LCMs) with proposed regulatory activity on inflammatory and atherogenic genes. In this study the LCM formation kinetics was characterized in HepG2 and HepaRG human hepatic cells, supplemented with RRR-α-tocopherol (α-TOH). α-13’OH was the main product of α-TOH metabolism, while α-13’COOH metabolite and the short chain metabolite α-CEHC, were detected only in traces, thus demonstrating the poor efficiency of vitamin E catabolism in these cells. However, this metabolism was significantly simulated when the hepatic cells were challenged with (lipo)toxic agents, such as ethanol or palmitate. Under such conditions a significant gene repression effect was observed for PPARγ nuclear receptor and the CYP-450 isoenzyme 4F2, which therefore appear to play a minor role in this metabolism. The upregulation of other CYPs such as 3A4 and 2E1 during these challenges, supports the previously proposed role of PXR as key responsive element in the hepatic metabolism of vitamin E. Worth of note, LCMs, similarly to their semi-synthesis natural precursor garcinoic acid, were found to be more potent than α-TOH to stimulate CYP4F2 and PPAR-γ protein expression, an original finding that may lead to speculate the importance of vitamin E metabolism in the gene regulation and lipid homeostasis of liver cells.

A sensitive and selective LC-MS/MS method for the quantification of vitamin E (α- and γ-tocopherol), polyunsaturated fatty acids (arachidonic, eicosapentaenoic, docosahexaenoic and α-linolenic acids) and their metabolites in human plasma and serum was developed and validated. The sample treatment consisted of an enzymatic hydrolysis followed by a deproteinization and a liquid-liquid extraction. The separation was achieved by reversed phase chromatography. For each analyte, two MRM transitions were monitored in order to confirm identity. All determined compounds showed good linearity over the investigated concentration range. Method accuracy was satisfactory according to the international guidelines. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids and tocopherols in human plasma of adult and children subjects. The determined levels are in agreement with the literature, thus providing a flexible method to explore pathophysiological processes in which changes of the investigated compounds are involved.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide in association with oxidative stress and insulin resistance. Steatosis can evolve to steatohepatitis (NASH) by lipotoxicity, inflammation, fibrosis and cell death leading to cirrhosis and hepatocellular carcinoma. No valid therapy has been developed so far to control inflammatory and fibrogenic pathways of NAFLD/NASH. α-Tocopherol (α-TOH) is an antioxidant that partially reverts the histopatological spectrum of NASH. However, the biological mechanism that may support a preventive or therapeutic role of vitamin E in NASH and chronic liver diseases remain elusive. In this study we explored in vitro and in vivo the metabolism of vitamin E in liver cells and in the whole liver exposed to some pathogenic stimuli associated with the progression of NAFLD/NASH. Protein levels of CYP4F2, the cytochrome P450 isoenzyme involved in the metabolism of vitamin E increased in HepG2 cells incubated with α-TOH. This effect increased with a synergic action by the treatment with the fatty acids oleic acid or palmitic acid, or with fructose, which are well-known NASH stimuli. In mice fed control or NAFLD/NASH diets, e.g. high-fat (HFD) or HFD plus fructose diet, the abnormal lipid metabolism of liver tissue was associated with a lowered expression of CYP4F2 protein. The same finding was observed in animals treated with CCl4, a free radical-generating hepatotoxic xenobiotic leading to hepatic fibrosis, in the presence of lowered levels of liver α-TOH. In conclusion, either acute or chronic challenges with NAFLD/NASH stimuli influence the liver expression of CYP4F2. The observed acute transcriptional upregulation and the in vivo downregulation of this protein, point to a role for CYP4F2 in the inflammatory and lipotoxicity signaling of NASH. Transcriptional effects of vitamin E on this gene may help to explain earlier and positive clinical findings, which are worth of further investigation.