Federico Leighton

The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer’s disease

One of the theories involved in the etiology of Alzheimers disease (AD) is the oxidative stress hypothesis. The amyloid beta-peptide (A beta), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of different antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in A beta-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis.

Fatty acid oxidation by human liver peroxisomes

Abstract A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C 12 –C 18 saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already established properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.

Myricetin, quercetin and catechin-gallate inhibit glucose uptake in isolated rat adipocytes

The facilitative glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in adipocytes and muscles, and the participation of GLUT4 in the pathogenesis of various clinical conditions associated with obesity, visceral fat accumulation and insulin resistance has been proposed. Glucose uptake by some members of the GLUT family, mainly GLUT1, is inhibited by flavonoids, the natural polyphenols present in fruits, vegetables and wine. Therefore it is of interest to establish if these polyphenolic compounds present in the diet, known to be effective antioxidants but also endowed with several other biological activities such as protein-tyrosine kinase inhibition, interfere with GLUT4 function. In the present study, we show that three flavonoids, quercetin, myricetin and catechin-gallate, inhibit the uptake of methylglucose by adipocytes over the concentration range of 10-100 microM. These three flavonoids show a competitive pattern of inhibition, with K(i)=16, 33.5 and 90 microM respectively. In contrast, neither catechin nor gallic acid inhibit methylglucose uptake. To obtain a better understanding of the interaction among GLUT4 and flavonoids, we have derived a GLUT4 three-dimensional molecular comparative model, using structural co-ordinates from a GLUT3 comparative model and a mechanosensitive ion channel [PDB (Protein Data Bank) code 1MSL] solved by X-ray diffraction. On the whole, the experimental evidence and computer simulation data favour a transport inhibition mechanism in which flavonoids and GLUT4 interact directly, rather than by a mechanism related to protein-tyrosine kinase and insulin signalling inhibition. Furthermore, the results suggest that GLUT transporters are involved in flavonoid incorporation into cells.

Inflammation, not hyperhomocysteinemia, is related to oxidative stress and hemostatic and endothelial dysfunction in uremia

BACKGROUND Several cardiovascular risk factors are present in patients with chronic renal failure (CRF), among which are systemic inflammation and hyperhomocysteinemia. Increased oxidative stress, endothelial activation/dysfunction, and coagulation activation are considered integral components of the inflammatory response, but have also been proposed as mediators of plasma homocysteine (tHcy)-induced cell damage. Using correlation analysis, we assessed the relative contributions of inflammation and hyperhomocysteinemia in the abnormal oxidative stress, endothelial activation/dysfunction, and hemostasis activation in patients with CRF. METHODS The relationships of inflammatory proteins and tHcy with plasma markers of these processes were studied in 64 patients with CRF (serum creatinine 526 +/- 319 micromol/L) on conservative treatment, comparing the results with healthy controls (N = 15 to 40, depending on the measured variable) of similar sex and age. RESULTS Patients had significant increases in inflammatory cytokines (TNF-alpha and IL-8) and acute-phase proteins (C-reactive protein, fibrinogen and alpha1-antitrypsin). tHcy was increased in 87.5% of patients (mean = 27.1 micromol/L, range 6.5 to 118). Patients had significant increases in (1) indices of oxidative stress: TBARS (thiobarbituric acid-reactive species), a marker of lipid peroxidation and AOPP (advanced oxidation protein products), a marker of protein oxidation; (2) endothelial cell markers such as von Willebrand factor (vWF:Ag), soluble ICAM-1 and soluble thrombomodulin (sTM); (3) markers of intravascular thrombin generation: thrombin-antithrombin complexes (TAT) and prothrombin fragment F(1+2) (PF(1+2)); and (4) indices of activation of fibrinolysis: plasmin-antiplasmin complexes (PAP), fibrin degradation products (FnDP) and fibrinogen degradation products (FgDP). tHcy was significantly correlated with plasma creatinine (r = 0.29, P < 0.018) and with serum folate (r = -0.38, P < 0.002). However, no significant correlations were observed between tHcy and TBARS, AOPP, vWF:Ag, sICAM-1, sTM, TAT, F(1+2), sTF, PAP, FnDP, and FgDP. Conversely, acute-phase proteins showed significant, positive correlations with most markers of oxidative stress, endothelial dysfunction and hemostatic activation. CONCLUSIONS Systemic inflammation, which is closely associated with augmented oxidative stress, endothelial cell dysfunction and hemostatic activation, emerges as a major cardiovascular risk factor in CRF. tHcy is unrelated to these events. Thus, alternative mechanisms through which hyperhomocysteinemia could predispose to vascular lesion and thrombotic events in CRF needs to be investigated.

A high-fat diet induces and red wine counteracts endothelial dysfunction in human volunteers

Endothelial dysfunction is associated with atherogenesis and oxidative stress in humans. In rat and rabbit blood vessels, wine polyphenol antioxidants induce vascular relaxationin vitro through the NO-cGMP pathway. To assess the effect of a regular high-fat diet (HFD) and moderate red wine consumption on endothelial function (EF), a study was performed in healthy male volunteers. EF was measured as flow-mediated dilatation of the brachial artery, employing high-resolution ultrasound after an overnight fast. Other clinical and biochemical parameters related to EF were also measured. Six volunteers received a control diet, rich in fruits and vegetables (27% calories as fat) and five volunteers received an HFD (39.5% calories as fat). Measurements were done twice on each volunteer: after a period of 30 d with diet plus 240 mL of red wine/d, and after a period of 30 d with diet, without wine. In the absence of wine, there is a reduction of EF with HFD when compared to the control diet (P=0.014). This loss of EF is not seen when both diets are supplemented with wine for 30 d (P=0.001). Plasma levels ofn−3 fatty acids (R2=0.232,P=0.023) and lycopene (R2=0.223,P=0.020) show a positive correlation with individual EF measurements, but they do not account for the significant differences observed among dietary groups or after wine supplementation. These results help elucidate the deleterious effect of a high-fat diet and the protective role of wine, n−3 fatty acids and dietary antioxidants in cardiovascular disease.

Complementary effects of Mediterranean diet and moderate red wine intake on haemostatic cardiovascular risk factors

Objectives: To compare the effect of alcohol-free Mediterranean-type diet (MD) and high-fat diet (HFD) on plasma concentration of emergent haemostatic cardiovascular risk factors (HCVRF). Also, to test if red wine supplementation modifies HCVRF, independent of diet.Design, subjects and intervention: Controlled prospective intervention study. Two groups, each of 21 healthy male university students (22±3.4 y), received either MD or HFD for 90 days. Between days 30 and 60, both diets were supplemented with 240 ml/day of red wine. Baseline and T30, T60 and T90-day samples were drawn. No drop out from the study was observed.Setting: University campus and outpatient nutrition clinic.Results: Volunteers on HFD at T30 had increases in pro-coagulants fibrinogen (22%), factor VIIc (9%), and factor VIIIc (4%), and decreases in natural anticoagulants antithrombin III (3%), protein C (11%) and protein S (6%) and of 20% in plasminogen activator inhibitor-1. At the same time, individuals on MD had increases in fibrinogen (4%), antithrombin III (5%), protein C (3%), protein S (2.7%), and decreases in factor VIIIc (9%), and plasminogen activator inhibitor-1 (21%). After adjusting by baseline values, MD was associated with lower plasma fibrinogen (P=0.03), factor VIIc (P=0.034) and factor VIIIc (P=0.0057) and with higher levels of protein S (P=0.013). Red wine supplementation, in both diets, resulted in decreased plasma fibrinogen (P=0.001) and factor VIIc (P=0.05), and increased tissue plasminogen activator antigen (P=0.01) and plasminogen activator inhibitor-1 antigen (P=0.0003). Wine consumption was also associated with significantly (P=0.01) divergent effects on antithrombin III: it decreased by 10% in individuals on HFD but increased slightly in those on MD. No effects of diet or wine were detected in plasma protein C and C-reactive protein.Conclusion: MD and moderate consumption of red wine have complementary, mostly beneficial effects on HCVRF.Sponsorship: P Catholic University of Chile.Descriptors: diet; wine; haemostasis; cardiovascular risk factorsEuropean Journal of Clinical Nutrition (2001) 55, 444–451

Endothelial cell oxidative stress and signal transduction

Endothelial dysfunction (ED) is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS) have been implicated as important mechanisms that contribute to ED, and ROSs may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1), tyrosine kinases (Src and Syk) and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC), we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy) and oxidized LDL (oxLDL) enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process.

Flavonoids as stabilizers of fish oil: An alternative to synthetic antioxidants

The antioxidant activities against fish oil oxidation of six commercially available flavonoids and of five flavonoids purified from two Chilean native plants were compared to those ofdl-α-tocopherol and of two synthetic antioxidants, butylated hydroxytoluene and butylated hydroxyanisole. Among the commercial flavonoids, catechin, morin and quercetin showed a higher activity when fish oil oxidation (either spontaneous or Fe2+-induced) was assessed from the formation of peroxides or thiobarbituric acid-reactive substances. Among the native flavonoids, the 5,3′,4′-trihydroxy-7-methoxy flavanone (designated as Pt-2) showed the highest antioxidant activity. Mixtures of quercetin or of Pt-2 withdl-α-tocopherol produced better inhibitory effects when compared to that of each substance assayed by itself. Also, when Pt-2 and quercetin were assayed in combination (0.3 g/kg oil and 0.7 g/kg oil, respectively), a synergistic antioxidant effect was observed. Results indicate that several flavonoids could be used as natural antioxidants as a means to replace those synthetic antioxidants, the use of which has been questioned.

Wine, Diet, Antioxidant Defenses, and Oxidative Damage

Abstract: Oxidative stress is a central mechanism for the pathogenesis of ischemic heart disease and atherogenesis, for cancer and other chronic diseases in general, and it also plays a major role in the aging process. Dietary antioxidants constitute a large group of compounds that differ in mechanism of action, bioavailability and side effects. A systematic analysis of the role of the various antioxidants in chronic diseases is hampered by the difficulty of employing death or clinical events as end points in intervention studies. Therefore, valid markers for oxidative stress, which show dose response and are sensitive to changes in dietary supply of antioxidants, are potentially of great value when trying to establish healthy dietary patterns, or when one component, like red wine, is evaluated specifically. To evaluate potential oxidative stress markers we have studied the effect of different diets plus wine supplementation on antioxidant defenses and oxidative damage. In three experimental series, four groups of young male university students, one of older men and other of older women, 20‐24 volunteers each, received Mediterranean or occidental (high‐fat) diets alone or supplemented with red wine, white wine, or fruits and vegetables. Measurements included, leukocyte DNA 8‐OH‐deoxyguanosine (8OHdG), plasma 7β‐hydroxycholesterol, TBARS and well‐characterized antioxidants, and plasma and urine polyphenol antioxidants. In all experimental groups that received red wine, consumption resulted in marked decrease in 8OHdG. The changes observed in 8OHdG correlate positively with the other markers of oxidative damage, and shows a clear inverse correlation with the plasma level of well established antioxidants and with measurements of total antioxidant capacity. Urinary total polyphenol content as well as the sum of some specific plasma species also correlate inversely with 8OHdG. In conclusion, the results identify 8OHdG as a very promising general marker of oxidative stress in nutrition intervention studies in humans, and red wine shows a remarkable protective effect.

Methionine sulfoxide reductase A expression is regulated by the DAF-16/FOXO pathway in Caenorhabditis elegans

The methionine sulfoxide reductase system has been implicated in aging and protection against oxidative stress. This conserved system reverses the oxidation of methionine residues within proteins. We analyzed one of the components of this system, the methionine sulfoxide reductase A gene, in Caenorhabditis elegans. We found that the msra‐1 gene is expressed in most tissues, particularly in the intestine and the nervous system. Worms carrying a deletion of the msra‐1 gene are more sensitive to oxidative stress, show chemotaxis and locomotory defects, and a 30% decrease in median survival. We established that msra‐1 expression decreases during aging and is regulated by the DAF‐16/FOXO3a transcription factor. The absence of this enzyme decreases median survival and affects oxidative stress resistance of long lived daf‐2 worms. A similar effect of MSRA‐1 absence in wild‐type and daf‐2 (where most antioxidant enzymes are activated) backgrounds, suggests that the lack of this member of the methionine repair system cannot be compensated by the general antioxidant response. Moreover, FOXO3a directly activates the human MsrA promoter in a cell culture system, implying that this could be a conserved mechanism of MsrA regulation. Our results suggest that repair of oxidative damage in proteins influences the rate at which tissues age. This repair mechanism, rather than the general decreased of radical oxygen species levels, could be one of the main determinants of organisms’ lifespan.