Folic acid supplementation during early hepatocarcinogenesis: cellular and molecular effects

Folic acid (FA) supplementation during carcinogenesis is controversial. Considering the impact of liver cancer as a public health problem and mandatory FA fortification in several countries, the role of FA supplementation in hepatocarcinogenesis should be elucidated. We evaluated FA supplementation during early hepatocarcinogenesis. Rats received daily 0.08 mg (FA8 group) or 0.16 mg (FA16 group) of FA/100 g body weight or water (CO group, controls). After a 2-week treatment, animals were subjected to the ""resistant hepatocyte"" model of hepatocarcinogenesis (initiation with diethylnitrosamine, selection/promotion with 2-acetylaminofluorene and partial hepatectomy) and euthanized after 8 weeks of treatment. Compared to the CO group, the FA16 group presented: reduced (p < 0.05) number of persistent and increased (p < 0.05) number of remodeling glutathione S-transferase (GST-P) positive preneoplastic lesions (PNL); reduced (p < 0.05) cell proliferation in persistent GST-P positive PNL; decreased (p < 0.05) hepatic DNA damage; and a tendency (p < 0.10) for decreased c-myc expression in microdissected PNL. Regarding all these parameters, no differences (p > 0.05) were observed between CO and FA8 groups. FA-treated groups presented increased hepatic levels of S-adenosylmethionine but only FA16 group presented increased S-adenosylmethionine/S-adenosylhomocysteine ratio. No differences (p > 0.05) were observed between experimental groups regarding apoptosis in persistent and remodeling GST-P positive PNL, and global DNA methylation pattern in microdissected PNL. Altogether, the FA16 group, but not the FA8 group, presented chemopreventive activity. Reversion of PNL phenotype and inhibition of DNA damage and of c-myc expression represent relevant FA cellular and molecular effects.

Effects of a Low-Protein Diet on Plasma Amino Acid and Homocysteine Levels and Oxidative Status in Rats

Background/Aims: Transmethylation reactions and antioxidant metabolism are linked by transsulfuration, where homocysteine (Hcy) is converted to cysteine and reduced glutathione (GSH). Low protein intake can modulate the balance of this metabolic reaction. The aim of the present investigation was to study the effect of a low-protein diet on Hcy metabolism by monitoring levels of the amino acids involved in these pathways, and relating these levels to GSH levels and lipid peroxidation in rats. Methods: Sixteen rats were divided into 2 groups: control (C; standard AIN-93 diet, 20% protein) and low-protein diet (LPD; 8% protein diet). Rats in both groups were placed on the diets for 28 days. Results: A significant reduction (p < 0.05) in plasma Hcy concentration was found in LPD rats (0.16 +/- 0.04 mu mol/mg protein) versus C rats (0.25 +/- 0.03 mu mol/mg protein). Methionine levels were not significantly different between the 2 groups (C: 1.24 +/- 0.22 mu mol/mg protein; LPD: 1.03 +/- 0.27 mu mol/mg protein). A significant reduction (p ! 0.05) in hepatic GSH concentrations (C: 44 8 10 mu mol/mg protein; LPD: 17.4 +/- 4.3 mu mol/mg protein) was accompanied by an increase in lipid peroxidation (C: 0.13 +/- 0.01 mu mol/mg protein; LPD: 0.17 +/- 0.02 mu mol/mg protein; r = -0.62, p < 0.01). Conclusion: Hcy levels were reduced under a low-protein diet, resulting in modulated methyl balance and reduced GSH formation leading to increased susceptibility of hepatic cells to oxidative events. Copyright (C) 2009 S. Karger AG, Basel

DOUBLE ANEUPLOIDY (48,XXY,+21) OF MATERNAL ORIGIN IN A CHILD BORN TO A 13-YEAR-OLD MOTHER: EVALUATION OF THE MATERNAL FOLATE METABOLISM

Double aneuploidy, (48,XXY,+21) of maternal origin in a child born to a 13-year-old mother: evoluation of the maternal folate metabolism: The occurrence of non-mosaic double trisomy is exceptional in newborns. In this paper, a 48,XXY,+21 child, the parental origin of the extra chromosomes and the evaluation of the maternal folate metabolism are presented. The infant was born to a 13-year-old mother and presented with the typical clinical features of Down syndrome (DS). The origin of the additional chromosomes was maternal and most likely resulted from errors during the first meiotic division. Molecular analysis of 12 genetic polymorphisms involved in the folate metabolism revealed that the mother is heterozygous for the MTHFR C677T and TC2 A67G polymorphisms, and homozygous for the mutant MTRR A66G polymorphism. The maternal homocysteine concentration was 4.7 mu mol/L, a value close to the one considered as a risk factor for DS in our previous study. Plasma methylmalonic acid and serum folate concentrations were 0.17 mu mol/L and 18.4 ng/mL, respectively. It is possible that the presence of allelic variants for the folate metabolism and Hey concentration might have favored errors in chromosomal disjunction (hiring gametogenesis in this young mother. To our knowledge, this is the first patient with non-mosaic Down-Klinefelter born to a teenage mother, resulting from a rare fertilization event combining an abnormal 25,XX,+21 oocyte and a 23,Y spermatozoon.

Discordant Nadir GH After Oral Glucose and IGF-I Levels on Treated Acromegaly: Refining the Biochemical Markers of Mild Disease Activity

Biochemical markers for remission on acromegaly activity are controversial. We studied a subset of treated acromegalic patients with discordant nadir GH levels after oral glucose tolerance test (oGTT) and IGF-I values to refine the current consensus on acromegaly remission. We also compared GH results by two GH immunoassays. From a cohort of 75 treated acromegalic patients, we studied 13 patients who presented an elevated IGF-I despite post-oGTT nadir GH of <= 1 mu g/l. The 12-h daytime GH profile (GH-12 h), nadir GH after oGTT, and basal IGF-I levels were studied in patients and controls. Bland-Altman method showed high concordance between GH assays. Acromegalic patients showed higher mean GH-12 h values (0.71+/-0.36 vs. 0.31+/-0.28 mu g/l; p<0.05) and nadir GH after oGTT (0.48+/-0.32 vs. 0.097+/-0.002 mu g/l; p<0.05) as compared to controls. Nadir GH correlated with mean GH-12 h (r=0.92, p<0.05). The mean GH-12 h value from upper 95% CI of controls (0.54 mu g/l) would correspond to a theoretical normal nadir GH of <= 0.27 mu g/l. Patients with GH nadir <= 0.3 mu g/l had IGF-I between 100-130% ULNR (percentage of upper limit of normal range) and mean GH-12 h of 0.35+/-0.15, and patients with GH nadir >0.3 and <= 1 mu g/l had IGF-I >130% ULNR and mean GH-12 h of 0.93+/-0.24 mu g/l. Our data integrate daytime GH secretion, nadir GH after oGTT, and plasma IGF-I concentrations showing a continuum of mild residual activity in a subgroup of treated acromegaly with nadir GH values <= 1 mu g/l. The degree of increased IGF-I levels and nadir GH after oGTT are correlated with the subtle abnormalities of daytime GH secretion.

Genetic polymorphisms involved in folate metabolism and concentrations of methylmalonic acid and folate on plasma homocysteine and risk of coronary artery disease

Objectives Alterations in the enzymes involved in homocysteine (Hcy) metabolism or vitamin deficiency could play a role in coronary artery disease (CAD) development. This study investigated the influence of MTHFR and MTR gene polymorphisms, plasma folate and MMA on Hcy concentrations and CAD development. MMA and folate concentrations were also investigated according to the polymorphisms. Methods Two hundred and eighty-three unrelated Caucasian individuals undergoing coronary angiography (175 with CAD and 108 non-CAD) were assessed in a case-control study. Plasma Hcy and MMA were measured by liquid chromatography/tandem mass spectrometry. Plasma folate was measured by competitive immunoassay. Dietary intake was evaluated using a nutritional questionnaire. Polymorphisms MTHFR and MTR were investigated by polymerase chain reaction (PCR) followed by enzyme digestion or allele-specific PCR. Results Hcy mean concentrations were higher in CAD patients compared to controls, but below statistical significance (P = 0.246). Increased MMA mean concentrations were frequently observed in the CAD group (P = 0.048). Individuals with MMA concentrations > 0.5 mu mol/l (vitamin B(12) deficiency) were found only in the CAD group (P = 0.004). A positive correlation between MMA and Hcy mean concentrations was observed in both groups, CAD (P = 0.001) and non-CAD (P = 0.020). MMA mean concentrations were significantly higher in patients with hyperhomocysteinemia in both groups, CAD and non-CAD (P = 0.0063 and P = 0.013, respectively). Folate mean concentration was significantly lower in carriers of the wild-type MTHFR 1298AA genotype (P = 0.010). Conclusion Our results suggest a correlation between the MTHFR A1298C polymorphism and plasma folate concentration. Vitamin B(12) deficiency, reflected by increased MMA concentration, is an important risk factor for the development both of hyperhomocysteinemia and CAD.

Antihypertensive effects exerted by enalapril in mild to moderate hypertension are not associated with changes in the circulating levels of nitric oxide-related markers

The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are explained, at least in part, by enhanced bradykinin-dependent nitric oxide (NO) formation and decreased angiotensin II-induced oxidative stress and vasoconstriction. We examined for the first time whether treatment with enalapril increases the plasma levels of markers of NO formation and decreases oxidative stress in mild to moderate hypertensive patients. Eighteen untreated hypertensive patients were treated with enalapril 10 mg/day (n = 10) or 20 mg/day (n = 8) for 60 days. Eighteen normotensive healthy controls were followed for the same period. Venous blood samples were collected at baseline and after 30/60 days of treatment with enalapril. Plasma NOx (nitrites + nitrates) concentrations were determined by using the Griess reaction. Plasma nitrite and whole blood nitrite concentrations were determined by using an ozone-based chemiluminescence assay. Plasma thiobarbituric acid-reactive species (TBARS) and 8-isoprostane concentrations were determined by a fluorimetric method and by ELISA, respectively. Treatment with enalapril decreased blood pressure in hypertensive patients. However, we found no significant changes in plasma NOx, nitrite, whole blood nitrite, and in the levels of markers of oxidative stress in both normotensive controls and hypertensive patients treated with enalapril. Our data show that enalapril 10-20 mg/day does not affect the concentrations of relevant markers of NO formation or markers of oxidative stress in mild to moderately hypertensive subjects, despite satisfactory blood pressure control. Our findings do not rule out the possibility that ACEi may produce such effects in more severely hypertensive patients treated with higher doses of ACEi.

Increases in circulating matrix metalloproteinase-9 levels following fibrinolysis for acute pulmonary embolism

Introduction: Fibrinolyis is one of the first line therapies in high risk pulmonary embolism (PE) according to current guidelines. Previous studies showed that brinolytic therapy with tPA (tissue plasminogen activator, or alteplase) upregulates the concentrations of matrix metalloproteinases (MMPs) and contributes to hemorrhagic transformation after cardioembolic stroke. However, no previous study has described the circulating MMPs levels following fibrinolysis for acute PE. Materials and Methods: We serially measured the circulating levels of MMPs (MMP-9 and MMP-2) and their endogenous inhibitors, the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in alteplase and in streptokinase-treated patients with acute PE by gelatin zymography and by enzyme-linked immunosorbent assays, respectively. Results: We found that therapy of PE streptokinase or with alteplase is associated increased pro-MMP-9, but not MMP-2, concentrations for up to 24 hours, whereas no significant changes were found in TIMP-1 or TIMP-2 concentrations. This alteration returned to normal 3 to 5 days after thrombolysis. This is the first study reporting on MMPs alterations following fibrinolysis for acute PE. Conclusions: We found transient increases in circulating pro-MMP-9 levels following fibrinolysis for acute PE. Our findings support the hypothesis that increased MMP-9 levels may underlie the risk of intracerebral hemorrhage or other bleeding complication of thrombolysis for acute PE, and the use of MMP inhibitors may decrease such risk. (C) 2010 Elsevier Ltd. All rights reserved.

Alterations in cyclic GMP levels in preeclampsia may reflect increased B-type natriuretic peptide levels and not impaired nitric oxide activity

Objectives: We compared nitrite, B-type natriuretic peptide (BNP), and cGMP levels in preeclamptic with those found in healthy pregnant. Methods: We studied 21 healthy pregnant and 27 preeclamptic. Plasma cGMP and BNP levels were determined by ELISA. Nitrite levels were determined by chemiluminescence. Results: Higher cGMP and BNP, and lower nitrite levels were found in preeclamptic versus healthy pregnant Conclusions: Altered cGMP levels reflect increased BNP levels and not impaired nitric oxide activity in preeclampsia. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Increased basal FSH levels as predictors of low-quality follicles in infertile women with endometriosis

Objective: To determine whether basal levels of follicle-stimulating hormone (FSH) and anti-mullerian hormone (AMH), antral follicle count (AFC), and the numbers of dominant follicles, oocytes, and mature oocytes retrieved after ovarian stimulation differed between infertile women with endometriosis and healthy women undergoing assisted reproduction techniques (ART). Method: Of 77 consecutive ART candidates, 27 were infertile and had endometriosis. A male factor caused the infertility of the other 50, who acted as controls. Results: The AMH and AFC levels were similar in the 2 groups. The FSH levels were higher (8.28 mIU/ML [range, 5.25-24.1 mIU/ML] vs 5.91 mIU/mL [range, 2.47-18.7 mIU/ML]; P<0.01) in the study group. And the numbers of retrieved (n = 5 [range, 0-12] vs n = 9 [range, 0-27]; P<0.05) and mature oocytes (n = 4 [range, 0-11] vs n = 5 [range, 0-16]; P<0.05) were less in the study group. Conclusion: Because AMH levels were unchanged, endometriosis seems not to damage the primordial pool of follicles and oocytes, but to lessen the quality of the ovarian response to the hCG injection. Basal FSH levels may be of value in predicting ART success in women with the disease. (C) 2010 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01: r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01: r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women. (C) 2010 Elsevier Inc. All rights reserved.

Basal levels of DNA damage detected by micronuclei and comet assays in untreated breast cancer patients and healthy women

Breast cancer is the second most frequent type of cancer worldwide and is the most common malignant disease among women. Risk factors for breast cancer include early menarche, late menopause, hormonal therapies, exposure to environmental pollutants, smoking and alcohol use. However, increased or prolonged exposure to estrogen is the most important risk factor. It has been suggested that accumulation of DNA damage may contribute to breast carcinogenesis. Epidemiological studies suggest that cytogenetic biomarkers such as micronuclei in peripheral blood lymphocytes may predict cancer risk because they indicate genomic instability in target tissues. The objective of the present study was to evaluate the frequencies of micronuclei and the extent of DNA damage detected by comet assay in peripheral blood lymphocytes of untreated breast cancer patients and healthy women. The study was conducted using peripheral blood lymphocytes from 45 women diagnosed for Ductal ""in situ"" or invasive breast carcinoma and 85 healthy control women. Micronuclei and comet assays were performed to detect spontaneous DNA damage. The results showed that micronuclei frequencies and tail intensity, detected by comet assay, were significantly higher in the breast cancer group than in controls. The levels of DNA damage were similar in smokers and non-smokers, and aging did not influence the frequencies of micronuclei or tail intensity values observed in either group. In conclusion, the present work demonstrates higher levels of DNA damage in untreated breast cancer patients than in healthy women.

High levels of anxiety and depression have a negative effect on quality of life of women with chronic pelvic pain

Chronic pelvic pain (CPP) is a common and complex disease whose cause is often clinically inexplicable, with consequent difficulty in diagnosis and treatment. Patients with CPP have high levels of anxiety and depression, with a consequent impairment of their quality of life. The objective of this study was to determine the prevalence of anxiety and depression and their impact on the quality of life of women with CPP. A cross-sectional controlled study was conducted on 52 patients with CPP and 54 women without pain. Depression and anxiety were evaluated by the Hospital Anxiety and Depression Scale, and quality of life was evaluated by the World Health Organization Quality of life Whoqol-bref questionnaire. Data were analysed statistically by the Mann-Whitney U-test, the Fisher exact test, chi-square test and Spearman correlation test. The prevalence of anxiety was 73% and 37% in the CPP and control groups, respectively, and the prevalence of depression was 40% and 30% respectively. Significant differences between groups were observed in the physical, psychological and social domains. Patients with higher anxiety and depression scores present lower quality of life scores. The fact that DPC is a syndromic complex, many patients enter a chronic cycle of search for improvement of medical symptoms. The constant presence of pain may be responsible for affective changes in dynamics, family, social and sexual. Initially the person is facing the loss of a healthy body and active, to a state of dependence and limitations. In this study, patients with higher scores of anxiety and depression scores had lower quality of life and patients with lower scores of anxiety and depression had scores of quality of life. These results show that perhaps the depression and anxiety may be related to the negative impact on quality of life of these patients. In view of this association, we emphasise the importance of a specific approach to the treatment of anxiety and depression together with clinical treatment to improve the quality of life of these patients.

Comparison between the reliability levels of manual palpation and pressure pain threshold in children who reported orofacial pain

The aim of this study was to compare the intra-and inter-rater reliability of pressure pain threshold (PPT) and manual palpation (MP) of orofacial structures in symptomatic and symptom-free children for temporomandibular disorders (TMD). Fourteen children reporting pain in masticatory muscles or the temporomandibular joint and 16 symptom-free children were randomly assessed on three different occasions: by rater-1 in the first and third session and by rater-2 in the second session. The trained raters applied algometry and MP as recommended by the Research Diagnostic Criteria for TMD. Intraclass correlation coefficients and the Kappa statistic were used to assess the levels of reliability of PPT and MP, respectively. Excellent intra-and inter-rater reliability levels were observed for PPT values at most of the examined sites for symptom-free children and excellent and moderate reliability levels for children reporting pain. For MP, moderate and poor intra-rater and inter-rater reliability levels were observed for most sites in both groups. Algometry showed higher reliability levels for both groups of children and is recommended for pain assessment in children in association with MP. (C) 2010 Elsevier Ltd. All rights reserved.

Administration of cannabidiol and imipramine induces antidepressant-like effects in the forced swimming test and increases brain-derived neurotrophic factor levels in the rat amygdala

Objective: Cannabidiol is a chemical constituent from Cannabis sativa and it has multiple mechanisms of action, including antidepressant effects. The main objective of the present study was to evaluate behavioural and molecular effects induced by administration of cannabidiol and imipramine in rats. Methods: In the present study, rats were acutely or chronically treated for 14 days once a day with saline, cannabidiol (15, 30 and 60 mg/kg) or imipramine (30 mg/kg) and the animals behaviour was assessed in forced swimming and open-field tests. Afterwards, the prefrontal cortex, hippocampus and amygdala brain-derived neurotrophic factor (BDNF) levels were assessed by enzyme-linked immunosorbent sandwich assay. Results: We observed that both acute and chronic treatments with imipramine at the dose of 30 mg/kg and cannabidiol at the dose of 30 mg/kg reduced immobility time and increased swimming time; climbing time was increased only with imipramine at the dose of 30 mg/kg, without affecting locomotor activity. In addition, chronic treatment with cannabidiol at the dose of 15 mg/kg and imipramine at the dose of 30 mg/kg increased BDNF levels in the rat amygdala. Conclusion: In conclusion, our results indicate that cannabidiol has an antidepressant-like profile and could be a new pharmacological target for the treatment of major depression.

Chronic administration of harmine elicits antidepressant-like effects and increases BDNF levels in rat hippocampus

A growing body of evidence has pointed to the beta-carboline harmine as a potential therapeutic target for the treatment of major depression. The present study was aimed to evaluate behavioural and molecular effects of the chronic treatment with harmine and imipramine in rats. To this aim, rats were treated for 14 days once a day with harmine (5, 10 and 15 mg/kg) and imipramine (10, 20 and 30 mg/kg) and then subjected to the forced swimming and open-field tests. Harmine and imipramine, at all doses tested, reduced immobility time of rats compared with the saline group. Imipramine increased the swimming time at 20 and 30 mg/kg and harmine increased swimming time at all doses. The climbing time increased in rats treated with imipramine (10 and 30 mg/kg) and harmine (5 and 10 mg/kg), without affecting spontaneous locomotor activity. Brain-derived neurotrophic factor (BDNF) hippocampal levels were assessed in imipramine and harmine-treated rats by ELISA sandwich assay. Interestingly, chronic administration of harmine at the higher doses (10 and 15 mg/kg), but not imipramine, increased BDNF protein levels in rat hippocampus. Finally, these findings further support the hypothesis that harmine could bring about behavior and molecular effects, similar to antidepressants drugs.