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Background: Adherence to a Mediterranean diet (MedDiet) is thought to reduce liver steatosis., Objectives: To explore the associations with liver steatosis of 3 different diets: a MedDiet + extra-virgin olive oil (EVOO), MedDiet + nuts, or a control diet., Methods: This was a subgroup analysis nested within a multicenter, randomized, parallel-group clinical trial, PREvención con DIeta MEDiterránea (PREDIMED trial: ISRCTN35739639), aimed at assessing the effect of a MedDiet on the primary prevention of cardiovascular disease. One hundred men and women (mean age: 64 ± 6 y), at high cardiovascular risk (62% with type 2 diabetes) from the Bellvitge-PREDIMED center were randomly assigned to a MedDiet supplemented with EVOO, a MedDiet supplemented with mixed nuts, or a control diet (advice to reduce all dietary fat). No recommendations to lose weight or increase physical activity were given. Main measurements were the percentage of liver fat and the diagnosis of steatosis, which were determined by NMR imaging. The association of diet with liver fat content was analyzed by bivariate analysis after a median follow-up of 3 y., Results: Baseline adiposity and cardiometabolic risk factors were similar among the 3 treatment arms. At 3 y after the intervention hepatic steatosis was present in 3 (8.8%), 12 (33.3%), and 10 (33.3%) of the participants in the MedDiet + EVOO, MedDiet + nuts, and control diet groups, respectively (P = 0.027). Respective mean values of liver fat content were 1.2%, 2.7%, and 4.1% (P = 0.07). A tendency toward significance was observed for the MedDiet + EVOO group compared with the control group. Median values of urinary 12(S)-hydroxyeicosatetraenoic acid/creatinine concentrations were significantly (P = 0.001) lower in the MedDiet + EVOO (2.3 ng/mg) than in the MedDiet + nuts (5.0 ng/mg) and control (3.9 ng/mg) groups. No differences in adiposity or glycemic control changes were seen between groups., Conclusions: An energy-unrestricted MedDiet supplemented with EVOO, a food with potent antioxidant and anti-inflammatory properties, is associated with a reduced prevalence of hepatic steatosis in older individuals at high cardiovascular risk., (Copyright © American Society for Nutrition 2019.)

Sofrito is a mix of tomato, onion, garlic, and olive oil, which contains phenolic compounds and carotenoids. Consumption of tomato-based sofrito has been related to a lower risk of cardiovascular events, but the mechanisms behind such beneficial effects remain unclear. This study aimed to analyze the effects of representative sofrito compounds such as naringenin, hydroxytyrosol, lycopene, and β-carotene on mechanisms involved in the pathogenesis of atherosclerosis. We demonstrated that both phenolic compounds and both carotenoids studied were able to inhibit low density lipoproteins (LDL) oxidation, as well as oxidative stress and eicosanoid production induced by oxidized LDL (oxLDL) in macrophage cultures. These effects were not the consequences of disturbing oxLDL uptake by macrophages. Finally, we observed an additive effect of these sofrito compounds, as well as the activity of a main naringenin metabolite, naringenin 7- O -β-d-glucuronide on LDL oxidation and oxidative stress.

Purpose: Serum nitric oxide (NO) reduction and increased endothelin-1 (ET-1) play a pivotal role in endothelial dysfunction and hypertension. Considering that traditional Mediterranean diet (TMD) reduces blood pressure (BP), the aim of this study was to analyze whether TMD induced changes on endothelial physiology elements such as NO, ET-1 and ET-1 receptors which are involved in BP control., Methods: Non-smoking women with moderate hypertension were submitted for 1 year to interventions promoting adherence to the TMD, one supplemented with extra virgin olive oil (EVOO) and the other with nuts versus a control low-fat diet (30 participants/group). BP, NO, ET-1 and related gene expression as well as oxidative stress biomarkers were measured., Results: Serum NO and systolic BP (SBP) or diastolic BP (DBP) were negatively associated at baseline, as well as between NO and ET-1. Our findings also showed a DBP reduction with both interventions. A negative correlation was observed between changes in NO metabolites concentration and SBP or DBP after the intervention with TMD + EVOO (p = 0.033 and p = 0.044, respectively). SBP reduction was related to an impairment of serum ET-1 concentrations after the intervention with TMD + nuts (p = 0.008). We also observed changes in eNOS, caveolin 2 and ET-1 receptors gene expression which are related to NO metabolites levels and BP., Conclusions: The changes in NO and ET-1 as well as ET-1 receptors gene expression explain, at least partially, the effect of EVOO or nuts on lowering BP among hypertensive women.

Epidemiological and clinical studies have reported that olive oil reduces the incidence of cardiovascular disease. However, the mechanisms involved in this beneficial effect have not been delineated. The endothelium plays an important role in blood pressure regulation through the release of potent vasodilator and vasoconstrictor agents such as nitric oxide (NO) and endothelin-1 (ET-1), respectively, events that are disrupted in type 2 diabetes. Extra virgin olive oil contains polyphenols, compounds that exert a biological action on endothelial function. This study analyzes the effects of olive oil polyphenols on endothelial dysfunction using an in vitro model that simulates the conditions of type 2 diabetes. Our findings show that high glucose and linoleic and oleic acids decrease endothelial NO synthase phosphorylation, and consequently intracellular NO levels, and increase ET-1 synthesis by ECV304 cells. These effects may be related to the stimulation of reactive oxygen species production in these experimental conditions. Hydroxytyrosol and the polyphenol extract from extra virgin olive oil partially reversed the above events. Moreover, we observed that high glucose and free fatty acids reduced NO and increased ET-1 levels induced by acetylcholine through the modulation of intracellular calcium concentrations and endothelial NO synthase phosphorylation, events also reverted by hydroxytyrosol and polyphenol extract. Thus, our results suggest a protective effect of olive oil polyphenols on endothelial dysfunction induced by hyperglycemia and free fatty acids., (© 2014 Published by Elsevier Ltd.)

Objectives: TNFRSF1A is involved in an autosomal dominant autoinflammatory disorder called TNFR-associated periodic syndrome (TRAPS). Most TNFRSF1A mutations are missense changes and, apart from those affecting conserved cysteines, their deleterious effect remains often questionable. This is especially true for the frequent R92Q mutation, which might not be responsible for TRAPS per se but represents a susceptibility factor to multifactorial inflammatory disorders. This study investigates TRAPS pathophysiology in a family exceptional by its size (13 members) and compares the consequences of several mutations affecting arginine 92., Methods: TNFRSF1A screening was performed by PCR-sequencing. Comparison of the 3-dimensional structure and electrostatic properties of wild-type and mutated TNFR1 proteins was performed by in silico homology modeling. TNFR1 expression was assessed by FACS analysis, western blotting and ELISA in lysates and supernatants of HEK293T cells transiently expressing wild-type and mutated TNFR1., Results: A TNFRSF1A heterozygous missense mutation, R92W (c.361C>T), was shown to perfectly segregate with typical TRAPS manifestations within the family investigated (p<5.10(-4)). It was associated with very high disease penetrance (0.9). Prediction of its impact on the protein structure revealed local conformational changes and alterations of the receptor electrostatic properties. R92W also impairs the TNFR1 expression at the cell surface and the levels of soluble receptor. Similar results were obtained with R92P, another mutation previously identified in a very small familial form with incomplete penetrance and variable expressivity. In contrast, TNFR1-R92Q behaves like the wild-type receptor., Conclusions: These data demonstrate the pathogenicity of a mutation affecting arginine 92, a residue whose involvement in inflammatory disorders is deeply debated. Combined with previous reports on arginine 92 mutations, this study discloses an unusual situation in which different amino acid substitutions at the same position in the protein are associated with a clinical spectrum bridging Mendelian to multifactorial conditions.

Background & Aims: Metabolic syndrome (MetS), in which a non-classic feature is an increase in systemic oxidative biomarkers, presents a high risk of diabetes and cardiovascular disease (CVD). Adherence to the Mediterranean Diet (MedDiet) is associated with a reduced risk of MetS. However, the effect of the MedDiet on biomarkers for oxidative damage has not been assessed in MetS individuals. We have investigated the effect of the MedDiet on systemic oxidative biomarkers in MetS individuals., Methods: Randomized, controlled, parallel clinical trial in which 110 female with MetS, aged 55-80, were recruited into a large trial (PREDIMED Study) to test the efficacy of the traditional MedDiet on the primary prevention of CVD. Participants were assigned to a low-fat diet or two traditional MedDiets (MedDiet + virgin olive oil or MedDiet + nuts). Both MedDiet group participants received nutritional education and either free extra virgin olive oil for all the family (1 L/week), or free nuts (30 g/day). Diets were ad libitum. Changes in urine levels of F2-Isoprostane (F2-IP) and the DNA damage base 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) were evaluated at 1-year trial., Results: After 1-year urinary F2-IP decreased in all groups, the decrease in MedDiet groups reaching a borderline significance versus that of the Control group. Urinary 8-oxo-dG was also reduced in all groups, with a higher decrease in both MedDiet groups versus the Control one (P < 0.001)., Conclusions: MedDiet reduces oxidative damage to lipids and DNA in MetS individuals. Data from this study provide evidence to recommend the traditional MedDiet as a useful tool in the MetS management., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Aims of the Study: Wine polyphenols attenuate the development of atherosclerosis, which involves an inflammatory process. We studied the beneficial effect of de-alcoholised white and red wines (DWW and DRW, respectively) on the development of atheroma plaques and on the expression of biomarkers., Methods: We administered control or de-alcoholised wine-rich diets to apoE-deficient mice for 12 or 20 weeks. We then used optical microscopy or immunofluorescence to examine atherosclerotic lesion development in the thoracic aorta and aortic root and assessed the presence of cytokines and adhesion molecules by qPCR and immunofluorescence in total aorta and aortic root, respectively., Results: Atherosclerotic lesions in thoracic aorta were significantly decreased in mice supplemented with DWW (30 %) and DRW (62 %) for 20 weeks. In addition, the expressions of interferon-γ, interleukin-1β, the monocyte chemoattractant protein-1 and CD68 were reduced by DRW. The adhesion molecule P-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 were decreased by 52, 76 and 45 %, respectively, in mice fed DRW for 12 weeks, whereas DWW reduced these parameters in a minor extent. The NF-κB expression in total aorta was significantly decreased in the mice treated with de-alcoholised wines for 12 weeks., Conclusions: DRW is shown to be more effective than DWW on cytokines and adhesion molecule expression, in the early stages of the inflammatory events associated with atherosclerosis development, probably due to the high phenolic content of red wine. Downregulation of NF-κB expression may be involved in the mechanism by which de-alcoholised wines modulate atherosclerosis.

Redox state unbalance and the activation of the arachidonic acid (AA) cascade, contribute to the pathogenesis of cardiovascular disease (CVD) and cancer. Inflammatory cells that infiltrate the atheroma plaque or tumor are a major source of reactive oxygen species and eicosanoids. The human antioxidant defense network is complex and interlocking and there is controversy surrounding the beneficial effects of diet-derived antioxidants in vivo. However, epidemiological studies indicate that populations that consume high levels of plant-derived foods containing phenolic compounds have low rates of CVD and cancer. The molecular mechanisms for these effects are multi-faceted. They include the regulation of transcription factors and consequently the modulation of genes (cytokines, growth factors and adhesion molecules), and growth factor-receptor interactions and cell signaling cascades, which determine the expression of genes involved in cell cycle, cell survival and apoptosis, as well as adhesiveness/invasiveness and angiogenesis. The present paper also focuses on the effects of phenolic compounds on AA cascade enzymes (cyclooxygenases and lipoxygenases) and the subsequent synthesis of eicosanoids, which are involved in CVD and cancer. A better understanding of these processes could explain the beneficial effects of polyphenols on the most prevalent diseases in Western societies. This commentary shows that antioxidants under evaluation include structural modifications of low-molecular-mass polyphenols, which could lead to a valuable strategy for modulating the generation of inflammatory mediators involved in these chronic diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Polyphenols exert beneficial effects in atherosclerosis. The crucial step in atherosclerosis is the recruitment of monocytes to the subendothelial space, induced by endothelial adhesion molecules through the activation of factors such as NF-κB. We studied the effect of a dealcoholised lager beer (DLB) and a dealcoholised dark beer (DDB) on atherosclerotic lesions, and the underlying mechanisms. Dealcoholised beers were administered in the diet (42 ml/kg body weight per d) to 4-week-old male apoE knockout (apoE - / - ) mice for 20 weeks. The atherosclerotic lesions in the thoracic aorta were reduced by 44 % (P = 0·003) and 51 % (P < 0·001) in DLB- and DDB-treated mice, respectively. Also, the mRNA expressions of the endothelial adhesion molecules in the total aorta were decreased: P-selectin showed a 17 % (P = 0·004) reduction in DDB-treated mice; vascular cell adhesion molecule-1 (VCAM-1) was decreased by 20 % (P = 0·012) and 32 % (P = 0·001) in DLB- and DDB-treated mice, respectively; intercellular adhesion molecule-1 (ICAM-1) showed a 14 % (P = 0·014) reduction in DLB-treated mice. The protein expressions of these molecules and NF-κB were studied in the aortic root. P-selectin was decreased by 37 % (P = 0·012) in DDB-treated mice; VCAM-1 was reduced by 48 % (P = 0·001) and 54 % (P < 0·001) in DLB- and DDB-treated mice, respectively; ICAM-1 was decreased by 25 % (P = 0·028) and 30 % (P = 0·018) in DLB- and DDB-treated mice, respectively; NF-κB was reduced by 46 % (P = 0·042) in DDB-treated mice. In conclusion, dealcoholised beers protected apoE - / -  mice against atherosclerosis, through the modulation of endothelial adhesion molecules, possibly induced by NF-κB.

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F(2)-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Background: Rhizophora mangle (L.) produce a variety of substances that possesses pharmacological actions. Although it shown antioxidant properties in some assays, there is no available information about its effect on some free radical species. So the objective of the present research is to evaluate the DPPH radical and superoxide anion scavenging properties of R. mangle extract and its polyphenol fraction., Methods: Rhizophora mangle (L.) bark aqueous extract and its major constituent, polyphenols fraction, were investigated for their antioxidant activities employing 2 in vitro assay systems: 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radicals scavenging., Results: IC(50) for DPPH radical-scavenging activity was 6.7 µg tannins/mL for extract and 7.6 µg tannins/mL for polyphenolic fraction. The extract showed better activity than its fraction (P < 0.05) in the DPPH radicals reducing power. Polyphenolic fraction exhibited better superoxide anion scavenging ability (IC(50) = 21.6 µg tannins/mL) than the extract (IC(50) = 31.9 µg tannins/mL). Antioxidant activities of both samples increased with the rise of tannins concentration. The comparison of regression lines showed significant differences (P < 0.05) between extract and its polyphenolic fraction in both assays, indicating that extract was more effective in DPPH radical scavenging than its fraction at tannin concentrations below the crossing point of both lines, while that fraction was more effective than extract inhibiting the superoxide anions generation., Conclusions: R. mangle aqueous extract showed a potent antioxidant activity, achieved by the scavenging ability observed against DPPH radicals and superoxide anions. Regarding its polyphenolic composition, the antioxidant effects observed in this study are due, most probably, to the presence of polyphenolic compounds.

Aim: To examine the relevance of hypoxia inducible factor (HIF-1) and nitric oxide (NO) on the preservation of fatty liver against cold ischemia-reperfusion injury (IRI)., Methods: We used an isolated perfused rat liver model and we evaluated HIF-1alpha in steatotic and non-steatotic livers preserved for 24 h at 4 degrees C in University of Wisconsin and IGL-1 solutions, and then subjected to 2 h of normothermic reperfusion. After normoxic reperfusion, liver enzymes, bile production, bromosulfophthalein clearance, as well as HIF-1alpha and NO [endothelial NO synthase (eNOS) activity and nitrites/nitrates] were also measured. Other factors associated with the higher susceptibility of steatotic livers to IRI, such as mitochondrial damage and vascular resistance were evaluated., Results: A significant increase in HIF-1alpha was found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage. Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters. These benefits were enhanced by the addition of trimetazidine (an anti-ischemic drug), which induces NO and eNOS activation, to IGL-1 solution. In normoxic reperfusion, the presence of NO favors HIF-1alpha accumulation, promoting also the activation of other cytoprotective genes, such as heme-oxygenase-1., Conclusion: We found evidence for the role of the HIF-1alpha/NO system in fatty liver preservation, especially when IGL-1 solution is used.

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE(-/-) mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O(2)(.-)) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22(phox) expression and O(2)(.-)production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.

Here we examine the effect of adding carvedilol (CVD) to University of Wisconsin (UW) solution on the preservation of steatotic and nonsteatotic livers during cold ischemia and after normothermic reperfusion. We used an isolated perfused rat liver model. The following protocols were evaluated. Protocol 1 concerned the effect of CVD after cold ischemia. Steatotic and nonsteatotic livers were preserved for 24 hours in UW solution alone or with CVD. Livers without cold ischemia were used as controls. Transaminases were evaluated in the flushing effluent. Protocol 2 involved the effect of CVD after reperfusion. Both liver types were preserved for 24 hours in UW solution alone or with CVD and then perfused ex vivo for 2 hours at 37 degrees C. Livers flushed and perfused without ischemia were used as controls. Hepatic injury and functionality [transaminases, bile production, and hepatic clearance of sulfobromophthalein (BSP)] were evaluated after reperfusion. In addition, factors potentially involved in hepatic ischemia-reperfusion injury, including oxidative stress (malondialdehyde and superoxide anion levels), mitochondrial damage (glutamate dehydrogenase activity), microcirculatory disorders (flow rate and vascular resistance), and adenosine triphosphate (ATP) depletion, were evaluated after reperfusion. After cold ischemia, steatotic livers preserved in UW solution showed higher transaminase levels than nonsteatotic livers. After reperfusion, steatotic livers preserved in UW solution showed higher transaminase levels and lower bile production and BSP clearance than nonsteatotic livers. Alterations in the perfusion flow rate and vascular resistance, mitochondrial damage, and reduced ATP content were more evident in steatotic livers preserved in UW solution. The addition of CVD to UW solution reduced hepatic injury, obstructed its mechanisms, and improved hepatic functionality in both liver types. We conclude that CVD is a useful additive for UW solution that improves the preservation of steatotic and nonsteatotic livers subjected to prolonged cold ischemia.

Oxidative stress plays a key role in the male reproductive function. Differences between rabbit breeds have been found for testis size, seminiferous tubule diameter, number and size of interstitial and germ cells, etc. Traits related to the redox system could also be affected by genetic factors. It is likely that differences between breeds for these traits would lead to differences in reproductive maturation and fertility. We have investigated in the present paper the age-related changes of the plasma testosterone (TST) concentration, superoxide anion (O(2)(-)) radical formation, superoxide dismutase (SOD) activity, catalase (CAT) activity and thiobarbituric acid-reactive substances (TBARs) level in testis of rabbits in two breeds selected for different aptitudes (Caldes for growth rate and Prat for litter size). The effect of birth season for these traits was also assessed. Major changes in parameters related to oxidative stress were observed at an early age and most probably can be explained by the concomitant changes in testicular structure and function. Both lines showed similar developmental profiles and levels for all the variables studied. There was no interaction between line and birth season, consequently environmental conditions affected both lines in the same manner. Significant differences between males born in different seasons were found for O(2)(-) (4.84+/-0.19RLU/mg tissuemin versus 5.67+/-0.19RLU/mg tissuemin), SOD (6.12+/-0.11U/mg protein versus 7.09+/-0.11U/mg protein) and CAT (0.058+/-0.002K/mg protein versus 0.040+/-0.002K/mg protein). Future studies should take into account differences between seasons for a more precise analysis.

Background/objectives: The aim of this study was to analyze the influence of a Mediterranean dietary pattern on plasma total antioxidant capacity (TAC) after 3 years of intervention and the associations with adiposity indexes in a randomized dietary trial (PREDIMED trial) with high cardiovascular risk patients., Subjects/methods: 187 subjects were randomly selected from the PREDIMED-UNAV center after they completed 3-year intervention program. Participants were following a Mediterranean-style diet with high intake of virgin olive oil or high intake of nuts, or a conventional low-fat diet. Adiposity indexes were measured at baseline and at year 3. Plasma TAC was evaluated using a commercially available colorimetric assay kit., Results: Plasma TAC in the control, olive oil and nuts groups was 2.01+/-0.15, 3.51+/-0.14 and 3.02+/-0.14 mM Trolox, respectively after adjusting for age and sex. The differences between the Mediterranean diet and control groups were statistically significant (P<0.001). Moreover higher levels of TAC were significantly associated with a reduction in body weight after 3 years of intervention among subjects allocated to the virgin olive oil group (B=-1.306; 95% CI=-2.439 to -0.173; P=0.025, after adjusting for age, sex and baseline body mass index)., Conclusions: Mediterranean diet, especially rich in virgin olive oil, is associated with higher levels of plasma antioxidant capacity. Plasma TAC is related to a reduction in body weight after 3 years of intervention in a high cardiovascular risk population with a Mediterranean-style diet rich in virgin olive oil.

Objectives: To evaluate the presence of oxidative stress and alterations in the levels of two cytoprotective agents, prostaglandin E2 and nitric oxide, in the gastrointestinal tract of aging rats., Methods: The production of superoxide anion, lipid peroxides, levels of superoxide dismutase and catalase, and production of prostaglandin E2 and nitric oxide in the stomach and duodenum of rats were determined at 1.5, 3, 12, 18 and 24 months of age., Key Findings: Oxidative stress was present in the stomach of the old rats (24 months), whereas prostaglandin E2 and nitric oxide production remained stable at 18 and 24 months. In the duodenum, no oxidative stress was observed at 24 months, but at 18 months, an increase in superoxide anion levels was detected. Prostaglandin E2 remained constant in the aged rats but nitric oxide decreased significantly at 24 months., Conclusions: The absence of macroscopic gastric injury throughout the gastrointestinal tract indicates that the oxidative stress in the stomach and the significant decrease of nitric oxide in the duodenum in the old rats are not sufficient to disrupt the mucosal defence network. The results support the notion that the disruption of the mucosal network is essentially regulated by the cytoprotective agents prostaglandin E2 and nitric oxide, and that injury appears only when both substances are concurrently reduced.

Dietary intake of long-chain n-3 polyunsaturated fatty acids (PUFA) reduces the risk for atherosclerosis. Here we examine the effect of a fish oil (FO)-rich diet on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. Mice were fed semi-purified diets containing 5% corn oil (CO), rich in n-6 PUFA or menhaden oil as FO, rich in long-chain n-3 PUFA and 0.15% cholesterol after reaching 4 weeks of age, and they were killed when they were 4 weeks, 12 weeks, 18 weeks or 24 weeks old. Oxidative stress in plasma and aortic tissue was not increased in mice fed the FO-rich diet, despite its high peroxidizability index. A reduction of stenosis and intrusion at the aortic root, a decrease in the surface area of atherosclerotic lesions at the aorta and a decrease in P-selectin, vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression were observed in FO-fed mice compared to CO-fed mice. It seems likely that the reduced expression of VCAM-1 and ICAM-1 could be transcriptionally regulated by nuclear factor-kappaB in the aortic root. The protective effect of FO against atherosclerosis was more evident at early ages. In conclusion, FO reduces adhesion molecule expression in lesions in apoE(-/-) mice. Because these molecules are involved in lesion progression the effect of FO may explain the observed decrease in atherogenesis.

The consumption of seaweeds has increased in recent years. However, their adverse and beneficial effects have scarcely been studied. Two extracts from the brown seaweed Fucus vesiculosus containing 28.8% polyphenols or 18% polyphenols plus 0.0012% fucoxanthin have been obtained and studied to determine their toxicity in mice and rats and also their antioxidant activity. Both extracts were shown to lack any relevant toxic effects in an acute toxicity test following a 4 week daily treatment in rats. The extracts exhibited antioxidant activity in noncellular systems and in activated RAW 264.7 macrophages, as well as in ex vivo assays in plasma and erythrocytes, after the 4 week treatment in rats. Our ex vivo results indicated that compounds from extract 2 may be more easily absorbed and that the antioxidants in their parent or metabolized form are more active. These findings support the view that the daily consumption of F. vesiculosus extract 2 (Healsea) would have potential benefits to humans.

In vitro experiments have demonstrated that polyphenols exhibit antioxidant and anti-inflammatory activities. The present study was designed to test whether dealcoholized red (DRW) and white (DWW) wines can decrease the oxidative stress associated with inflammation in vivo. Rats were fed for 15 d either a control diet or one supplemented with DRW or DWW. Finally, a granuloma was induced by subcutaneous administration of carrageenan. Although DRW showed higher antioxidant activity in vitro than DWW, both wines decreased the number of cells recruited into the granuloma pouch. Malondialdehyde decreased in plasma and inflammatory exudate from rats fed with DRW- and DWW-rich diets. Moreover, the concentration of NO increased in exudate, which correlates with the increase in the citrulline:arginine ratio. Polymorphonuclear leucocytes from the inflammatory exudate of rats fed dealcoholized wines showed decreased superoxide anion (O*2) production and increased NO production ex vivo. This change in NO production resulted from increased expression and activity of inducible NO synthase (EC 1.14.13.39). Moreover, the up regulation of cyclo-oxygenase-2 (EC 1.14.99.1) protein expression observed in rats fed the DRW-rich diet was not related to a direct effect of NO. The present results indicate that the non-alcoholic compounds of wines not only improve antioxidant status in an inflammatory situation, but also limit cell infiltration, possibly through a decrease in O*2 and an increase in NO production.

Cells are armed with a vast repertoire of antioxidant defence mechanisms to prevent the accumulation of oxidative damage. The cellular adaptive response is an important antioxidant mechanism against physiological and pathophysiological oxidative alterations in a cell's microenvironment. The aim of this paper was to study, in the rat aorta, whether this adaptive response and the inflammation associated with oxidative stress were expressed throughout the aging process. We examined the rat aorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12, 18 and 24 months of age were used. Superoxide anion (O2(-)) generation; levels of two antioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels of prostaglandin E2 (PGE2), an inflammatory marker, were measured. The results for rats at different ages were compared with those for 3 months of age. A balance between production of O2(-) and SOD activity was found in the aorta of rats from 1.5 to 12 months old. Oxidative stress was present in the aorta of old animals (18-24 months), due to a failure in the mechanisms of adaptation to oxidative stress. The observed increase in PGE2 levels in these rats reflected an inflammatory response. All together suggest that vascular oxidative stress and the inflammatory process observed in the old groups of rats could be closely related to vascular aging. Our results also remark the importance of the adaptative response to oxidative stress.

Flavonoids are ingested with vegetables and beverages and exert a beneficial effect on cardiovascular disease. Studies in animals in vitro and in humans ex vivo on the resistance of lipoproteins to oxidation are not consistent and the mechanisms by which flavonoids protect against atherosclerosis are a matter of debate. In the present study, we investigated the effects of administering diets containing 0.3% (wt/wt) quercetin, 0.3% (wt/wt) catechin, or 35% (vol/wt) dealcoholated red wine (DRW) for 10 days in healthy rats on markers of oxidative damage in lipoproteins and in plasma. The antioxidant levels in low-density lipoproteins (LDL) or the lag phase, oxidation rate, and maximum level of conjugated dienes during ex vivo LDL oxidation did not differ between control and treated rats. Plasma levels of alpha-tocopherol and retinol were similar in all groups. The total antioxidant status of the plasma from rats fed either quercetin or DRW diet was higher than in control rats. Only glucuronide and sulfate compounds of quercetin were detected in plasma from rats fed the quercetin-rich diet, and no flavonoids or their metabolites were detected in plasma or LDL from rats fed the catechin- or the DRW-rich diet. No significant differences in malondialdehyde or in conjugated dienes in plasma were observed. These results indicate that although metabolites from quercetin are present in plasma, they are not detected in lipoproteins and do not modify the level of other antioxidants. In conclusion, in the absence of any pathology or of oxidative stress the intake of quercetin, catechin, or DRW did not protect lipoproteins from oxidation ex vivo.

A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the.NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular.NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, l-arginine, and l-citrulline were also evaluated in homogenates. Superoxide anion (O(2)(-).) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%);.NO production increased by 90%, cGMP levels increased by 100%, and l-arginine decreased by 30%. No change in aortic O(2)(-). production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), the neuronal blocker tetrodotoxin (TTX, 10(-6) M), or both on spontaneous contractions and on the production of basal NO. Both L-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by L-NAME and was inhibited by 38% by TTX. When tested together, L-NAME in the presence of TTX or TTX in the presence of L-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.

Inflammatory disorders are associated with a decrease in iron absorption because of the blockade of iron in storage organs. Fish oils, rich in n-3 PUFA, are used to prevent and treat inflammatory disorders and increase iron absorption in noninflamed rats. Here we examine whether n-3 PUFA prevent inflammation-related hypoferremia. Two groups of rats were fed isoenergetic semipurified diets (a standard diet and a sardine oil-rich diet). A carrageenan granuloma was induced in half of the rats of each dietary group. Ferrokinetic studies using 59Fe, hematological analyses, and iron store evaluation were performed in noninflamed and inflamed rats. Although dietary n-3 PUFA increased 59Fe absorption in carageenan-treated rats, they did not restore the hypoferremia associated with inflammation, which is due to several mechanisms. Among these mechanisms, the blockade of iron in storage organs is relevant. However, this blockade was less evident in the spleen of inflamed rats fed the sardine oil diet.

Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene and loss of function in the extremities. It results from an excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall. Atherosclerotic lesions develop fundamentally in three stages: dysfunction of the vascular endothelium, fatty streak formation and fibrous cap formation. Each stage is regulated by the action of vasoactive molecules, growth factors and cytokines. This multifactorial etiology can be modulated through the diet. The degree of unsaturation of dietary fatty acids affects lipoprotein composition as well as the expression of adhesion molecules and other pro-inflammatory factors, and the thrombogenicity associated with atherosclerosis development. Thus, the preventive effects of a monounsaturated-fatty acid-rich diet on atherosclerosis may be explained by the enhancement of high-density lipoprotein-cholesterol levels and the impairment of low-density lipoprotein-cholesterol levels, the low-density lipoprotein susceptibility to oxidation, cellular oxidative stress, thrombogenicity and atheroma plaque formation. On the other hand, the increase of high-density lipoprotein cholesterol levels and the reduction of thrombogenicity, atheroma plaque formation and vascular smooth muscle cell proliferation may account for the beneficial effects of polyunsaturated fatty acid on the prevention of atherosclerosis. Thus, the advantages of the Mediterranean diet rich in olive oil and fish on atherosclerosis may be due to the modulation of the cellular oxidative stress/antioxidant status, the modification of lipoproteins and the down-regulation of inflammatory mediators.

Many clinical studies have indicated that diets rich in fish oil (FO) reduce the risk of cardiovascular disease and have anti-inflammatory and antithrombotic properties. Although the therapeutic effects of FO have been well described, their impact on iron metabolism remains unclear. The aim of this work was to study the activity and expression of IRP1 in the liver and the spleen of rats fed FO-rich diets with 0 (FO-0) or 100 (FO-1) mg/kg of all-rac-alpha-tocopherol acetate. We also measured nonheme iron, alpha-tocopherol and retinol concentrations, and superoxide (SOD) and catalase activity in these organs. Rats fed FO were compared to rats fed a corn oil (CO)-rich diet with 100 mg/kg all-rac-alpha-tocopherol acetate. The activity and expression of IRP1 in both the liver and the spleen of rats fed FO diets were greater than in those fed the CO diet. FO-fed rats also had lower nonheme iron concentrations in these organs. Hepatic alpha-tocopherol and retinol concentrations and SOD activity were lower in FO-0-fed rats compared to those fed the CO diet. In the spleen, alpha-tocopherol and retinal concentrations were not altered but SOD activity was lower in FO-0- fed rats, whereas catalase activity was greater than in rats fed CO. The results indicate that there is an increase in oxidative stress in the liver and in the spleen of rats fed FO diets. These changes, together with the reduction of nonheme iron concentrations in both FO-0- and FO-1-fed rats, may explain the increase in activity and expression of IRP1. Therefore, the ingestion of FO-rich diets should be monitored under close supervision.

The objective of the present study was to examine the effects of fish oil (FO)- and olive oil (OO)-rich diets on Fe metabolism and oxidative stress. Rats were fed for 16 weeks with diets containing 50 g lipids/kg; either OO, maize oil (MO) or FO. OO or MO diets contained a standard amount (100 mg/kg) of all-rac-alpha-tocopheryl acetate. FO diets were supplemented with 0, 100 or 200 mg all-rac-alpha-tocopheryl acetate/kg (FO-0, FO-1 or FO-2 diets, respectively). At the end of the feeding period, we measured non-haem Fe stores in liver and spleen, and erythrocyte and reticulocyte count. We also determined antioxidants and products derived from lipid peroxidation in plasma and erythrocytes. Our results showed reduced non-haem Fe stores in rats fed any of the FO diets. Reticulocyte percentage was higher in the rats fed FO-0 and FO-1. Plasma alpha-tocopherol was very low in rats fed the FO-0 diet. Rats fed the FO-1 and FO-2 diets showed higher alpha-tocopherol in plasma than the FO-0 group but lower than the MO or OO groups. We did not observe such differences in the alpha-tocopherol content in erythrocyte membranes. Superoxide dismutase and glutathione peroxidase activities were lower in the erythrocytes of rats fed the FO-0 diet. The products derived from lipid peroxidation were also higher in the FO groups. The administration of FO-rich diets increased lipid peroxidation and affected Fe metabolism. On the other hand, the OO-rich diet did not increase oxidative stress and did not alter Fe metabolism. Based on these results, we conclude that FO supplementation should be advised carefully.

Previous research has described how high cellular metabolism creates an acidic environment in inflammatory cells during respiratory burst. The aim of our work was to describe the acid-base dependence of exudate in superoxide (O2.-) and nitric oxide (NO.) generation by inflammatory cells from a carrageenan-granuloma. Although the carrageenan solution was alkaline (pH 7.74 when equilibrated with air) the exudate showed an acidification that stabilised at around 7 units of pH. A notable hypercapnia, but not hypoxia, was found in the exudate at up to 24 h. The effect of extracellular acidosis on O2.- and NO. production by inflammatory cells was also studied. The maximum O2.- production and the lowest levels of NO. were found at pH 7, which was closer to the pH of the granuloma-pouch. These results suggest that experiments with inflammatory cells ex vivo should be carried out at an identical pH to that found in vivo in order to reproduce the physiological mechanisms of free radical generation during inflammatory processes.

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.

Fish oil has a preventive role in cardiovascular and inflammatory diseases, but little is known about the effect of olive oil, which is widely consumed in Mediterranean regions. We examined the influence of dietary olive oil, corn oil and fish oil-rich diets on the production of superoxide anion (O2-) and nitric oxide (.NO) by resident macrophages stimulated by phorbol 12-myristate 13-acetate (PMA) and their effect on arachidonic acid release, prostaglandin G/H synthase-2 (PGHS-2) expression and the subsequent prostaglandin E(2) production. Resident peritoneal macrophages stimulated by PMA from rats fed with olive oil or corn oil had the same level of O2- production, but these levels were increased by the fish oil diet. Olive oil and the fish oil diets increased .NO and decreased arachidonic acid mobilization and the production of prostaglandin E(2). PGHS-2 expression, however, was not affected by diet. We conclude that although olive oil and fish oil reduce arachidonic acid mobilization and subsequent metabolism through the PGHS-2 pathway in PMA-stimulated macrophages, only olive oil offers an additional beneficial effect by increasing .NO/O2- production.

To study the effect of the degree of unsaturation of dietary fatty acids on the production of free radicals and on the vascular smooth muscle tone in rings of the aorta, Sprague-Dawley rats were fed a semipurified diet containing 5% lipids from either corn oil (CO) or menhaden oil (MO) for 8 wk. The MO diet did not change the basal or NADPH-dependent superoxide anion (O2-*) release. There were no significant differences in phenylephrine-induced contractions between the two groups in intact rings. However, these contractions increased in endothelium-intact aortic rings from the MO group after addition of the nitric oxide (*NO) synthase inhibitor NG-nitro-L-arginine and in endothelium-denuded rings, both indicating a greater endothelial basal *NO production in rats fed with the MO diet. Endothelium-dependent relaxations in response to acetylcholine were more prominent in rings from the MO group. These differences were not due to an increased smooth muscle response to.NO, because relaxations were the same using an exogenous *NO donor. Our results indicate that dietary MO did not modify O2-* release by the vessel wall or relaxation due to the cyclooxygenase pathway, but it potentiated endothelial production of *NO.

We studied the roles of nitrogen monoxide (NO&z.rad;) and peroxynitrite produced by the polymorphonuclear leukocytes (PMNs) isolated from an inflammatory exudate. PMNs were incubated either in a medium with a submicromolar concentration of iron or in a diethylenetriaminepenta-acetic acid (DTPA)-containing medium, and stimulated with phorbol 12-myristate 13-acetate (PMA) to generate free radicals. In both conditions superoxide anion (O(2)(*)(-)), NO&z.rad; and peroxynitrite were produced. In the presence of arachidonic acid, malondialdehyde (MDA) was generated. This MDA was generated in one of two way; the peroxynitrite iron-independent mechanism (40%) and the Fenton reaction, caused by free iron (60%). We also observed that the addition of L-arginine was followed by a 42% reduction in MDA, which can be explained by the antioxidant effect of NO&z.rad;. These results indicate that lipid peroxidation can occur in the absence of iron, through a peroxynitrite-mediated mechanism, and that NO&z.rad; may act as an antioxidant when it is produced in large amounts.

Many inflammatory processes are accompanied by anemia and repeated hemorrhages, but the local and systemic effect of the iron present in the inflamed area and the availability of this iron are not known. The experimental model used to mimic the above situation was the carrageenan-induced granuloma in rats with simultaneous iron-dextran injection into the granuloma pouch. We studied the effect of iron-dextran on leukocytes from the inflammatory exudate and the location of iron in the granuloma tissue. We also evaluated the systemic responses by studying several iron parameters in blood and in iron-storage organs. The results showed a reduction in the number of leukocytes present in the exudate and a reduction in their viability and also extensive damage to the granuloma tissue, essentially to macrophages, caused by local iron-induced oxidative stress. A small percentage of iron reaches the systemic circulation, and this is eventually stored in the liver and spleen as hemosiderin, which is unlikely to have any effect on anemia. In spite of its local toxicity, the accumulation of iron in inflamed areas can be interpreted as a protective mechanism against systemic oxidative radical reactions induced by iron mobilization.

Nitric oxide (NO.) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO. in vivo in an acute model of inflammation and evaluated the interactions between NO. and superoxide anion (O2-.) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 mumol of L-N5-(1-iminoethyl)ornithine (L-NIO group), or 6 mumol of L-arginine (L-arginine group) into the granuloma pouch induced by carrageenan in rats. NO2- plus NO3- (indicative of NO. generation) was 188 nmol in the exudate of the control group, but it decreased in the L-NIO group (P < 0.05) and increased in the L-arginine group (P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion (O2-.) decreased by approximately 46% in the L-arginine group. Furthermore, O2-. was inhibited in PMNs when L-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO. in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of O2-. production for cell-mediated injury.