Caterina Manna

Olive oil hydroxytyrosol protects human erythrocytes against oxidative damages

Hydroxytyrosol, the major representative phenolic compound of virgin olive oil, is a dietary component. Its possible protective effect on hydrogen peroxide (H(2)O(2))-induced oxidative alterations was investigated in human erythrocytes. Cells were pretreated with micromolar hydroxytyrosol concentrations and then exposed to H(2)O(2) over different time intervals. Subsequently, erythrocytes were analyzed for oxidative hemolysis and lipid peroxidation. Our data demonstrate that hydroxytyrosol prevents both oxidative alterations, therefore, providing protection against peroxide-induced cytotoxicity in erythrocytes. The effect of oxidative stress on erythrocyte membrane transport systems, as well as the protective role of hydroxytyrosol, also were investigated in conditions of nonhemolytic mild H(2)O(2) treatment. Under these experimental conditions, a marked decrease in the energy-dependent methionine and leucine transport is observable; this alteration is quantitatively prevented by hydroxytyrosol pretreatment. On the other hand, the energy-independent glucose transport is not affected by the oxidative treatment. The reported data give new experimental support to the hypothesis of a protective role played by nonvitamin antioxidant components of virgin olive oil on oxidative stress in human systems.

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4‐Dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco‐2 cell monolayers as the model system. The kinetic data demonstrate that [14C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3‐hydroxy‐4‐methoxyphenylethanol, the product of catechol‐O‐methyltransferase; when DPE is assayed in vitro with the purified enzyme a K m value of 40 μM has been calculated.

Biological Effects of Hydroxytyrosol, a Polyphenol from Olive Oil Endowed with Antioxidant Activity

A number of epidemiological studies indicate that dietary factors may influence the development of some types of cancer and degenerative pathologies, including cardiovascular diseases and cataract. In this respect, it is well documented that daily consumption of fruits and vegetables is associated with a lowered risk of these diseases.1 Polyphenols are bioactive substances that are widely distributed in the vegetable kingdom2,3 and therefore are present in high concentrations in typical components of the Mediterranean diet, such as fruit, vegetables, red wine, and olive oil. The aim of this article is to overview the most recent data on the nutritional value of the phenolic fraction of virgin olive oil in the ongoing studies on its beneficial effects on human health.

Clofazimine induced suicidal death of human erythrocytes

Background/Aims: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A2 and to induce apoptosis. In erythrocytes phospholipase A2 stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A2 is in part effective by fostering formation of prostaglandin E2, which triggers Ca2+ entry. Stimulators of Ca2+ entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca2+ activity ([Ca2+]i) from Fluo3-fluorescence, reactive oxygen species (ROS) from 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. Results: A 24-48 hours exposure of human erythrocytes to clofazimine (≥1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca2+]i, significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca2+, and by phospholipase A2 inhibitor quinacrine (25 µM). Clofazimine further augmented the effect of Ca2+ ionophore ionomycin (0.1 µM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca2+ removal and addition of quinacrine. Conclusion: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca2+, paralleled by cellular energy depletion and sensitive to phospholipase A2 inhibitor quinacrine.

Bromfenvinphos induced suicidal death of human erythrocytes

The organophosphorus pesticide bromfenvinphos ((E,Z)-O,O-diethyl-O-[1-(2,4-dichlorophenyl)-2-bromovinyl] phosphate) has been shown to decrease hematocrit and hemoglobin levels in blood presumably by triggering oxidative stress of erythrocytes. Oxidative stress is known to activate erythrocytic Ca(2+) permeable unselective cation channels leading to Ca(2+) entry and increase of cytosolic Ca(2+) activity ([Ca(2+)]i), which in turn triggers eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The present study explored, whether and how bromfenvinphos induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. As a result, a 48hour exposure of human erythrocytes to bromfenvinphos (≥100μM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. The effect of bromfenvinphos on annexin-V-binding and forward scatter was significantly blunted, but not abolished by removal of extracellular Ca(2+). In conclusion, bromfenvinphos triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca(2+) entry.

Olive oil phenolic compounds inhibit homocysteine-induced endothelial cell adhesion regardless of their different antioxidant activity.

In this study, we examine the effect of extra virgin olive oil phenolic compounds on homocysteine-induced endothelial dysfunction and whether the protective effects are related to their different scavenging activities. Structurally related compounds have been assayed for their ability to reduce homocysteine-induced monocyte adhesion as well as the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in EA.hy.926 cells. As well-known, among the selected phenolic compounds, hydroxytyrosol, homovanillyl alcohol, and the hydroxycinnamic acid derivatives caffeic and ferulic acid display high scavenging activities, while tyrosol and p-coumaric acid are poorly active. All of the tested compounds, approaching potential in vivo concentrations, significantly reduce homocysteine-induced cell adhesion and ICAM-1 expression. Interestingly, we report the first evidence that monophenols tyrosol and p-coumaric acid are selectively protective only in homocysteine-activated cells, while they are ineffective in reducing ICAM-1 expression induced by TNFalpha. Finally, we report the synergistic effect of o-diphenolic and monophenolic compounds.

Hydroxytyrosol inhibits phosphatidylserine exposure and suicidal death induced by mercury in human erythrocytes: Possible involvement of the glutathione pathway

Hydroxytyrosol (HT) is a phenolic antioxidant naturally occurring in virgin olive oil. In this study, we investigated the possible protective effects of HT on programmed suicidal death (eryptosis) induced by mercury (Hg) treatment in intact human erythrocytes (RBC). Our study confirms that the Hg-eryptosis is characterized by phosphatidylserine (PS) exposure at the cell surface, with cell shrinkage and ATP and glutathione depletion; calcium influx is also a key event that triggers eryptosis. Here we report that cell preconditioning with an optimal dose (1-5 μM) of HT prior to exposure to 2.5 μM HgCl2 causes a noteworthy decrease in PS-exposing RBC, almost restoring ATP and GSH content. Conversely, HT shows no effect against decrease in cell volume nor against influx of extracellular calcium. Taken together our data provide the first experimental evidence of the efficacy of HT in modulating the programmed suicidal death in non nucleated cells; the reported findings also confirm that the prevention of Hg toxicity should be regarded as an additional mechanism responsible for the health-promoting potential of this dietary phenol. Finally, virgin olive oil would appear to be a promising healthy food to reduce the adverse effects of chronic mercury exposure in humans.

Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients.

Several investigators have observed abnormalities of plasma DHA in various common forms of retinitis pigmentosa. Erythrocyte membranes are an accessible tissue which may more closely reflect neural membrane fatty acid levels, but little data is yet available on their fatty acid composition in retinitis pigmentosa patients. We have evaluated the DHA levels in red blood cells membrane phospholipids in various genetic forms of retinitis pigmentosa and relative controls. The results obtained indicate significantly lower DHA levels in red blood cell membrane phospholipids in retinitis pigmentosa patients with respect to controls (p < 0.01). This data is significant for autosomal dominant forms only (p < 0.01). Fatty acid content alterations in membrane phospholipids of red blood cells may be considered as markers for abnormalities in the lipid metabolism which disturb the retina integrity.

Effect of 5'-methylthioadenosine on in vivo methyl esterification of human erythrocyte membrane proteins

1. Introduction The enzyme S-adenosylmethionine:protein car- boxyl-U-methyl transferase (EC 2.1 .1.24; protein methylase II) methyl esterifies the aspartyl and/or glutamyl residues of preformed proteins, using S-adenosylmethionine as the methyl donor [ 141. The protein carboxyl methyl esters formed are un- stable and undergo rapid hydrolysis at physiological pH and temperature yielding methanol [5,6]. Several membrane proteins were shown to be the endogenous substrates for the enzyme in both prokaryotic and eukaryotic cells [ 1,7--91 and a number of studies were done to determine the physiological roles of this selec- tive, post-translational, protein methylation in mem- brane functions. The high activity of PM II and the large amounts of its membrane-bound endogenous substrates in nervous and endocrine tissues suggest a role of this reaction in the synaptic function [lo] and neurosecretory processes [ 1 O-l 21. In

Protein methylation in Caldariella acidophila, an extreme thermo-acidophilic archaebacterium

The enzymatic methylation of proteins occurs in both prokaryotes and eukaryotes by a group of enzymes highly specific with regard to the amino acid sidechain and protein involved [ 11. Three methylating enzymes have been identified so far and characterized on the basis of the methylaccepting protein moiety: the guanidino-groups of arginine residues are the methyl acceptors for protein methylase I (PM I; S-adenosylmethionine: proteinarginine-N-methyl transferase, EC 2.1 .1.23) [l-3]; protein methylase II (PM II; S-adenosylmethionine: protein carboxyl-@methyl transferase, EC 2.1.1.24) methylates the free carboxyl groups of aspartic and glutamic acid residues [ 1,3,4], while the e-amino groups of lysine residues are methylated by protein methylase III (PM III; S-adenosylmethionine: protein e-lysine-N-methyl transferase, EC 2.1 .1.43) [1,3,5]. Despite the large number of reports on protein methylation in mesophilic organisms, no data are available on the occurrence of these biochemical processes in thermophiles. It is well known that thermophilic microorganisms are capable of proliferating at elevated temperatures and the majority of their marcromolecules are heat stable. The mechanisms underlying the thermostability of cell constituents and the structural alterations in cellular components which allow the survival at high temperatures are not completely known at present. It is generally accepted that the primary structure of thermophilic proteins is similar to the mesophilic proteins with the same function [6]; however, thermophilic proteins are generally more stable to denaturing conditions than their mesophilic counterparts [6].