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.

Apoptosis induction by (+)alpha-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58 mu M, for NB4 and NB4-R2, respectively. a-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24 h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis. (C) 2008 Elsevier Ltd. All rights reserved.

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

Obese Patients Have Stronger Peristalsis and Increased Acid Exposure in the Esophagus

Obesity is a risk factor for GERD and a potential modulator of esophageal motility. To assess whether obese patients differ from non-obese patients in terms of esophageal motility and reflux. Patients (n = 332) were categorized in GERD and controls after clinical assessment, esophageal manometry, and pH monitoring. Non-obese (BMI 16-29.9) and obese (BMI 30-68) were compared in regard of distal esophageal amplitude (DEA), LES pressure (LESP), manometric diagnosis, and esophageal acid exposure (EAE). Obese showed higher DEA in both controls (122 +/- A 53 vs. 97 +/- A 36 mmHg, p = 0.041) and GERD patients (109 +/- A 38 vs. 94 +/- A 46 mmHg, p < 0.001), higher LESP in GERD patients (20.5 +/- A 10.6 vs. 18.2 +/- A 10.6 mmHg, p = 0.049), higher frequency of nutcracker esophagus in controls (30 vs. 0%, p = 0.001), lower frequency of ineffective motility in GERD patients (6 vs. 20%, p = 0.001), and higher EAE in both controls [total EAE: 1.6% (0.7-5.1) vs. 0.9% (0.2-2.4), p = 0.027] and GERD patients [upright EAE: 6.5% (3.8-11.1) vs. 5.2% (1.5-10.6), p = 0.048]. Multiple linear regression showed that BMI was associated either with EAE (p < 0.001), DEA (p = 0.006), or LESP (in men, p = 0.007). Obese patients differed from non-obese in terms of esophageal motility and reflux, regardless of the presence of GERD. Obese patients showed stronger peristalsis and increased acid exposure in the esophagus.

Bronchial responsiveness during esophageal acid infusion

Among the possible mechanisms explaining the worsening of asthma due to gastroesophageal reflux disease (GERD) is the increase in bronchial hyperresponsiveness. The effects of GERD on bronchial hyperresponsiveness in patients with bronchial asthma have yet to be studied in significant detail. The aim of this study was to determine the effects of esophageal acid perfusion on bronchial responsiveness to bradykinin in patients with both asthma and GERD. In 20 patients with asthma and GERD disease, esophageal pH was monitored with a pH meter and bronchial responsiveness was evaluated by aerosol inhalation of bradykinin during esophageal acid perfusion and, 24 h earlier or later the patients were submitted to another bronchial provocation test without acid infusion. No significant changes were observed in FEV1, FEF25-75%, FVC, or PEF during acid perfusion. The response to the bronchial provocation test did not differ between the control day and the day of acid infusion (p = 0.61). The concentration provoking a 20% fall in FEV1 (geometric mean +/- geometric SD) was 1.09 +/- 5.84 on the day of acid infusion and 0.98 +/- 5.52 on the control day. There is no evidence that acid infusion changes bronchial responsiveness to bradykinin. These findings strongly question the significance of acid infusion as a model to study the pathogenesis of GERD-induced asthma.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

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.

Role of homocysteic acid in the guinea pig (Cavia porcellus) anterior cingulate cortex in tonic immobility and the influence of NMDA receptors on the dorsal PAG

Tonic immobility (TI) is an innate defensive behaviour elicited by physical restriction and Postural inversion, and is characterised by a profound and temporary state of akinesis. Our previous studies demonstrated that glutamatergic stimulation of the dorsomedial/dorsolateral Portion of periaqueductal gray matter (dPAG) decreases the duration of TI in guinea pigs (Cavia porcellus). Furthermore, evidence suggests that the anterior cingulate cortex (ACC) constitutes an important Source of glutamate for the dPAG. Hence, in the current study, we investigated the effects of microinjection of the excitatory amino acid (EAA) agonist DL-homocysteic acid (DLH) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 into the ACC on the duration of TI in guinea pigs. We also assessed the effect of the NMDA receptor antagonist (MK-801) into the dorsal periaqueductal gray matter (dPAG) prior to DLH microinjection into the ACC on the TI duration in the guinea pig. Our results demonstrated that DLH microinjections into the ACC decreased the duration of TI. This effect was blocked by previous MK-801 microinjections into the ACC or into the dPAG. The MK-801 microinjections alone did not influence TI duration. These results provide the new insight that EAAs in the ACC can decrease the duration of TI. The mechanism seems to be dependent on the NMDA receptors present in the ACC and in the dPAG. (C) 2009 Elsevier B.V. All rights reserved.

Modulation of tonic immobility in guinea pig PAG by homocysteic acid, a glutamate agonist

Tonic immobility (TI) is an innate defensive behavior elicited by physical restriction and postural inversion, and is characterized by a profound and temporary state of motor inhibition. The participation of the periaqueductal gray matter (PAG) in TI modulation has previously been described. In addition, the excitatory amino acids (EAA) are important mediators involved in the adjustment of several defensive responses produced by PAG. In the present study, we investigated the effect of microinjection of the EAA agonist DL-homocysteic acid (DLH) and the N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801) into the ventrolateral and dorsal PAG over the duration of TI in guinea pigs. Microinjection of 15 nmol/0.2 mu l of DLH into the ventrolateral PAG (vlPAG) and 30 nmol/0.2 mu l of DLH into the dorsal PAG (dPAG) promoted an increase and decrease in TI duration, respectively. These responses were blocked by prior microinjection of the NMDA receptor antagonist, MK-801 (3.6 nmol/0.2 mu l) at the same site. Microinjection of MK-801 alone into the APAG and dPAG did not alter the duration of TI episodes. These results suggest that NMDA receptors are involved in the modulation of TI in both the vlPAG and dPAG. In addition, PAC excitatory amino acids modulate the TI response via columnar organization of the PAC. In this manner, the vlPAG facilitates TI modulation whereas dPAG has an inhibitory role in TI. (C) 2008 Elsevier Inc. All rights reserved.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G -> A change in lipA1 allele, which results in a Glu(214) -> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.

In vitro susceptibility to antimycotic drug undecanoic acid, a medium-chain fatty acid, is nutrient-dependent in the dermatophyte Trichophyton rubrum

The antimycotic activity of fatty acids has long been known, and their presence in human skin and sweat appears to protect the host against superficial mycoses. Undecanoic acid is a medium-chain fatty acid that has been used in the treatment of dermatophytoses in humans. In this study, we selected one Trichophyton rubrum undecanoic acid-resistant strain that showed a marked reduction in its capacity to grow on human nail fragments, which correlated with the reduced activity of secreted keratinolytic proteases. Moreover, the susceptibility of T. rubrum to undecanoic acid is also dependent on the carbon source utilized by both control and resistant strains. The growth of the control strain was strongly inhibited by undecanoic acid in Sabouraud medium or in cultures supplemented with low-fat milk, whereas it was ineffective when the cultures were supplemented with Tween 20 or keratin as the carbon source, suggesting that nutrient conditions are crucial in establishing a susceptibility to antifungal drugs, which is helpful for the isolation and characterization of resistant strains, and in the screening for new antifungal drugs.

Ultrasonography in pregnant women with antiphospholipid syndrome using salicylic acid and heparin

The objective of the present study was to evaluate fetal biometry, Doppler values, and perinatal outcomes in pregnant women with antiphospholipid syndrome treated with acetylsalicylic acid and heparin. Twenty-five pregnant women with antiphospholipid syndrome using 100 mg/day acetylsalicylic acid and 5,000 IU heparin every 12 h were evaluated in this prospective observational study. Ultrasonography was performed between 24 and 38 weeks of gestational age to assess estimated fetal weight, placental thickness, amniotic fluid index, fetal biophysical profile and Doppler evaluation of maternal uterine arteries, and fetal middle cerebral and umbilical arteries. Data regarding Apgar score, gender, delivery mode, and birth weight and length were recorded after birth. The observed values for ultrasonographic assessment and perinatal outcomes were not very different from the expected values for normal pregnancies. The birth weight was 2863.3 +/- A 737.7 g (mean +/- A SD) and length was 46.8 +/- A 4.2 cm. Only one newborn (4%) had the 1-min Apgar score < 7 and all had the 5-min Apgar score > 7. Gestational and perinatal evaluation of pregnant women with antiphospholipid syndrome using both acetylsalicylic acid and heparin was reassuring.