Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes.

BACKGROUND/AIMS: Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. METHODS: For this aim, human RBC were treated in vitro with different HgCl2 concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. RESULTS: Findings presented in this paper indicate that RBC exposure to HgCl2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl2 treatment in a dose dependent manner. CONCLUSION: Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges.

Olive Oil Phenols Prevent Mercury-Induced Phosphatidylserine Exposure and Morphological Changes in Human Erythrocytes Regardless of Their Different Scavenging Activity.

Phosphatidylserine (PS) translocation to the external membrane leaflet represents a key mechanism in the pathophysiology of human erythrocytes (RBC) acting as an "eat me" signal for the removal of aged/stressed cells. Loss of physiological membrane asymmetry, however, can lead to adverse effects on the cardiovascular system, activating a prothrombotic activity. The data presented indicate that structurally related olive oil phenols prevent cell alterations induced in intact human RBC exposed to HgCl2 (5-40 µM) or Ca2+ ionophore (5 µM), as measured by hallmarks including PS exposure, reactive oxygen species generation, glutathione depletion and microvesicles formation. The protective effect is observed in a concentration range of 1-30 µM, hydroxytyrosol being the most effective; its in vivo metabolite homovanillic alcohol still retains the biological activity of its dietary precursor. Significant protection is also exerted by tyrosol, in spite of its weak scavenging activity, indicating that additional mechanisms are involved in the protective effect. When RBC alterations are mediated by an increase in intracellular calcium, the protective effect is observed at higher concentrations, indicating that the selected phenols mainly act on Ca2+-independent mechanisms, identified as protection of glutathione depletion. Our findings strengthen the nutritional relevance of olive oil bioactive compounds in the claimed health-promoting effects of the Mediterranean Diet.

Erythrocytes as a Model for Heavy Metal-Related Vascular Dysfunction: The Protective Effect of Dietary Components.

Heavy metals are toxic environmental pollutants associated with severe ecological and human health risks. Among them is mercury (Hg), widespread in air, soil, and water, due to its peculiar geo-biochemical cycle. The clinical consequences of Hg exposure include neurotoxicity and nephrotoxicity. Furthermore, increased risk for cardiovascular diseases is also reported due to a direct effect on cardiovascular tissues, including endothelial cells, recently identified as important targets for the harmful action of heavy metals. In this review, we will discuss the rationale for the potential use of erythrocytes as a surrogate model to study Hg-related toxicity on the cardiovascular system. The toxic effects of Hg on erythrocytes have been amply investigated in the last few years. Among the observed alterations, phosphatidylserine exposure has been proposed as an underlying mechanism responsible for Hg-induced increased proatherogenic and prothrombotic activity of these cells. Furthermore, following Hg-exposure, a decrease in NOS activity has also been reported, with consequent lowering of NO bioavailability, thus impairing endothelial function. An additional mechanism that may induce a decrease in NO availability is the generation of an oxidative microenvironment. Finally, considering that chronic Hg exposure mainly occurs through contaminated foods, the protective effect of dietary components is also discussed.

New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury-A Possible Risk to Sperm Chromatin Structure?

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl2 similar to those found in seawater (range 1-100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl2, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl2 at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.

Spermatozoa Transcriptional Response and Alterations in PL Proteins Properties after Exposure of Mytilus galloprovincialis to Mercury.

Mercury (Hg) is an environmental pollutant that impacts human and ecosystem health. In our previous works, we reported alterations in the properties of Mytilus galloprovincialis protamine-like (PL) proteins after 24 h of exposure to subtoxic doses of toxic metals such as copper and cadmium. The present work aims to assess the effects of 24 h of exposure to 1, 10, and 100 pM HgCl2 on spermatozoa and PL proteins of Mytilus galloprovincialis. Inductively coupled plasma-mass spectrometry indicated accumulation of this metal in the gonads of exposed mussels. Further, RT-qPCR analyses showed altered expression levels of spermatozoa mt10 and hsp70 genes. In Mytilus galloprovincialis, PL proteins represent the major basic component of sperm chromatin. These proteins, following exposure of mussels to HgCl2, appeared, by SDS-PAGE, partly as aggregates and showed a decreased DNA-binding capacity that rendered them unable to prevent DNA damage, in the presence of CuCl2 and H2O2. These results demonstrate that even these doses of HgCl2 exposure could affect the properties of PL proteins and result in adverse effects on the reproductive system of this organism. These analyses could be useful in developing rapid and efficient chromatin-based genotoxicity assays for pollution biomonitoring programs.

Novel Insights into Mercury Effects on Hemoglobin and Membrane Proteins in Human Erythrocytes.

Mercury (Hg) is a global environmental pollutant that affects human and ecosystem health. With the aim of exploring the Hg-induced protein modifications, intact human erythrocytes were exposed to HgCl2 (1-60 µM) and cytosolic and membrane proteins were analyzed by SDS-PAGE and AU-PAGE. A spectrofluorimetric assay for quantification of Reactive Oxygen Species (ROS) generation was also performed. Hg2+ exposure induces alterations in the electrophoretic profile of cytosolic proteins with a significant decrease in the intensity of the hemoglobin monomer, associated with the appearance of a 64 kDa band, identified as a mercurized tetrameric form. This protein decreases with increasing HgCl2 concentrations and Hg-induced ROS formation. Moreover, it appears resistant to urea denaturation and it is only partially dissociated by exposure to dithiothreitol, likely due to additional protein-Hg interactions involved in aggregate formation. In addition, specific membrane proteins, including band 3 and cytoskeletal proteins 4.1 and 4.2, are affected by Hg2+-treatment. The findings reported provide new insights into the Hg-induced possible detrimental effects on erythrocyte physiology, mainly related to alterations in the oxygen binding capacity of hemoglobin as well as decreases in band 3-mediated anion exchange. Finally, modifications of cytoskeletal proteins 4.1 and 4.2 could contribute to the previously reported alteration in cell morphology.

Hydroxytyrosol Decreases Phosphatidylserine Exposure and Inhibits Suicidal Death Induced by Lysophosphatidic Acid in Human Erythrocytes.

BACKGROUND/AIMS: Lysophosphatidic acid (LPA) is a phospholipid signal molecule that regulates many cellular processes both physiological and pathological. Moreover, its high plasma concentrations are toxic for several cellular types, including erythrocytes (RBC), as it acts as a pro-thrombotic and pro-atherogenic agent. It is therefore essential to explore the potential protective role of nutrition in protecting cells from the possible toxic effects of high plasma concentrations of LPA by testing bioactive nutrients. In particular, our focus was on hydroxytyrosol (HT), a phenolic antioxidant occurring naturally in virgin olive oil, investigating its possible protective effect in preventing LPA-induced programmed cell death (eryptosis) in human RBC. METHODS: Intact RBC were incubated in the presence of 2.5 µM LPA and increasing concentrations of HT. Phosphatidylserine (PS) exposure with cell shrinkage, influx of extracellular calcium (Ca2+), adenosine triphosphate (ATP) and glutathione levels were measured by FACS analysis. In addition, confocal laser scanning microscopy was used to determine RBC morphological alterations, as well as microvesicle formation. RESULTS: Our study confirms that LPA-induced eryptosis is characterized by PS exposure at the cell surface, with cell shrinkage and ATP and glutathione depletion; (Ca2+) influx is also a key event that triggers eryptosis. Here we report for the first time that cell co-incubation with LPA and in quantities as low as 0.1 µM HT causes a significant decrease in PS-exposing RBC, in addition to providing significant protection from the decrease in cell volume. Moreover, treatment of RBC with HT counters the influx of extracellular Ca2+ and completely restores ATP and glutathione content at 1 µM. Finally, under the same experimental conditions, HT exerts a protective effect on RBC morphological changes and microvescicle release, completely restoring the typical biconcave shape at 1 µM. CONCLUSION: Taken together, the findings reported in this paper point to a novel biological effect for HT in preventing programmed suicidal death in anucleated cells and indicate that prevention from LPA toxic effects may represent an additional mechanism responsible for the health-promoting effect of this dietary phenol which has been claimed, particularly related to cardiovascular diseases.

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.

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 Ca(2+) entry. Stimulators of Ca(2+) 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 Ca(2+) activity ([Ca(2+)]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 [Ca(2+)]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 Ca(2+), and by phospholipase A2 inhibitor quinacrine (25 µM). Clofazimine further augmented the effect of Ca(2+) ionophore ionomycin (0.1 µM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca(2+) removal and addition of quinacrine. CONCLUSION: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca(2+), paralleled by cellular energy depletion and sensitive to phospholipase A2 inhibitor quinacrine.

Mercury Chloride Affects Band 3 Protein-Mediated Anionic Transport in Red Blood Cells: Role of Oxidative Stress and Protective Effect of Olive Oil Polyphenols.

Mercury is a toxic heavy metal widely dispersed in the natural environment. Mercury exposure induces an increase in oxidative stress in red blood cells (RBCs) through the production of reactive species and alteration of the endogenous antioxidant defense system. Recently, among various natural antioxidants, the polyphenols from extra-virgin olive oil (EVOO), an important element of the Mediterranean diet, have generated growing interest. Here, we examined the potential protective effects of hydroxytyrosol (HT) and/or homovanillyl alcohol (HVA) on an oxidative stress model represented by human RBCs treated with HgCl2 (10 µM, 4 h of incubation). Morphological changes as well as markers of oxidative stress, including thiobarbituric acid reactive substance (TBARS) levels, the oxidation of protein sulfhydryl (-SH) groups, methemoglobin formation (% MetHb), apoptotic cells, a reduced glutathione/oxidized glutathione ratio, Band 3 protein (B3p) content, and anion exchange capability through B3p were analyzed in RBCs treated with HgCl2 with or without 10 µM HT and/or HVA pre-treatment for 15 min. Our data show that 10 µM HT and/or HVA pre-incubation impaired both acanthocytes formation, due to 10 µM HgCl2, and mercury-induced oxidative stress injury and, moreover, restored the endogenous antioxidant system. Interestingly, HgCl2 treatment was associated with a decrease in the rate constant for SO42- uptake through B3p as well as MetHb formation. Both alterations were attenuated by pre-treatment with HT and/or HVA. These findings provide mechanistic insights into benefits deriving from the use of naturally occurring polyphenols against oxidative stress induced by HgCl2 on RBCs. Thus, dietary supplementation with polyphenols might be useful in populations exposed to HgCl2 poisoning.

Morphological, Gene, and Hormonal Changes in Gonads and In-Creased Micrococcal Nuclease Accessibility of Sperm Chromatin Induced by Mercury.

Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of Mytilus galloprovincialis to 1, 10 and 100 pM HgCl2 for 24 h on the gonadal morphology and on the expression level of three stress genes: mt10, hsp70 and πgst. In this tissue we also evaluated the level of steroidogenic enzymes 3ß-HSD and 17ß-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3ß-HSD and 17ß-HSD antibodies was observed following exposure to 1 and 10 pM HgCl2 and complete absence at 100 pM HgCl2 exposure. Gonads of mussels exposed to all HgCl2 doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl2 exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.

Phenol-Rich Feijoa sellowiana (Pineapple Guava) Extracts Protect Human Red Blood Cells from Mercury-Induced Cellular Toxicity.

Plant polyphenols, with broadly known antioxidant properties, represent very effective agents against environmental oxidative stressors, including mercury. This heavy metal irreversibly binds thiol groups, sequestering endogenous antioxidants, such as glutathione. Increased incidence of food-derived mercury is cause for concern, given the many severe downstream effects, ranging from kidney to cardiovascular diseases. Therefore, the possible beneficial properties of Feijoa sellowiana against mercury toxicity were tested using intact human red blood cells (RBC) incubated in the presence of HgCl2. Here, we show that phenol-rich (10-200 µg/mL) extracts from the Feijoa sellowiana fruit potently protect against mercury-induced toxicity and oxidative stress. Peel and pulp extracts are both able to counteract the oxidative stress and thiol decrease induced in RBC by mercury treatment. Nonetheless, the peel extract had a greater protective effect compared to the pulp, although to a different extent for the different markers analyzed, which is at least partially due to the greater proportion and diversity of polyphenols in the peel. Furthermore, Fejioa sellowiana extracts also prevent mercury-induced morphological changes, which are known to enhance the pro-coagulant activity of these cells. These novel findings provide biochemical bases for the pharmacological use of Fejioa sellowiana-based functional foods in preventing and combating mercury-related illnesses.

Salicylic Acid and Melatonin Alleviate the Effects of Heat Stress on Essential Oil Composition and Antioxidant Enzyme Activity in Mentha × piperita and Mentha arvensis L.

The aim of this study was to evaluate changes in the chemical profile of essential oils and antioxidant enzymes activity (catalase CAT, superoxide dismutase SOD, Glutathione S-transferases GST, and Peroxidase POX) in Mentha × piperita L. (Mitcham variety) and Mentha arvensis L. (var. piperascens), in response to heat stress. In addition, we used salicylic acid (SA) and melatonin (M), two brassinosteroids that play an important role in regulating physiological processes, to assess their potential to mitigate heat stress. In both species, the heat stress caused a variation in the composition of the essential oils and in the antioxidant enzymatic activity. Furthermore both Salicylic acid (SA) and melatonin (M) alleviated the effect of heat stress.

In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats.

BACKGROUND: Cyclosporine A (CsA) is the first-line immunosuppressant used in transplant patients and in auto- immune diseases. Nephrotoxicity is the major limitation of CsA use. Although the mechanisms of nephrotoxicity have not been completely defined, some evidence suggests that reactive oxygen species (ROS) play a causal role. The present study was designed to investigate in vivo effects of hydroxytyrosol (DOPET), a natural olive oil antioxidant, on oxidative stress, renal histology and haemodynamic alterations induced in rats by CsA treatment. METHODS: Adult Sprague-Dawley rats were treated i.p. with CsA (15 mg/kg) alone or in combination with DOPET (20 mg/kg) for 3 weeks. At the end of the treatment, superoxide concentration within the cells of the abdominal aorta and renal artery was quantified from the oxidation of dihydroethidium (DHE) using fluorescence microscopic imaging analysis. In kidney tissues, lipid peroxidation was measured by thiobarbituric acid-reacting substances (TBARS) assay, glutathione level was assessed enzymatically and the expression of haem oxygenase-1 (HO-1) gene was evaluated by semiquantitative RT-PCR. Renal morphology was studied by classical histological techniques, while the glomerular filtration rate (GFR) was estimated by inulin clearance. Systemic blood pressure was monitored by the tail method and through the catheterization of the carotid artery. RESULTS: CsA administration increased superoxide concentration both in the aorta and in the renal artery, while DOPET completely prevented this effect. Higher levels of TBARS, a significant decrease in GSH and an upregulation of HO-1 mRNA were observed in the kidneys of CsA-treated rats. DOPET treatment reversed quantitatively these effects. However, CsA-dependent changes in renal histology were only partially reversed by DOPET. Finally, CsA induced a severe reduction in GFR and a significant increase in both systolic and diastolic blood pressure; the DOPET treatment had no significant effect on these haemodynamic alterations. CONCLUSION: The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.

Comparative Analysis of the Effects of Olive Oil Hydroxytyrosol and Its 5-S-Lipoyl Conjugate in Protecting Human Erythrocytes from Mercury Toxicity.

Oxidative stress is one of the underlying mechanisms of the toxic effects exerted by mercury (Hg) on human health. Several antioxidant compounds, including the olive oil phenol hydroxytyrosol (HT), were investigated for their protective action. Recently, we have reported that 5-S-lipoylhydroxytyrosol (Lipo-HT) has shown increased antioxidant activities compared to HT and exerted potent protective effects against reactive oxygen species (ROS) generation and oxidative damage in human hepatocellular carcinoma HepG2 cell lines. In this study, the effects of Lipo-HT and HT on oxidative alterations of human erythrocytes induced by exposure to 40 µM HgCl2 were comparatively evaluated. When administered to the cells, Lipo-HT (5-20 µM) proved nontoxic and it decreased the Hg-induced generation of ROS, the hemolysis, and the depletion of intracellular GSH levels. At all tested concentrations, Lipo-HT exhibited higher ability to counteract Hg-induced cytotoxicity compared to HT. Model studies indicated the formation of a mercury complex at the SH group of Lipo-HT followed by a redox reaction that would spare intracellular GSH. Thus, the enhanced erythrocyte protective action of Lipo-HT from Hg-induced damage with respect to HT is likely due to an effective chelating and reducing ability toward mercury ions. These findings encourage the use of Lipo-HT in nutraceutical strategies to contrast heavy metal toxicity in humans.

Relevance of 4-F4t-neuroprostane and 10-F4t-neuroprostane to neurological diseases.

F4-neuroprostanes (F4-NeuroPs) are non-enzymatic oxidized products derived from docosahexaenoic acid (DHA) and are suggested to be oxidative damage biomarkers of neurological diseases. However, 128 isomers can be formed from DHA oxidation and among them, 4(RS)-4-F4t-NeuroP (4-F4t-NeuroP) and 10(RS)-10-F4t-NeuroP (10-F4t-NeuroP) are the most studied. Here, we report the identification and the clinical relevance of 4-F4t-NeuroP and 10-F4t-NeuroP in plasma of four different neurological diseases, including multiple sclerosis (MS), autism spectrum disorders (ASD), Rett syndrome (RTT), and Down syndrome (DS). The identification and the optimization of the method were carried out by gas chromatography/negative-ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) using chemically synthesized 4-F4t-NeuroP and 10-F4t-NeuroP standards and in oxidized DHA liposome. Both 4-F4t-NeuroP and 10-F4t-NeuroP were detectable in all plasma samples from MS (n = 16), DS (n = 16), ASD (n = 9) and RTT (n = 20) patients. While plasma 10-F4t-NeuroP content was significantly higher in patients of all diseases as compared to age and gender matched healthy control subjects (n = 61), 4-F4t-NeuroP levels were significantly higher in MS and RTT as compared to healthy controls. Significant positive relationships were observed between relative disease severity and 4-F4t-NeuroP levels (r = 0.469, P <0.0001), and 10-F4t-NeuroP levels (r = 0.757, P < 0.0001). The study showed that the plasma amount ratio of 10-F4t-NeuroP to 4-F4t-NeuroP and the plasma amount as individual isomer can be used to discriminate between different brain diseases. Overall, by comparing the different types of disease, our plasma data indicates that 4-F4t-NeuroP and 10-F4t -NeuroP: i) are biologically synthesized in vivo and circulated, ii) are related to clinical severity of neurological diseases, iii) are useful to identify shared pathogenetic pathways in distinct brain diseases, and iv) appears to be distinctive for different neurological conditions, thus representing potentially new biological disease markers. Our data strongly suggest that in vivo DHA oxidation follows preferential chemical rearrangements according to different brain diseases.

Homocysteine-induced endothelial cell adhesion is related to adenosine lowering and is not mediated by S-adenosylhomocysteine.

Hyperhomocysteinemia is a cardiovascular risk factor and may contribute to the pathogenesis of atherosclerosis by altering endothelial functions. The mechanism of homocysteine-induced cell adhesion has been here investigated using EA.hy 926 cells. Homocysteine induces a stereospecific, time- and dose-dependent cell adhesion which is prevented by adenosine. The dramatic increase of S-adenosylhomocysteine induced by adenosine-2',3'-dialdehyde does not cause cell adhesion, indicating that no apparent relationship exists between this process and intracellular S-adenosylhomocysteine content. Homocysteine-induced cell adhesion is abolished by pre-treatment with adenosine-2',3'-dialdehyde, demonstrating that the adenosine depletion caused by reversal of S-adenosylhomocysteine hydrolase reaction is responsible for homocysteine-induced cell damage.

Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bioantioxidant.

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.

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.