Impact of Heavy Metal Exposure on Mytilus galloprovincialis Spermatozoa: A Metabolomic Investigation.

Metabolomics is a method that provides an overview of the physiological and cellular state of a specific organism or tissue. This method is particularly useful for studying the influence the environment can have on organisms, especially those used as bio-indicators, e.g., Mytilus galloprovincialis. Nevertheless, a scarcity of data on the complete metabolic baseline of mussel tissues still exists, but more importantly, the effect of mussel exposure to certain heavy metals on spermatozoa is unknown, also considering that, in recent years, the reproductive system has proved to be very sensitive to the effects of environmental pollutants. In order to fill this knowledge gap, the similarities and differences in the metabolic profile of spermatozoa of mussels exposed to metallic chlorides of copper, nickel, and cadmium, and to the mixture to these metals, were studied using a metabolomics approach based on GC-MS analysis, and their physiological role was discussed. A total of 237 endogenous metabolites were identified in the spermatozoa of these mussel. The data underwent preprocessing steps and were analyzed using statistical methods such as PLS-DA. The results showed effective class separation and identified key metabolites through the VIP scores. Heatmaps and cluster analysis further evaluated the metabolites. The metabolite-set enrichment analysis revealed complex interactions within metabolic pathways and metabolites, especially involving glucose and central carbon metabolism and oxidative stress metabolism. Overall, the results of this study are useful to better understand how some pollutants can affect the specific physiological functions of the spermatozoa of this mussel, as well as for further GC-MS-based metabolomic health and safety studies of marine bivalves.

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.

Molecular Alterations and Severe Abnormalities in Spermatozoa of Young Men Living in the "Valley of Sacco River" (Latium, Italy): A Preliminary Study.

The Valley of Sacco River (VSR) (Latium, Italy) is an area with large-scale industrial chemical production that has led over time to significant contamination of soil and groundwater with various industrial pollutants, such as organic pesticides, dioxins, organic solvents, heavy metals, and particularly, volatile organic compounds (VOCs). In the present study, we investigated the potential impact of VOCs on the spermatozoa of healthy young males living in the VSR, given the prevalent presence of several VOCs in the semen of these individuals. To accomplish this, spermiograms were conducted followed by molecular analyses to assess the content of sperm nuclear basic proteins (SNBPs) in addition to the protamine-histone ratio and DNA binding of these proteins. We found drastic alterations in the spermatozoa of these young males living in the VSR. Alterations were seen in sperm morphology, sperm motility, sperm count, and protamine/histone ratios, and included significant reductions in SNBP-DNA binding capacity. Our results provide preliminary indications of a possible correlation between the observed alterations and the presence of specific VOCs.

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.