Indicaxanthin Induces Autophagy in Intestinal Epithelial Cancer Cells by Epigenetic Mechanisms Involving DNA Methylation.

Autophagy is an evolutionarily conserved process critical in maintaining cellular homeostasis. Recently, the anticancer potential of autophagy inducers, including phytochemicals, was suggested. Indicaxanthin is a betalain pigment found in prickly pear fruit with antiproliferative and pro-apoptotic activities in colorectal cancer cells associated with epigenetic changes in selected methylation-silenced oncosuppressor genes. Here, we demonstrate that indicaxanthin induces the up-regulation of the autophagic markers LC3-II and Beclin1, and increases autophagolysosome production in Caco-2 cells. Methylomic studies showed that the indicaxanthin-induced pro-autophagic activity was associated with epigenetic changes. In addition to acting as a hypermethylating agent at the genomic level, indicaxanthin also induced significant differential methylation in 39 out of 47 autophagy-related genes, particularly those involved in the late stages of autophagy. Furthermore, in silico molecular modelling studies suggested a direct interaction of indicaxanthin with Bcl-2, which, in turn, influenced the function of Beclin1, a key autophagy regulator. External effectors, including food components, may modulate the epigenetic signature of cancer cells. This study demonstrates, for the first time, the pro-autophagic potential of indicaxanthin in human colorectal cancer cells associated with epigenetic changes and contributes to outlining its potential healthy effect in the pathophysiology of the gastrointestinal tract.

Endogenous retrotransposons cause catastrophic deoxyribonucleic acid damage in human trophoblasts.

OBJECTIVE: To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts. DESIGN: Experimental ex vivo study. SETTING: Hospital-affiliated University. PATIENT(S): Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n = 10). INTERVENTION(S): Biochemical and genetic analysis and modification of primary human trophoblasts. MAIN OUTCOME MEASURE(S): To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNA sequencing, quantitative polymerase chain reaction, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed. RESULT(S): Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNA sequencing was notable for markedly elevated LINE-1 expression, confirmed with quantitative polymerase chain reaction, and that resulted in elevated expression of LINE-1-encoded proteins, as shown by immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis. CONCLUSION(S): Derepression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.

Nutritional epigenomic and DNA-damage modulation effect of natural stilbenoids.

The aim of the present work is the evaluation of biological effects of natural stilbenoids found in Vitis vinifera, with a focus on their activity as epigenetic modulators. In the present study, resveratrol, pterostilbene and for the first time their dimers (±)-trans-δ-viniferin, (±)-trans-pterostilbene dehydrodimer were evaluated in Caco-2 and HepG-2 cell lines as potential epigenetic modulators. Stilbenoids were added in a Caco-2 cell culture as a model of the intestinal epithelial barrier and in the HepG-2 as a model of hepatic environment, to verify their dose-dependent toxicity, ability to interact with DNA, and epigenomic action. Resveratrol, pterostilbene, and (±)-trans-pterostilbene dehydrodimer were found to have no toxic effects at tested concentration and were effective in reversing arsenic damage in Caco-2 cell lines. (±)-trans-δ-viniferin showed epigenomic activity, but further studies are needed to clarify its mode of action.

The Multiplicity of Argonaute Complexes in Mammalian Cells.

Argonautes (AGOs) are a highly conserved family of proteins found in most eukaryotes and involved in mechanisms of gene regulation, both at the transcriptional and post-transcriptional level. Among other functions, AGO proteins associate with microRNAs (miRNAs) to mediate the post-transcriptional repression of protein-coding genes. In this process, AGOs associate with members of the trinucleotide repeat containing 6 protein (TNRC6) family to form the core of the RNA-induced silencing complex (RISC), the effector machinery that mediates miRNA function. However, the description of the exact composition of the RISC has been a challenging task due to the fact the AGO's interactome is dynamically regulated in a cell type- and condition-specific manner. Here, we summarize some of the most significant studies that have identified AGO complexes in mammalian cells, as well as the approaches used to characterize them. Finally, we discuss possible opportunities to exploit what we have learned on the properties of the RISC to develop novel anti-cancer therapies. SIGNIFICANCE STATEMENT: The RNA-induced silencing complex (RISC) is the molecular machinery that mediates miRNA function in mammals. Studies over the past two decades have shed light on important biochemical and functional properties of this complex. However, many aspects of this complex await further elucidation, mostly due to technical limitations that have hindered full characterization. Here, we summarize some of the most significant studies on the mammalian RISC and discuss possible sources of biases in the approaches used to characterize it.

Efficacy and Safety of Varenicline for Smoking Cessation in Patients With Type 2 Diabetes: A Randomized Clinical Trial.

Importance: Evidence of effective smoking cessation interventions in patients with diabetes is limited. The unique behavioral and metabolic characteristics of smokers with type 2 diabetes warrants a randomized clinical trial of the smoking cessation drug varenicline. Objective: To evaluate the efficacy and safety of varenicline in patients with type 2 diabetes with an intention to quit smoking. Design, Setting, and Participants: This multicenter, double-blind, placebo-controlled randomized clinical trial recruited patients from 6 outpatient clinics in 5 hospitals in Catania, Italy. Patients with type 2 diabetes, who were smoking at least 10 cigarettes a day, and who intended to quit smoking were screened for eligibility. Eligible patients were randomized to either varenicline or placebo treatment. The trial consisted of a 12-week treatment phase followed by a 40-week follow-up, nontreatment phase. Intention-to-treat data analysis was performed from December 2020 to April 2021. Interventions: Varenicline, 1 mg, twice daily or matched placebo administered for 12 weeks. Patients in both treatment groups also received smoking cessation counseling. Main Outcomes and Measures: The primary efficacy end point of the study was the continuous abstinence rate (CAR) at weeks 9 to 24. Secondary efficacy end points were the CAR at weeks 9 to 12 and weeks 9 to 52 as well as 7-day point prevalence of abstinence at weeks 12, 24, and 52. Results: A total of 300 patients (mean [SD] age, 57.4 [0.8] years; 117 men [78.0%] in varenicline group and 119 men [79.3%] in placebo group) were randomized to receive varenicline (n = 150) or placebo (n = 150). The CAR at weeks 9 to 24 was significantly higher for the varenicline than placebo group (24.0% vs 6.0%; odds ratio [OR], 4.95; 95% CI, 2.29-10.70; P < .001). The CARs at weeks 9 to 12 (31.3% vs 7.3%; OR, 5.77; 95% CI, 2.85-11.66; P < .001) and weeks 9 to 52 (18.7% vs 5.3%; OR, 4.07; 95% CI, 1.79-9.27; P < .001) as well as the 7-day point prevalence of abstinence at weeks 12, 24, and 52 were also significantly higher for the varenicline vs placebo group. The most frequent adverse events occurring in the varenicline group compared with the placebo group were nausea (41 [27.3%] vs 17 [11.4%]), insomnia (29 [19.4%] vs 19 [12.7%]), abnormal dreams (19 [12.7%] vs 5 [3.4%]), anxiety (17 [11.4%] vs 11 [7.3%]), and irritability (14 [9.4%] vs 8 [5.4%]). Serious adverse events were infrequent in both groups and not treatment-related. Conclusions and Relevance: Results of this trial showed that inclusion of varenicline in a smoking cessation program is efficacious in achieving long-term abstinence without serious adverse events. Varenicline should be routinely used in diabetes education programs to help patients with type 2 diabetes stop smoking. Trial Registration: ClinicalTrials.gov Identifier: NCT01387425.

Alcoholic Consumption of Young Italians During the SARS-CoV-2 Pandemic.

BACKGROUND: The international health emergency caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which, at the end of 2019, hit the world, forced the governments of all countries to adopt stringent restrictive measures to contain the spread of the virus. Several studies have revealed worsening levels of anxiety, depression and perceived stress related to these restrictions and the resulting lifestyle changes. Some studies have also confirmed the presence of a relationship between SARS-CoV-2-related emotional distress and drinking behavior. Indeed, is a wellknown fact that alcohol consumption is one of the behavioral strategies used to reduce negative emotional states. However, it was documented that young people developed different responses to alcohol use during the pandemic than adults. OBJECTIVE: The aim of this work was to investigate the consumption habits of young Italians and how the consumption and purchase of alcoholic beverages have changed following the pandemic. New ways of drinking alcohol were also interesting to observe, such as online. METHODS: Young people between 18 and 35 years old were subjected to an anonymous questionnaire of 22 questions on the adoption of forms of behavior at risk through alcohol consumption, the quantity and occasions of preferential consumption, and on the methods and quantities of alcoholic beverage purchase, before and during the SARS-CoV-2 pandemic. The subjects who declared themselves "non-drinkers" were not included in the statistical survey. RESULTS: About 33% of the enrolled "drinkers" (268/823), adopted risky forms of alcoholic behavior. Males reported a higher average habit of drinking wine or alcohol (M = 1.9953 ± 1.39743, F = 1.7373 ± 1.36688, p <0.005); an increased frequency of drinking (M = 2.3025 ± 0.80610 F = 2.0494 ± 0.75043 p <0.001); a higher average number of drinks consumed (M = 1.5182 ± 0.85646, F = 1.2618 ± 0.53292, p <0.001) and binge drinking to the greatest extent (M = 1.1933 ± 0.96522 F = 0.8176 ± 0.85446 p <0.001). Education and employment were significantly correlated with the frequency of alcohol consumption (r = 0.107 p <0.005 and r = 0.120 p = 0.001 respectively). Subjects reported buying alcoholic beverages during the pandemic with a frequency of "less than once a month" (N = 291, 35.36%) and mainly in shops (N = 556, 67.56%), while before the pandemic they mainly bought alcohol once a week (N = 431, 52.37%) and predominantly in bars / clubs (N = 619, 75.21%). New ways of drinking alcohol such as online drinking, have not been significantly identified. CONCLUSION: A change in alcohol consumed and alcohol purchased before and during the SARSCoV- 2 pandemic was revealed.

The Lesson from the First Italian Lockdown: Impacts on Anxiety and Depressive Symptoms and Sleep Quality in Patients with Remission of Inflammatory Bowel Disease.

BACKGROUND: During the COVID-19 pandemic in Italy, decisions were taken to adopt restrictive legislative measures, such as the first half of the 2020 lockdown. In those months, patients with inflammatory bowel disease experienced social isolation and reduced access to health care. OBJECTIVE: We aimed to evaluate, in this condition, the presence of remission subgroups that were most impacted by the lockdown. METHODS: During the first Italian lockdown, we recruited patients with remission of inflammatory bowel disease by administering an online questionnaire including patient demographics, the Beck Anxiety Questionnaire Inventory, the Beck Depression Inventory questionnaire, and the Pittsburg Sleep Quality Index, all validated standardized questionnaires for anxiety symptom levels, depression, and sleep quality. RESULTS: Our results showed how female patients (p<0.0001) with Crohn's disease (p<0.001) experienced worse levels of anxiety symptoms. Female patients (p<0.0001) between 50 and 60 years of age (p=0.013) with Crohn's disease (p=0.047) experienced worse levels of depressive symptoms. Females also experienced significantly worse sleep levels (p<0.001). We found a correlation between the number of sleeping hours (p<0.001) and the time taken to fall asleep (p<0.001) and the Beck Anxiety Questionnaire Inventory,which showed a linear worsening of the number of minutes it took to fall asleep, and the Beck Depression Inventory questionnaire. CONCLUSION: Among patients with remission of inflammatory bowel disease, female patients, patients with Crohn's disease, and people aged between 50 and 70 years should be considered for screening for anxiety and depression disorders and an assessment of sleep quality.

The analysis of alcohol consumption during the severe acute respiratory syndrome Coronavirus 2 Italian lockdown.

BACKGROUND: The SARS-CoV-2 lockdown resulted in deep changes of lifestyles, promoting in many people the onset of psychological symptoms generally associated with drug and alcohol abuse. The aim of this study was to assess the variation of alcohol drinking habits in a sample of Italian citizens during lockdown and to identify the psychosocial factors surrounding it. METHODS: An online anonymous questionnaire was created and submitted from April 9 to April 28, 2020. Questions were related to personal psychosocial details and alcohol drinking habits during the lockdown, including Alcohol Use Disorders Identification Test (AUDIT C) questions. RESULTS: On a total of 1234 surveys the increase of both anxiety and fear was largely detected (63% and 61% respectively). The 18% increased alcohol consumption during the lockdown and it showed a significant correlation with anxiety and fear experienced (both P<0.001). The relative risk for 7 to 9 and more than 10 drinks per day consumption were directly linked to these symptoms (P<0.001). The most involved categories of participants showed this harmful association were self-employed workers and participants who live alone, subject aged 30-50 with high level of instruction or students, and not occupied people in the age range 18-19 (all P<0.001). Additionally, the subset of the study population that showed low alcohol consumption before the lockdown has demonstrated the worsening of alcohol assumption during the quarantine (P<0.0001). CONCLUSIONS: Several psychosocial factors are involved in determining the increase of alcohol consumption during lockdown and need the healthcare support to avoid awful impact on human life.

International randomised controlled trial evaluating metabolic syndrome in type 2 diabetic cigarette smokers following switching to combustion-free nicotine delivery systems: the DIASMOKE protocol.

INTRODUCTION: Reducing exposure to cigarette smoke is an imperative for public health and for patients with diabetes. Increasingly, combustion-free nicotine delivery systems (C-F NDS) such as e-cigarettes and heated tobacco products are substituting conventional cigarettes and accelerating the downward trends in smoking prevalence. However, there is limited information about the long-term health impact in patients with diabetes who use C-F NDS. This randomised trial of type 2 diabetic cigarette smokers will test the hypothesis that following a switch from conventional cigarettes to C-F NDS a measurable improvement in metabolic syndrome (MetS) factors will be shown over the course of 2 years. METHODS AND ANALYSIS: The study is multicentre and thus will take place in five locations in four countries in an ambulatory setting. A total of 576 patients with diabetes will be randomised (1:2 ratio) to either a control arm (Study Arm A), in which they will be offered referral to smoking cessation programmes or to an intervention arm (Study Arm B) assigned to C-F NDS use. Participants will be at least 23 years old and of any gender. Patient recruitment will start in February 2021 and is expected to be completed by December 2021. Primary outcome measures include fasting plasma glucose, blood pressure, triglycerides, high-density lipoprotein and waist circumference, while secondary feature absolute change in the sum of the individual factors of MetS and change in each individual factor of MetS measured at each study time point. ETHICS AND DISSEMINATION: The approval of research ethics committee (REC) regarding the trial protocol, informed consent forms and other relevant documents is required to commence the study. Substantial amendments to the study protocol cannot be implemented until the REC grants a favourable opinion. The results of the study are intended to be published as articles in high quality peer-reviewed journals and disseminated through conference papers. TRIAL REGISTRATION NUMBER: NCT04231838. Pre-results stage.

Gut Microbiota in Alzheimer's Disease, Depression, and Type 2 Diabetes Mellitus: The Role of Oxidative Stress.

Gut microbiota consists of over 100 trillion microorganisms including at least 1000 different species of bacteria and is crucially involved in physiological and pathophysiological processes occurring in the host. An imbalanced gastrointestinal ecosystem (dysbiosis) seems to be a contributor to the development and maintenance of several diseases, such as Alzheimer's disease, depression, and type 2 diabetes mellitus. Interestingly, the three disorders are frequently associated as demonstrated by the high comorbidity rates. In this review, we introduce gut microbiota and its role in both normal and pathological processes; then, we discuss the importance of the gut-brain axis as well as the role of oxidative stress and inflammation as mediators of the pathological processes in which dysbiosis is involved. Specific sections pertain the role of the altered gut microbiota in the pathogenesis of Alzheimer's disease, depression, and type 2 diabetes mellitus. The therapeutic implications of microbiota manipulation are briefly discussed. Finally, a conclusion comments on the possible role of dysbiosis as a common pathogenetic contributor (via oxidative stress and inflammation) shared by the three disorders.

Direct toxicity of insulin on the human placenta and protection by metformin.

OBJECTIVE: To study the effects of insulin and metformin on primary trophoblasts from early pregnancies. DESIGN: Experimental in vitro study. SETTING: Academic research institute. PATIENT(S): Trophoblasts from healthy patients undergoing first trimester elective termination of pregnancy and primary lung fibroblasts (IMR-90). INTERVENTION(S): Culture and treatment with insulin and metformin of primary trophoblasts and primary lung fibroblasts (IMR-90). MAIN OUTCOME MEASURE(S): DNA damage measured by expression of γ-H2AX with immunofluorescence and Western blot. Apoptosis measured by expression of cleaved caspase-3 by Western blot. Cell survival measured by cell proliferation assay. RESULT(S): Culture of purified primary trophoblast cells in the presence of insulin at levels as low as 1 nM resulted in a 386% increase in the number of cell with elevated γ-H2AX expression, a 66% reduction in cell survival and a marked increase of cleaved caspase-3 expression. Pretreatment of trophoblasts with therapeutic doses of metformin prevented the detrimental effects of insulin. Treatment with insulin and/or metformin had no effects on primary fibroblasts. CONCLUSION(S): Elevated insulin levels are directly toxic to first trimester trophoblasts and result in increased DNA damage, apoptosis, and decreased cell survival. These effects are prevented by metformin. Trophoblast cells from early pregnancy are uniquely vulnerable to elevated levels of insulin. These findings, if confirmed in vivo, suggest that there may be a role for insulin resistance screening before attempting pregnancy and for focusing on prevention of hyperinsulinemia during early pregnancy.

MiR-27a-3p and miR-124-3p, upregulated in endometrium and serum from women affected by Chronic Endometritis, are new potential molecular markers of endometrial receptivity.

PROBLEM: Chronic endometritis (CE) is usually asymptomatic and different studies demonstrated the relation with infertility and recurrent pregnancy loss. Altered regulation of protein-encoding genes in CE has been demonstrated, but no evidence about the involvement of microRNAs in the pathology is present in literature. METHOD OF STUDY: In the endometrium from 15 women with CE and 15 healthy women, by RT-qPCR single assays, we investigated some microRNAs targeting IL11, CCL4, IGF1, and IGFBP1, which mRNAs had been found differentially expressed in endometrium of women affected by CE. The expression of IGF1 and IL11, targets of the deregulated microRNAs, has been analyzed in the same endometrium samples. We assessed the expression profiles of the deregulated microRNAs in the serum of the same patients validating their ability as biomarkers by statistical analysis. RESULTS: We demonstrated the upregulation of miR-27a-3p and miR-124-3p in the endometrium and serum from women with CE and found an anticorrelation relationship between miR-27a-3p and IGF1 in endometrium. ROC curve analysis suggested that miRNA investigation in endometrium and serum could discriminate women with CE. CONCLUSION: MiR-27a-3p and miR-124-3p could represent non-invasive markers of CE and, in a near future, could be used to assess the endometrial quality in IVF.

Sirolimus induces depletion of intracellular calcium stores and mitochondrial dysfunction in pancreatic beta cells.

Sirolimus (rapamycin) is an immunosuppressive drug used in transplantation. One of its major side effects is the increased risk of diabetes mellitus; however, the exact mechanisms underlying such association have not been elucidated. Here we show that sirolimus impairs glucose-stimulated insulin secretion both in human and murine pancreatic islets and in clonal ß cells in a dose- and time-dependent manner. Importantly, we demonstrate that sirolimus markedly depletes calcium (Ca2+) content in the endoplasmic reticulum and significantly decreases glucose-stimulated mitochondrial Ca2+ uptake. Crucially, the reduced mitochondrial Ca2+ uptake is mirrored by a significant impairment in mitochondrial respiration. Taken together, our findings indicate that sirolimus causes depletion of intracellular Ca2+ stores and alters mitochondrial fitness, eventually leading to decreased insulin release. Our results provide a novel molecular mechanism underlying the increased incidence of diabetes mellitus in patients treated with this drug.

Modulation of the Fanconi anemia pathway via chemically induced changes in chromatin structure.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.

Whole Chromosome Instability induces senescence and promotes SASP.

Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.

Role of the antioxidant defence system and telomerase in arsenic-induced genomic instability.

Arsenic (AS) is a reactive oxygen species (ROS)-inducer carcinogen, whose mode of action is still unclear. To defend against ROS, cells use enzymatic and non-enzymatic antioxidants, such as superoxide dismutase (SOD) and catalase. Failure of antioxidant systems (AXS) can result in dicentric chromosomes formation as well as telomere associations for the reduced activity of telomerase. In order to clarify the long-term effects of a past AS exposure, we evaluated the efficiency of the AXS and the telomerase activity in the progeny of arsenite-treated cells named ASO (arsenic shake-off) cells, previously obtained from arsenite-treated V79 cells and selected by shake-off. Despite SOD1 expression level correlated to the level of ROS observed over time, no changes of the relative amount of antioxidant activities were observed in ASO cells. Moreover, we found that clones characterised by low levels of SOD1 and high levels of ROS acquired a transformed phenotype. Treatment with 5-azacytidine determined an increase of SOD1 expression in a clone and decrease in one other, suggesting that aberrant DNA methylation may be responsible for the abnormal expression of SOD 1 or SOD1 inhibitor genes in different clones. TRAP assay results showed that the progeny of arsenite-treated cells were characterised by a time-dependent decrease of telomerase activity. Integrated results suggest that the increases of ROS levels are accompanied by defective telomerase activity. Finally, we propose that cells escaping the arsenite-induced death perpetuated the memory of past exposure via ROS likely because antioxidant and telomerase activity impairment and ultimately acquire a transformed phenotype.

Effects of tadalafil treatment combined with physical activity in patients with low onset hypogonadism: results from a not-randomized single arm phase 2 study.

PURPOSE: To investigate a possible relation between penile Doppler ultrasound examination (PDUE) parameters and efficacy of chronic therapy with tadalafil (TAD) combined with a protocol of aerobic physical activity (PA) in patients with late onset hypogonadism (LOH). METHODS: The study evaluated 30 patients consecutively enrolled with LOH and erectile dysfunction which present contraindication to hormonal replacement therapy for concomitant prostate disease. These patients were subjected to a combined protocol with phosphodiesterase V selective inhibitors (TAD 5 mg daily) and aerobic PA. RESULTS: After three months, we observed significant improvements in erectile function [IIEF-5, median (IQR) = 13.0 (7.0-18.0) versus 6.0 (5.0-6.75); p < 0.01] and of the main metabolic [homeostatic model assessment index, median (IQR) = 2.5 (1.62-3.37) versus 3.0 (2.0-3.75); p < 0.01; body mass index, median (IQR) = 27.0 (24.0-28.75) versus 27.5 (24.0-29.5)] and vascular parameters [peak systolic velocity, median (IQR) = 29.5 (24.25-31.0) versus 28.0 (23.0-24.25); acceleration time, median (IQR) = 114 (105.25-134.0) versus 115.0 (106.5-134.0)], assessed by PDUE. CONCLUSION: PA in association with phosphodiesterase V inhibitors could compensate the effects of hypogonadism on erectile function and facilitate the clinical response to these drugs even in the absence of adequate serum concentrations of total testosterone.

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects.