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.
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 ...HgCl
(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. Hg
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 HgCl
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 Hg
-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.
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.
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 HgCl
(5-40 µM) or Ca
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 Ca
-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.
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.
For this aim, human RBC were treated in vitro with different HgCl
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.
Findings presented in this paper indicate that RBC exposure to HgCl
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 HgCl
treatment in a dose dependent manner.
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.
Mercury (Hg) is an environmental pollutant that impacts human and ecosystem health. In our previous works, we reported alterations in the properties of
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 HgCl
on spermatozoa and PL proteins of
. 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
and
genes. In
, PL proteins represent the major basic component of sperm chromatin. These proteins, following exposure of mussels to HgCl
, 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 CuCl
and H
O
. These results demonstrate that even these doses of HgCl
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.
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.
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 (Ca
), 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.
Our study confirms that LPA-induced eryptosis is characterized by PS exposure at the cell surface, with cell shrinkage and ATP and glutathione depletion; (Ca
) 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 Ca
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.
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.
Folk medicine uses wild herbs, especially from the Lamiaceae family, such as oregano and lavender, in the treatment of many diseases. In the present study, we investigated the antibacterial activity ...of the essential oils of Origanum glandulosum Desf. and Lavandula dentata L. against multidrug-resistant Klebsiella pneumoniae strains. The chemical composition of essential oils and their effect on the ultrastructure of the tested bacteria and on the release of cellular components that absorb at 260 nm were studied. Furthermore, the cytotoxicity and the production of reactive oxygen species in human lymphocytes treated with essential oils were evaluated. Thymol (33.2%) was the major constituent in O. glandulosum, and β-pinene (17.3%) was the major constituent in L. dentata. We observed ultrastructural damage in bacteria and increased release of cellular material. Furthermore, ROS production in human lymphocytes treated with essential oils was lower than in untreated lymphocytes and no cytotoxicity was observed. Therefore, the essential oils of lavender and oregano could be used as a source of natural antibacterial and antioxidant agents with potential pharmacological applications.
Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present ...work, we analyzed PLs from
by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl
for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl
, while, at all NiCl
doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl
, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl
. A possible molecular mechanism for the toxic reproductive effects of nickel in
is discussed.
Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of
to 1, 10 and 100 pM HgCl
for 24 h on the gonadal morphology and on the expression ...level of three stress genes:
,
and
. In this tissue we also evaluated the level of steroidogenic enzymes 3β-HSD and 17β-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 HgCl
and hypo-expression of the three stress genes, particularly for
. Furthermore, decreased labelling with both 3β-HSD and 17β-HSD antibodies was observed following exposure to 1 and 10 pM HgCl
and complete absence at 100 pM HgCl
exposure. Gonads of mussels exposed to all HgCl
doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl
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.
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