Effect of n-3 fatty acids and statins on oxidative stress in statin-treated hypercholestorelemic and normocholesterolemic women

Introduction: Although the combination of statins with n-3 fatty acids seems to be beneficial under the lipid profile aspect, there is little information about the interaction of these two compounds on oxidative stress. Objective: Evaluate the interaction between statins and n-3 fatty acids on oxidative stress in women, using a 2(2) factorial design. Methods: Forty-three women participated in this crossover design. They were separated into two groups in which 20 were under statin treatment for more than 6 months, and 23 were normolipidemic. Within each group, half of the patients received capsules containing 2.4 g/day of a mixture of EPA and DHA for 6 weeks, while the other half received a mixture of soya and corn oil. After a period of 90 days of washout, the groups were switched, and received the supplementation for 6 weeks more. Results: Statins reduced serum LDL and increased SOD expression. n-3 fatty acids increased the plasma malondialdehyde and SOD activity but reduced catalase expression (p < 0.05). The interaction involving statins and n-3 fatty acids was nearly significant to the serum triacylglycerol reduction (p = 0.054). Conclusion: Combining statins and n-3 fatty acids is an excellent strategy to reduce plasma cholesterol and triacylglycerol concentration in women. However, n-3 fatty acids increased the oxidative stress and the pleiotropic effect of statins seemed to be not enough to counterbalance this result. Our data also suggested that the mechanism by which n-3 fatty acids interfere in oxidative stress can be associated with antioxidant enzymes expression and activity. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients

Background: p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype. Aim: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. Materials and methods: Fifty-one patients with primary iron overload (transferrin saturation >= 50% in females and >= 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. Results: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. Conclusions: The HE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G2045 mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated. (C) 2011 Elsevier Inc. All rights reserved.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation >50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and beta 2-microglobulin ((beta 2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. (C) 2010 Elsevier Inc. All rights reserved.

Hemojuvelin and Hepcidin Genes Sequencing in Brazilian Patients with Primary Iron Overload

Background: Most hereditary hemochromatosis (HH) patients are homozygous for the p. C282Y mutation in the HFE gene. Some studies reported that HH phenotypic expression could be modulated by genetic factors such as HJV and HAMP gene mutations. Aims: The aims of this study were to identify HJV and HAMP mutations and to analyze their impact on HH phenotype in non-p. C282Y homozygous individuals. Methods: Twenty-four Brazilian patients with primary iron overload and non-p. C282Y homozygous genotype (transferrin saturation >50% in women and >60% in men and absence of secondary causes) were selected. Subsequent bidirectional sequencing of the HJV and HAMP exons was performed. Results: Sequencing revealed a substitution in heterozygosis, c. 929C>G, which corresponds to p.A310G polymorphism in HJV exon 4 (rs7540883). In the same gene, in another individual, an IVS1-36C>G intronic variant was detected in heterozygosis. In the HAMP gene, an IVS3 + 42G>A intronic variant was identified. There were six (25.0%) patients carrying a heterozygous genotype for the HFE p. C282Y and nine (37.5%) patients carrying a heterozygous genotype for the HFE p. H63D. Conclusion: HJV p.A310G polymorphism and two intronic variants were found, but none of these alterations were associated with digenic inheritance with the HFE gene. Our data indicate that HJV and HAMP functional mutations are not frequent in these patients.

Classification of individuals with dyslipidaemia controlled by statins according to plasma biomarkers of oxidative stress using cluster analysis

Oxidative stress is a physiological condition that is associated with atherosclerosis. and it can be influenced by diet. Our objective was to group fifty-seven individuals with dyslipidaemia controlled by statins according to four oxidative biomarkers, and to evaluate the diet pattern and blood biochemistry differences between these groups. Blood samples were collected and the following parameters were evaluated: diet intake; plasma fatty acids; lipoprotein concentration; glucose; oxidised LDL (oxLDL); malondialdehyde (MDA): total antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing ability power assays. Individuals were separated into five groups by cluster analysis. All groups showed a difference with respect to at least one of the four oxidative stress biomarkers. The separation of individuals in the first axis was based upon their total antioxidant activity. Clusters located on the right side showed higher total antioxidant activity, higher myristic fatty acid and lower arachidonic fatty acid proportions than clusters located on the left side. A negative correlation was observed between DPPH and the peroxidability index. The second axis showed differences in oxidation status as measured by MDA and oxLDL concentrations. Clusters located on the Upper side showed higher oxidative status and lower HDL cholesterol concentration than clusters located on the lower side. There were no differences in diet among the five clusters. Therefore, fatty acid synthesis and HDL cholesterol concentration seem to exert a more significant effect on the oxidative conditions of the individuals with dyslipidaemia controlled by statins than does their food intake.

HJV Hemochromatosis, Iron Overload, and Hypogonadism in a Brazilian Man: Treatment with Phlebotomy and Deferasirox

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil[2008/54131-0]

Iron deficiency and frequency of HFE C282Y gene mutation in Brazilian blood donors

Limited data are available about iron deficiency (ID) in Brazilian blood donors. This study evaluated the frequencies of ID and iron-deficiency anaemia (IDA) separately and according to frequency of blood donations. The protective effect of the heterozygous genotype for HFE C282Y mutation against ID and IDA in female blood donors was also determined. Five hundred and eight blood donors were recruited at the Blood Bank of Santa Casa in Sao Paulo, Brazil. Haemoglobin and serum ferritin concentrations were measured. The genotype for HFE C282Y mutation was determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis. The ID was found in 21 center dot 1% of the women and 2 center dot 6% of the men whereas the IDA was found in 6 center dot 8 and 0 center dot 3%, respectively. The ID was found in 11 center dot 9% of the women in group 1 (first-time blood donors) and the frequency increased to 38 center dot 9% in women of the group 3 (blood donors donating once or more times in the last 12 months). No ID was found in men from group 1; however the ID frequency increased to 0 center dot 9% in group 2 (who had donated blood before but not in the last 12 months) and 5 center dot 0% in group 3. In summary, the heterozygous genotype was not associated with reduction of ID or IDA frequencies in both genders, but in male blood donors it was associated with a trend to elevated ferritin levels (P = 0 center dot 060). ID is most frequent in Brazilian women but was also found in men of group 3.

Biomarkers of oxidative stress and endothelial dysfunction in glucose intolerance and diabetes mellitus

Objectives: To evaluate biomarkers of endothelial dysfunction and oxidative stress in glucose intolerance (GI) compared to overt diabetes (DM2). Design and methods: 140 volunteers including 96 with DM2, 32 with GI and 12 controls (C) were Studied. NO metabolites, NO synthase inhibitors. thiols and N-acetyl-beta-glucosaminidase (NAGase) activity were analyzed by chemiluminescence, capillary electrophoresis, ELISA and colorimetric assay, respectively. Results: (center dot)NO metabolites were higher in GI (NOx: P=0.03 S-nitrosothiols: p=0.001) and DM2 (p=0.006; p=0.0006) groups in relation to group C, while nitrotyrosine was higher only in the DM2 group in comparison 10 the other groups. NAGase activity was elevated in GI (p=0.003) and DM2 (p=0.0004) groups in relation to group C, as well as, ADMA (p=0.01: p=0.003) and GSSG (p=0.01 p=0.002). Conclusions: (center dot)NO metabolites. (center dot)NO synthase inhibitors. thiols and NAGase are biomarkers Suitable to indicate endothelial dysfunction and oxidative stress in the early stages of impaired response to insulin. (c) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Iron bioavailability from ferric pyrophosphate in rats fed with fructan-containing yacon (Smallanthus sonchifolius) flour

The effects of inulin-type fructans (ITF)-containing yacon flour (YF) on Fe bioavailability from ferric pyrophosphate (FP) were evaluated in Fe-deficient rats using the Hb repletion efficiency (HRE) assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulphate (FS) or FP, supplemented with 7.5% ITF as either YF or Raftilose (RAF), a purified ITF. ITF increased caecal fermentation, whereas YF was more butyrogenic than RAF. ITF improved FIRE in FP-fed rats, and those fed YF had a higher relative biological value compared with those fed FP and RAF. Liver Fe was increased by ITF, but only YF led to values similar to those in the FS group. It is observed that ITF increased caecal fermentation and Fe bioavailability. These effects were more pronounced when YF was the ITF source. (C) 2010 Elsevier Ltd. All rights reserved.

Involvement of oxidative stress in the hepatotoxicity induced by aromatic antiepileptic drugs

The use of the classic aromatic antiepileptic drugs (AAEDs) has recently been expanded to a broad spectrum of psychiatric and neurological disorders. However, the clinical use of these drugs is limited by several adverse effects, mainly idiosyncratic hepatotoxicity. AAED-induced hepatotoxicity has been attributed to a defective detoxification by the epoxide hydrolase and accumulation of arene oxides. The underlying mechanism has been proposed as immune-mediated, but direct toxicity has also been suggested. In general, idiosyncratic drug-induced hepatotoxicity may be mediated, at least in part, by oxidative stress. On the other hand, the oxidative stress induced by the AAED metabolites has not been demonstrated yet. Therefore, in the present study we have evaluated the induction of oxidative stress by three classical AAEDs: carbamazepine. phenytoin and phenobarbital as well as by their metabolites. The toxic effects of the metabolites were evaluated by incubating the drug with rat liver microsomes. The AAED-induced oxidative stress was demonstrated by the increased malondialdehyde levels, oxidation of cardiolipin; oxidation of sulfhydryl proteins and alteration of the cellular redox status. Results suggest that the hepatotoxicity associated with AAED might be mediated by the oxidative stress induced by the drugs metabolites. (C) 2008 Elsevier Ltd. All rights reserved

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.

Iron chelating-mediated antioxidant activity of Plectranthus barbatus extract on mitochondria

Plectranthus barbatus Andrews (Lamiaceae) is a popular medicinal plant used to treat gastrointestinal and hepatic ailments. In this work, we assessed the antioxidant activity of the aqueous extract of P. barbatus leaves on Fe(2+)-citrate-mediated membrane lipid peroxidation in isolated rat liver mitochondria, as well in non-mitochondrial systems: DPPH reduction, (center dot)OH scavenging activity, and iron chelation by prevention of formation of the Fe(2+)-bathophenanthroline disulfonic acid (BPS) complex. Within all the tested concentrations (15-75 mu g/ml), P. barbatus extract presented significant free radical-scavenging activity (IC(50) = 35.8 +/- 0.27 mu g/ml in the DPPH: assay and IC(50) = 69.1 +/- 0.73 mu g/ml in the (center dot)OH assay) and chelated iron (IC(50) = 30.4 +/- 3.31 mu g/ml). Over the same concentration range, the plant extract protected mitochondria against Fe(2+)/citrate-mediated swelling and malondialdehyde production, a property that persisted even after simulation of its passage through the digestive tract. These effects could be attributed to the phenolic compounds, nepetoidin - caffeic acid esters, present in the extract. Therefore, P. barbatus extract prevents mitochondrial membrane lipid peroxidation, probably by chelation of iron, revealing potential applicability as a therapeutic source of molecules against diseases involving mitochondrial iron overload. (C) 2010 Elsevier Ltd. All rights reserved.

Pre and post-natal exposure to ambient level of air pollution impairs memory of rats: the role of oxidative stress

The aims of this study were to evaluate whether air pollution during pre-natal and post-natal phases change habituation and short-term discriminative memories and if oxidants are involved in this process. As secondary objectives, it was to evaluate if the change of filtered to nonfiltered environment could protect the cortex of rats against oxidative stress as well as to modify the behavior of these animals. Wistar, male rats were divided into four groups (n = 12/group): pre and post-natal exposure until adulthood to filtered air (FA); pre-natal period to nonfiltered air (NFA-FA); until (21st post-natal day) and post-natal to filtered air until adulthood (PND21); prenatal to filtered air until PND21 and post-natal to nonfiltered air until adulthood (FA-NFA); pre and post-natal to nonfiltered air (NFA). After 150 days of air pollution exposure, animals were tested in the spontaneous object recognition test to evaluate short-term discriminative and habituation memories. Rats were euthanized; blood was collected for metal determination; cortex dissected for oxidative stress evaluation. There was a significant increase in malondialdehyde (MDA) levels in the NFA group when compared to other groups (FA: 1.730 +/- 0.217; NFA-FA: 1.101 +/- 0.217; FA-NFA: 1.014 +/- 0.300; NFA: 5.978 +/- 1.920 nmol MDA/mg total proteins; p = 0.007). NFA group presented a significant decrease in short-term discriminative (FA: 0.603 +/- 0.106; NFA-FA: 0.669 +/- 0.0666; FA-NFA: 0.374 +/- 0.178; NFA: -0.00631 +/- 0.106 sec; p = 0.006) and an improvement in habituation memories when compared to other groups. Therefore, exposure to air pollution during both those periods impairs short-term discriminative memory and cortical oxidative stress may mediate this process.

Mercury exposure and oxidative stress in communities of the Brazilian Amazon

This study was designed to assess possible associations between biomarkers of mercury (Hg) exposure and oxidative stress in fish-eating Amazonian communities. Clinical samples were obtained from riparians living in the Brazilian Amazon. Biomarkers of oxidative stress (glutathione - GSH, glutathione peroxidase - GSH-Px, catalase - CAT, activity and reactivation index of delta-aminolevulinate dehydratase - ALA-D (R%) were determined in blood. Total Hg was measured in whole blood (B-Hg), plasma (P-Hg) and hair (H-Hg). Association between biomarkers of Hg exposure and oxidative stress were examined using multiple regression models, including age, gender, alcohol consumption, smoking status, fish consumption and then stratified for gender. Significant inverse relations were observed between GSH-Px, GSH, CAT, ALA-D activity and B-Hg or H-Hg (p<0.05). ALA-D reactivation index was positively related to B-Hg (p<0.0001). P-Hg was directly related to ALA-D reactivation index and inversely associated with GSH-Px, GSH, and ALA-D activity (p<0.05). When stratified for gender, women showed significant inverse associations between all biomarkers of Hg exposure and CAT (p<0.05) or GSH (p<0.05), while for men only P-Hg showed a significant inverse relation with GSH (p<0.001). Our results clearly demonstrated an association between Hg exposure and oxidative stress. Moreover, for B-Hg, P-Hg and H-Hg gender differences were present. (C) 2009 Elsevier B.V. All rights reserved.