Tissue factor pathway-related biomarkers in liver cancer: activated factor VII-antithrombin complex and tissue factor mRNA levels are associated with mortality.

Background: Tissue factor (TF), the main initiator of the coagulation cascade, plays a role in cancer progression and prognosis. Activated factor VII-antithrombin complex (FVIIa-AT) is considered an indirect marker of TF exposure by reflecting TF-FVIIa interaction. Objectives: To assess the link between FVIIa-AT plasma levels, TF messenger RNA (mRNA) expression, and survival in cancer. Methods: TF pathway-related coagulation biomarkers were assessed in 136 patients with cancer (52 with hepatocellular carcinoma, 41 with cholangiocarcinoma, and 43 with colon cancer) undergoing surgical intervention with curative intent. TF mRNA expression analysis in neoplastic vs nonneoplastic liver tissues was evaluated in a subgroup of 91 patients with primary liver cancer. Results: FVIIa-AT levels were higher in patients with cancer than in 136 sex- and age-matched cancer-free controls. In patients with cancer, high levels of FVIIa-AT and total TF pathway inhibitor were associated with an increased mortality risk after adjustment for confounders, but only FVIIa-AT remained a predictor of mortality by including both FVIIa-AT and total TF pathway inhibitor in Cox regression (hazard ratio, 2.80; 95% CI, 1.23-6.39; the highest vs the lowest quartile). This association remained significant even after adjustment for extracellular vesicle-associated TF-dependent procoagulant activity. In the subgroup of patients with primary liver cancer, patients with high TF mRNA levels had an increased mortality risk compared with that for those with low TF mRNA levels (hazard ratio, 1.92; 95% CI, 1.03-3.57), and there was a consistent correlation among high FVIIa-AT levels, high TF mRNA levels, and increased risk of mortality. Conclusion: High FVIIa-AT levels may allow the identification of patients with cancer involving high TF expression and predict a higher mortality risk in liver cancer.

Modulation of DNA methylation by one-carbon metabolism: a milestone for healthy aging.

Healthy aging can be defined as an extended lifespan and health span. Nutrition has been regarded as an important factor in healthy aging, because nutrients, bioactive food components, and diets have demonstrated beneficial effects on aging hallmarks such as oxidative stress, mitochondrial function, apoptosis and autophagy, genomic stability, and immune function. Nutrition also plays a role in epigenetic regulation of gene expression, and DNA methylation is the most extensively investigated epigenetic phenomenon in aging. Interestingly, age-associated DNA methylation can be modulated by one-carbon metabolism or inhibition of DNA methyltransferases. One-carbon metabolism ultimately controls the balance between the universal methyl donor S-adenosylmethionine and the methyltransferase inhibitor S-adenosylhomocysteine. Water-soluble B-vitamins such as folate, vitamin B6, and vitamin B12 serve as coenzymes for multiple steps in one-carbon metabolism, whereas methionine, choline, betaine, and serine act as methyl donors. Thus, these one-carbon nutrients can modify age-associated DNA methylation and subsequently alter the age-associated physiologic and pathologic processes. We cannot elude aging per se but we may at least change age-associated DNA methylation, which could mitigate age-associated diseases and disorders.

Proteomic analysis of urinary extracellular vesicles highlights specific signatures for patients with primary aldosteronism.

Background: Urinary extracellular vesicles (uEVs) can be released by different cell types facing the urogenital tract and are involved in cellular trafficking, differentiation and survival. UEVs can be easily detected in urine and provide pathophysiological information "in vivo" without the need of a biopsy. Based on these premises, we hypothesized that uEVs proteomic profile may serve as a valuable tool in the differential characterization between Essential Hypertension (EH) and primary aldosteronism (PA). Methods: Patients with essential hypertension (EH) and PA were enrolled in the study (EH= 12, PA=24: 11 Bilateral Primary Aldosteronism subtype (BPA) and 13 Aldosterone Producing Adenoma (APA)). Clinical and biochemical parameters were available for all the subjects. UEVs were isolated from urine by ultracentrifugation and analysed by Transmission Electron Microscopy (TEM) and nanotrack particle analysis (NTA). UEVs protein content was investigated through an untargeted MS-based approach. Statistical and network analysis was performed to identify potential candidates for the identification and classification of PA. Results: MS analysis provided more than 300 protein identifications. Exosomal markers CD9 and CD63 were detected in all samples. Several molecules characterizing EH vs PA patients as well as BPA and APA subtypes were identified after statistical elaboration and filtering of the results. In particular, some key proteins involved in water reabsorption mechanisms, such as AQP1 and AQP2, were among the best candidates for discriminating EH vs PA, as well as A1AG1 (AGP1). Conclusion: Through this proteomic approach, we identified uEVs molecular indicators that can improve PA characterization and help in the gain of insights of the pathophysiological features of this disease. In particular, PA was characterized by a reduction of AQP1 and AQP2 expression as compared with EH.

Urinary extracellular vesicles carry valuable hints through mRNA for the understanding of endocrine hypertension.

Urinary extracellular vesicles (uEVs), released from cells of the urogenital tract organs, carry precious information about originating tissues. The study of molecules transported through uEVs such as proteins, lipids and nucleic acids provides a deeper understanding of the function of the kidney, an organ involved in the pathogenesis of hypertension and a target of hypertension-mediated organ damage. Molecules derived from uEVs are often proposed for the study of disease pathophysiology or as possible disease diagnostic and prognostic biomarkers. Analysis of mRNA loading within uEVs may be a unique and readily obtainable way to assess gene expression patterns of renal cells, otherwise achievable only by an invasive biopsy procedure. Interestingly, the only few studies investigating transcriptomics of hypertension-related genes through the analysis of mRNA from uEVs are inherent to mineralocorticoid hypertension. More specifically, it has been observed that perturbation in human endocrine signalling through mineralcorticoid receptors (MR) activation parallels changes of mRNA transcripts in urine supernatant. Furthermore, an increased copy number of uEVs-extracted mRNA transcripts of the 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene were detected among subjects affected by apparent mineralocorticoid excess (AME), a hypertension-inducing autosomal recessive disorder due to a defective enzyme function. Moreover, by studying uEVs mRNA, it was observed that the renal sodium chloride cotransporter (NCC) gene expression is modulated under different conditions related to hypertension. Following this perspective, we illustrate here the state of the art and the possible future of uEVs transcriptomics towards a deeper knowledge of hypertension pathophysiology and ultimately more tailored investigational, diagnostic-prognostic approaches.

Changes of gene expression in peripheral blood mononuclear cells of lung cancer patients with or without anorexia.

BACKGROUND & AIMS: Anorexia is a disabling symptom in cancer and we aimed at investigating the role of changes in gene expression in lung cancer patients presenting with anorexia. METHODS: Genome-wide transcriptomic profiling was assessed in PBMCs RNA from newly diagnosed lung cancer patients and in a control group. RT-qPCR was used for selected genes. RESULTS: RNA-Seq analysis revealed among groups a large number of differentially expressed genes mainly implicated in immune system regulation, oxidative stress and cytokine-mediated inflammation signaling pathways. In particular, we identified a total of 983 DEGs (843 up-regulated; 140 down-regulated) in anorexic cancer compared to controls. A selected number of DEGs including ADAM8, SMAD4, CCR4 and CLU were differentially expressed within cancer group according to the presence/absence of anorexia. In terms of RT-qPCR, ADAM8 was less expressed in cancer patients than controls (p < 0.001), and in anorexic patients vs controls (p = 0.001). The expression of SMAD4 was lower in cancer vs controls (p = 0.005), and in anorexic patients vs controls (p = 0.009). We observed lower CCR4 expression in both anorexic and non-anorexic vs control (p = 0.004, p = 0.011, respectively) and a similar trend was present for CLU. CONCLUSIONS: Our data shed new light on the role of specific genes and their associated molecular pathways as potential key mechanisms for the development of anorexia and may represent a novel landmark for understanding the complex pathophysiology of impaired appetite in cancer.

Lifestyle and dietary factors, iron status and one-carbon metabolism polymorphisms in a sample of Italian women and men attending a Transfusion Medicine Unit: a cross-sectional study.

Iron (Fe) status among healthy male and female blood donors, aged 18-65 years, is estimated. General characteristics and lifestyle factors, dietary habits and major one-carbon metabolism-related polymorphisms were also investigated. An explorative cross-sectional study design was used to examine a sample of blood donors attending the Transfusion Medicine Unit of the Verona University Hospital, Italy. From April 2016 to May 2018, 499 subjects were enrolled (255 men, 244 women, 155 of whom of childbearing age). Major clinical characteristics including lifestyle, dietary habits and Fe status were analysed. The MTHFR 677C > T, cSHMT 1420C > T, DHFR 19bp ins/del and RFC1 80G > A polymorphisms were also assayed. Mean plasma concentrations of Fe and ferritin were 16·6 µmol/l (95 % CI 16·0, 17·2) and 33·8 µg/l (95 % CI 31·5, 36·2), respectively. Adequate plasma Fe concentrations (> 10·74 µmol/l) were detected in 84·3 % and adequate ferritin concentrations (20-200 µg/l) was found in 72·5 % of the whole cohort. Among the folate-related polymorphisms analysed, carriers of the DHFR 19bp del/del mutant allele showed lower ferritin concentration when compared with DHFR 19bp ins/del genotypes. In a sample of Italian healthy blood donors, adequate plasma concentrations of Fe and ferritin were reached in a large proportion of subjects. The relationship of Fe status with lifestyle factors and folate-related polymorphisms requires more investigation to clarify further gene-nutrient interactions between folate and Fe metabolism.

Urinary extracellular vesicle mRNA analysis of sodium chloride cotransporter in hypertensive patients under different conditions.

Urinary extracellular vesicles (UEV) mainly derive from cells of the urogenital tract and their cargo (proteins, nucleic acids, lipids, etc.) reflects their cells of origin. Na chloride cotransporter (NCC) is expressed at the kidney level in the distal convoluted tubule, is involved in salt reabsorption, and is the target of the diuretic thiazides. NCC protein has been recognized and quantified in UEV in previous studies; however, UEV NCC mRNA has never been studied. This study aimed to identify and analyze NCC mRNA levels in primary aldosteronism (PA). The rationale for this investigation stems from previous observations regarding NCC (protein) as a possible biomarker for the diagnosis of PA. To evaluate modulations in the expression of NCC, we analyzed NCC mRNA levels in UEV in PA and essential hypertensive (EH) patients under different conditions, that is, before and after saline infusion, anti-aldosterone pharmacological treatment, and adrenal surgery. NCC mRNA was measured by RT-qPCR in all the samples and was regulated by volume expansion. Its response to mineralocorticoid receptor antagonist was correlated with renin, and it was increased in PA patients after adrenalectomy. NCC mRNA is evaluable in UEV and it can provide insights into the pathophysiology of distal convolute tubule in different clinical conditions including PA.

Unravelling the Complex Relationship between Diet and Nephrolithiasis: The Role of Nutrigenomics and Nutrigenetics.

Nephrolithiasis is an increasingly prevalent condition, especially in high income countries, and is associated with high morbidity. Extraordinary progress in genetics made the identification of genetic forms of nephrolithiasis possible. These genetic diseases are usually rare and do not account for the most common forms of nephrolithiasis that are the result of several factors such as environment, dietary habits, and predisposing genes. This knowledge has shaped what we classify as nephrolithiasis, a condition that is now recognized as systemic. How and to what extent all these factors interact with one another and end in kidney stone formation, growth, and recurrence is not completely understood. Two new research fields have recently been trying to give some answers: nutrigenomics and nutrigenetics. These fields have the aim of understanding the intricate diet/genome interface that influences gene expression regulation mainly through epigenetic mechanisms and results in specific medical conditions such as cancer, metabolic syndrome, and cardiovascular diseases. Epigenetics seems to play a crucial role and could represent the link between environmental factors, that we are constantly exposed to, and risk factors for nephrolithiasis. In this systematic review, we summarize all the available evidence of proven or hypothesized epigenetic mechanisms related to nephrolithiasis.

Case Report: Microangiopathic Hemolytic Anemia With Normal ADAMTS13 Activity.

Thrombotic microangiopathies (TMAs) include a heterogeneous group of diseases characterized by abnormalities in the vessel walls of arterioles and capillaries resulting in microvascular thrombosis that typically presents with a microangiopathic hemolytic anemia (MAHA) and severe thrombocytopenia. We describe here the case of an 82-year-old woman, who came to our attention for a clinical condition consistent with thrombotic microangiopathy. Even if initially highly suggestive for a thrombotic thrombocytopenic purpura (TTP), the elevated ADAMTS13 activity together with the alteration of the main coagulation parameters (D-dimer elevation, fibrinogen consumption, slightly prolonged prothrombin time), induced us to consider several other diseases in the differential diagnostic process. The case evolved toward a suspected overlapped secondary hemophagocytic syndrome, though the hyperferritinemia was finally interpreted within the frame of a cytokine storm. After a complex diagnostic workup, the clinical and biochemical parameters guided us toward the diagnosis of a cancer-related microangiopathic hemolytic anemia (CR-MAHA) secondary to a relapsing breast cancer with multiple metastatic localizations. Prednisone 1 mg/kg body weight was started, and several units of fresh frozen plasma were infused, obtaining a good control of the hemolysis. No specific oncological therapies were, however, possible, due to the older age and the critically compromised general condition of the patient; therefore, after clinical stabilization, the patient was discharged for treatment in a palliative care Hospital.

The Positive Association between Plasma Myristic Acid and ApoCIII Concentrations in Cardiovascular Disease Patients Is Supported by the Effects of Myristic Acid in HepG2 Cells.

BACKGROUND: In the settings of primary and secondary prevention for coronary artery disease (CAD), a crucial role is played by some key molecules involved in triglyceride (TG) metabolism, such as ApoCIII. Fatty acid (FA) intake is well recognized as a main determinant of plasma lipids, including plasma TG concentration. OBJECTIVES: The aim was to investigate the possible relations between the intakes of different FAs, estimated by their plasma concentrations, and circulating amounts of ApoCIII. METHODS: Plasma samples were obtained from 1370 subjects with or without angiographically demonstrated CAD (mean ± SD age: 60.6 ± 11.0 y; males: 75.8%; BMI: 25.9 ± 4.6 kg/m2; CAD: 73.3%). Plasma lipid, ApoCIII, and FA concentrations were measured. Data were analyzed by regression models adjusted for FAs and other potential confounders, such as sex, age, BMI, diabetes, smoking, and lipid-lowering therapies. The in vitro effects of FAs were tested by incubating HepG2 hepatoma cells with increasing concentrations of selected FAs, and the mRNA and protein contents in the cells were quantified by real-time RT-PCR and LC-MS/MS analyses. RESULTS: Among all the analyzed FAs, myristic acid (14:0) showed the most robust correlations with both TGs (R = 0.441, P = 2.6 × 10-66) and ApoCIII (R = 0.327, P = 1.1 × 10-31). By multiple regression analysis, myristic acid was the best predictor of both plasma TG and ApoCIII variability. Plasma TG and ApoCIII concentrations increased progressively at increasing concentrations of myristic acid, independently of CAD diagnosis and gender. Consistent with these data, in the in vitro experiments, an ∼2-fold increase in the expression levels of the ApoCIII mRNA and protein was observed after incubation with 250 µM myristic acid. A weaker effect (∼30% increase) was observed for palmitic acid, whereas incubation with oleic acid did not affect ApoCIII protein or gene expression. CONCLUSIONS: Plasma myristic acid is associated with increased ApoCIII concentrations in cardiovascular patients. In vitro experiments indicated that myristic acid stimulates ApoCIII expression in HepG2 cells.

Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein.

We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.

Trace Elements Status and Metallothioneins DNA Methylation Influence Human Hepatocellular Carcinoma Survival Rate.

BACKGROUND: Mechanisms underlying hepatocellular carcinoma (HCC) development are largely unknown. The role of trace elements and proteins regulating metal ions homeostasis, i.e. metallothioneins (MTs), recently gained an increased interest. Object of the study was to investigate the role of promoter DNA methylation in MTs transcriptional regulation and the possible prognostic significance of serum trace elements in HCC. METHODS: Forty-nine HCC patients were enrolled and clinically characterized. Cu, Se, and Zn contents were measured by Inductively Coupled Plasma Mass Spectrometry in the serum and, for a subset of 27 patients, in HCC and homologous non-neoplastic liver (N) tissues. MT1G and MT1H gene expression in hepatic tissues was assessed by Real-Time RT-PCR and the specific promoter DNA methylation by Bisulfite-Amplicon Sequencing. RESULTS: Patients with Cu serum concentration above the 80th percentile had a significantly decreased survival rate (P < 0.001) with a marked increased hazard ratio for mortality (HR 6.88 with 95% CI 2.60-18.23, P < 0.001). Se and Zn levels were significantly lower in HCC as compared to N tissues (P < 0.0001). MT1G and MT1H gene expression was significantly down-regulated in HCC as compared to N tissues (P < 0.05). MTs promoter was hypermethylated in 9 out of the 19 HCC tissues showing MTs down-regulation and methylation levels of three specific CpGs paralleled to an increased mortality rate among the 23 patients analyzed (P = 0.015). CONCLUSIONS: MT1G and MT1H act as potential tumor suppressor genes regulated through promoter DNA methylation and, together with serum Cu concentrations, be related to survival rate in HCC.

Muscle derangement and alteration of the nutritional machinery in NSCLC.

Weight loss and depletion of nutritional status are frequent presentation hallmarks in non-small cell lung cancer (NSCLC). Decline in muscle mass is a major component in weight loss and may have both a prognostic and predictive value for survival and treatment-related toxicities. Recent findings suggest that weight and skeletal muscle mass gain during treatment may represent surrogate markers for outcome in advanced NSCLC patients. Herein we present an in-depth view of the impact of nutritional status derangements on NSCLC patients' outcome, focusing on lean body mass variations during disease course. We explored the impact of malnutrition with a major attention on novel treatment options. We reviewed molecular, metabolic and immunological mechanisms underlying muscle-wasting condition, which may exhibit a meaningful targeting potential. Incorporating a specialized and accurate body composition assessment into a comprehensive, patient-centered and tailored intervention will facilitate the achievement of nutritional goals and optimal care for lung cancer patients.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

BACKGROUND: The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. MATERIALS AND METHODS: Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An in vitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. RESULTS: Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. CONCLUSIONS: This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

One-carbon genetic variants and the role of MTHFD1 1958G>A in liver and colon cancer risk according to global DNA methylation.

Several polymorphic gene variants within one-carbon metabolism, an essential pathway for nucleotide synthesis and methylation reactions, are related to cancer risk. An aberrant DNA methylation is a common feature in cancer but whether the link between one-carbon metabolism variants and cancer occurs through an altered DNA methylation is yet unclear. Aims of the study were to evaluate the frequency of one-carbon metabolism gene variants in hepatocellular-carcinoma, cholangiocarcinoma and colon cancer, and their relationship to cancer risk together with global DNA methylation status. Genotyping for BHMT 716A>G, DHFR 19bp ins/del, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, TCII 776C>G and TS 2rpt-3rpt was performed in 102 cancer patients and 363 cancer-free subjects. Methylcytosine (mCyt) content was measured by LC/MS/MS in peripheral blood mononuclear cells (PBMCs) DNA. The MTHFD1 1958AA genotype was significantly less frequent among cancer patients as compared to controls (p = 0.007) and related to 63% reduction of overall cancer risk (p = 0.003) and 75% of colon cancer risk (p = 0.006). When considering PBMCs mCyt content, carriers of the MTHFD1 1958GG genotype showed a lower DNA methylation as compared to carriers of the A allele (p = 0.048). No differences were highlighted by evaluating a possible relationship between the other polymorphisms analyzed with cancer risk and DNA methylation. The MTHFD1 1958AA genotype is linked to a significantly reduced cancer risk. The 1958GG genotype is associated to PBMCs DNA hypomethylation as compared to the A allele carriership that may exert a protective effect for cancer risk by preserving from DNA hypomethylation.

DNA Methylation and Hydroxymethylation in Primary Colon Cancer and Synchronous Hepatic Metastasis.

Colon cancer is one of the most frequent solid tumor and simultaneous diagnosis of primary colon cancer and liver metastases occurs in about one fourth of cases. The current knowledge on epigenetic signatures, especially those related to hydroxymethylation in primary cancer tissue, synchronous metastasis, and blood circulating cells is lacking. This study aimed to investigate both methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) status in the DNA of individual patients from colon cancer tissue, synchronous liver metastases, and in cancer-free colon and liver tissues and leukocytes. Patients undergoing curative surgery (n = 16) were enrolled and their laboratory and clinical history data collected. The contents of mCyt and hmCyt were determined by a liquid chromatography/mass spectrometry (LC/MS/MS) method in DNA extracted from primary colon cancer, synchronous hepatic metastatic tissues and homologous cancer-free tissues, i.e., colon and liver tissues as well as leukocytes. The mCyt and hmCyt levels were compared between cancerous and cancer-free tissues, and correlations between leukocytes and colon/liver tissues for both the mCyt and hmCyt levels were evaluated. The mCyt levels were similar in primary colon cancer and liver metastasis tissues (4.69 ± 0.37% vs. 4.77 ± 0.38%, respectively, p = 0.535), and both primary and metastatic tissues were hypomethylated compared to cancer-free colon (4.98 ± 0.26%). The difference in the mCyt content between cancerous and cancer-free colon tissues was significantly lower in primary colon cancer (p = 0.004), but not in liver metastasis (p = 0.148). The hmCyt content was similar in primary colon cancer compared to liver metastasis (0.035%, C.I. 0.024-0.052% versus 0.035%, C.I. 0.021-0.058%, respectively, p = 0.905) and markedly depleted compared to the cancer-free colon (0.081%, C.I. 0.055-0.119%) with a statistically significant difference (p < 0.05) for both comparisons. The mCyt levels showed a borderline correlation between leukocytes and colon cancer tissue (Pearson's correlation coefficient = 0.51, p = 0.052) while no correlations were detected for the hmCyt levels. In conclusion, primary colon cancer and synchronous liver metastasis tissues showed a similar epigenetic status but were significantly hypomethylated and hypohydroxymethylated as compared to homologous cancer-free colon tissues.

Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging.

PURPOSE: Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging. METHODS: Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber-DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA. RESULTS: Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment. CONCLUSIONS: These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver's response to aging and alcohol.

Epigenetics in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), a common hepatic disorder ranging from simple steatosis through steatohepatitis to fibrosis and cirrhosis, is an emerging health concern. NAFLD is a pathologic condition characterized by the buildup of extra fat in liver cells that is not caused by alcohol consumption. Excess hepatic fat accumulation results from increased delivery of triglycerides (TG) to the liver or conversion of surplus carbohydrates to TG. Importantly, a subgroup of NAFLD results in hepatocellular injury and inflammation, which is referred to as non-alcoholic steatohepatitis (NASH), and may progress to irreversible cirrhosis and hepatocellular carcinoma (HCC). NAFLD shares, in part, the common pathogenesis of metabolic syndrome including obesity, hyperlipidemia, insulin resistance, mitochondrial damage, oxidative stress response, and the release of inflammatory cytokines. Epigenetics, an inheritable phenomenon that affects gene expression without altering the DNA sequence, provides a new perspective on the pathogenesis of NAFLD. Reversible epigenetic changes take place at the transcriptional level and provide a phenotypic connection between the host and environment. An accumulating body of evidence suggests the importance of epigenetic roles in NAFLD, which in turn can be identified as potential therapeutic targets and non-invasive biomarkers of NAFLD. It is anticipated that the epigenetic modifiers in NAFLD may provide novel molecular indicators that can determine not only the initial risk but also the disease progression and prognosis. In the present review, we update the roles of epigenetics as pathologic mechanisms, therapeutic targets and biomarkers in NAFLD.

The RFC1 80G>A, among Common One-Carbon Polymorphisms, Relates to Survival Rate According to DNA Global Methylation in Primary Liver Cancers.

Polymorphisms within one-carbon metabolism genes have been largely studied in relation to cancer risk for the function of this pathway in nucleotide synthesis and DNA methylation. Aims of this study were to explore the possible link among several common functional gene polymorphisms within one-carbon metabolism and survival rate in primary liver cancers, i.e., hepatocellular carcinoma and cholangiocarcinoma, and to assess the additional effect of global DNA methylation on survival rate and mortality risk. Forty-seven primary liver cancer patients were genotyped for ten polymorphisms: DHFR 19bp ins/del, TS 2rpt-3rpt, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, BHMT 716 A>G, TC II 776C>G. Methylation was determined in peripheral blood mononuclear cells (PBMCs) DNA as methylcytosine (mCyt) content using LC/MS/MS. Among the polymorphisms analysed, the RFC1 80G>A (rs1051266) influenced the survival rate in primary liver cancers. The RFC1 80AA was associated to a significantly reduced survival rate (22.2%) as compared to both GG and GA genotypes (61.5% and 76% respectively, p = 0.005). When the cancer patients were stratified according to the mCyt median value as high (>5.34%) or low (≤5.34%), the concomitant presence of AA genotype and low mCyt level led to a significantly worse survival rate as compared to the G allele carriership (p<0.0001) with a higher Hazard Ratio (HR = 6.62, p = 0.001). The subjects carrying the AA genotype in association with high mCyt did not show a significant difference in survival rate as compared with the G allele carriers (p = 0.919). The RFC1 80G>A polymorphism influenced the survival rate, and the presence of RFC1 80AA genotype with low global methylation in PBMCs DNA was associated with poorer prognosis and higher mortality risk, therefore highlighting novel molecular signatures potentially helpful to define prognostic markers for primary liver cancers.

Iron Supplementation Reverses the Reduction of Hydroxymethylcytosine in Hepatic DNA Associated With Chronic Alcohol Consumption in Rats.

BACKGROUND: Alcohol is known to affect two epigenetic phenomena, DNA methylation and DNA hydroxymethylation, and iron is a cofactor of ten-eleven translocation (TET) enzymes that catalyze the conversion from methylcytosine to hydroxymethylcytosine. In the present study we aimed to determine the effects of alcohol on DNA hydroxymethylation and further effects of iron on alcohol associated epigenetic changes. METHODS: Twenty-four male Sprague-Dawley rats were fed either Lieber-DeCarli alcohol diet (36% calories from ethanol) or Lieber-DeCarli control diet along with or without iron supplementation (0.6% carbonyl iron) for 8 weeks. Hepatic non-heme iron concentrations were measured by colorimetric assays. Protein levels of hepatic ferritin and transferrin receptor were determined by Western blotting. Methylcytosine, hydroxymethylcytosine and unmodified cytosine in DNA were simultaneously measured by liquid chromatography/mass spectrometry method. RESULTS: Iron supplementation significantly increased hepatic non-heme iron contents (P < 0.05) but alcohol alone did not. However, both alcohol and iron significantly increased hepatic ferritin levels and decreased hepatic transferrin receptor levels (P < 0.05). Alcohol reduced hepatic DNA hydroxymethylation (0.21% ± 0.04% vs. 0.33% ± 0.04%, P = 0.01) compared to control, while iron supplementation to alcohol diet did not change DNA hydroxymethylation. There was no significant difference in methylcytosine levels, while unmodified cytosine levels were significantly increased in alcohol-fed groups compared to control (95.61% ± 0.08% vs. 95.26% ± 0.12%, P = 0.03), suggesting that alcohol further increases the conversion from hydroxymethylcytosine to unmodified cytosine. CONCLUSIONS: Chronic alcohol consumption alters global DNA hydroxymethylation in the liver but iron supplementation reverses the epigenetic effect of alcohol.