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

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

A Molecular Mechanism to Explain the Nickel-Induced Changes in Protamine-like Proteins and Their DNA Binding Affecting Sperm Chromatin in Mytilus galloprovincialis: An In Vitro Study

: 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 Mytilus galloprovincialis 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 NiCl2 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 NiCl2, while, at all NiCl2 doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl2, 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 NiCl2. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed

The current spectrum and prevalence of intestinal parasitosis in Campania (region of southern Italy) and their relationship with migration from endemic countries

Background: In Italy, the current clinical–epidemiological features of intestinal parasitosis and the impact of recent massive migration flows from endemic areas on their distribution are not very well known. Methods: An analysis was carried out involving 1766 patients (720 natives and 1046 immigrants) observed during the period 2009–2010 (the 'current group') and 771 native patients observed during the period 1996–1997 (the 'historical group'), a time at which immigration in the area was minimal. Patients were analyzed for intestinal parasitosis at four healthcare centres in Campania. Results: A wide variety of intestinal parasites was detected in the study subjects. Immigrants had a significantly higher prevalence of parasitosis and multiple simultaneous infections than natives in both groups. In both study groups of natives, the detection of at least one parasite was significantly associated with a history of travel to endemic areas. Among immigrants, we found an inverse correlation between the frequency of parasite detection and the amount of time spent in Italy. No circulation of parasites was found among contacts of parasitized patients. Conclusions: Intestinal parasites are still a cause of intestinal infection in Campania. Although immigrants have a significantly higher prevalence of parasitosis than natives, this does not increase the risk of infection for that population. This is likely due to the lack of suitable biological conditions in our area

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 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 HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3β-HSD and 17β-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

Biological Responses to Cadmium Stress in Liverwort Conocephalum conicum (Marchantiales)

Oxidative damage (production and localization of reactive oxygen species) and related response mechanisms (activity of antioxidant enzymes), and induction of Heat Shock Protein 70 expression, have been studied in the toxi-tolerant liverwort Conocephalum conicum (Marchantiales) in response to cadmium stress using two concentrations (36 and 360 µM CdCl2). Cadmium dose-dependent production of reactive oxygen species (ROS) and related activity of antioxidant enzymes was observed. The expression level of heat shock protein (Hsp)70, instead, was higher at 36 µM CdCl2 in comparison with the value obtained after exposure to 360 µM CdCl2, suggesting a possible inhibition of the expression of this stress gene at higher cadmium exposure doses. Biological responses were related to cadmium bioaccumulation. Since C. conicum was able to respond to cadmium stress by modifying biological parameters, we discuss the data considering the possibility of using these biological changes as biomarkers of cadmium pollution

MOLECULAR EFFECTS ON SPERMATOZOA OF MYTILUS GALLOPROVINCIALIS EXPOSED TO SEVERE HYPOSALINE CONDITIONS: A CASE OF FERTILITY PRESERVATION STRATEGY DUE TO GAMETE PLASTICITY.

Climate changes are affecting species physiology, pushing environmental tolerance limits and shifting biogeographic distribution ranges of marine organisms. In addition to temperature and ocean acidification, global climate changes can also occur through changes in seawater salinity. Salinity levels may change also by anthropogenic factors such as mining activity and agricultural and industrial processes. Salinity is considered one of the most significant environmental stressors for marine bivalves. Mussels, in close proximity to coasts and in estuaries, in fact, periodically experience hypo-saline stress, particularly during intense precipitations. Many bivalve species, when experiencing water with high temperatures and low salinity, have the skill to acclimatize, or migrate to deeper water which is cooler and more saline. Mytilus galloprovincialis, is distributed in the north Atlantic Ocean, Mediterranean Sea and Black Sea and in recent years has invaded new places, including South Africa, Japan and California. This mussel is a common member of the intertidal estuarine and coastal areas where it can meet different salinities. Mytilus galloprovincialis can survive at elevated heat exposure, but it is susceptible to hyposalinity, while the native Pacific coast M. trossulus is more tolerant to hypo-saline conditions but vulnerable to heat stress. Since salinity represents a critical factor in reproduction of marine species, we analyzed the responses of Mytilus galloprovincialis spermatozoa to hyposaline stress. We exposed mussels, in laboratory tanks, for 24 hours at 18°C to control (35.9 psu) and three hyposaline (17.1; 22.6 and 26.2 psu) conditions, and evaluated the expression of sperm hsp70 and protamine-like proteins genes. Further we analyzed the electrophoretic pattern, the DNA binding and the release from sperm nuclei of protamine-like proteins. For all experimental approaches used, the results obtained at 17.1 psu condition were very similar to those obtained in control condition, while alterations were always recorded at 22.6 and 26.2 psu conditions. Particularly, at 22.6 and 26.2 psu, was observed: 42.5 and 17.1-fold increase in hsp70 expression, respectively and hypo-expression of PL-II/PLIV protamine-like proteins genes. Further, electrophoretic mobility shift assays and salt-induced release of nuclear proteins from sperm nuclei, revealed alterations in the PL proteins/DNA binding, in these two hyposaline conditions. The similarity between the results obtained in control and in the more severe hyposaline condition (17.1 psu) could indicate a phenomenon of fertility preservation strategy due to gamete plasticity

CONTRIBUTION TO THE STUDY OF MOLECULAR EFFECTS OF COPPER ON MYTILUS GALLOPROVINCIALIS DURING REPRODUCTIVE PERIOD

Successful reproduction is a determining factor for the survival of species. In this century, major risks affecting reproductive health are for those species that fertilize externally, such as several marine invertebrates, since several marine ecosystems are polluted by different types of xenobiotics. Among the xenobiotics present in seawater, a relevant role is played by heavy metals whose release into the marine environment has increased their levels to large extents within the past few decades. Copper is one of the most interesting heavy metals, because in small quantities, it is essential, being involved in several physiological functions, such as redox reactions, oxygen transport, cellular respiration, free radical defense, neurotransmitter synthesis, and neuronal myelination, but can be toxic above certain threshold concentrations. In this study, we have evaluated the effects induced by 24 hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine‐like proteins) of Mytilus galloprovincialis. Protamine‐like proteins represent the main basic protein component of sperm chromatin of this organism. After exposure, we found accumulation of this metal in gonads, spermatozoa, and protamine‐like proteins of exposed mussels, as indicated by inductively coupled plasma–mass spectrometry analyses. Moreover, altered expression levels of mt10 and protamine‐like proteins genes in spermatozoa and gonads, respectively, of exposed mussels were registered by real‐time polymerase chain reaction analyses. We analyzed also the DNA binding affinity of protamine‐like proteins extracted from spermatozoa of exposed mussels. The results showed a higher DNA binding affinity and a different DNA binding mode in exposed mussels. Moreover, an increased amount of NaCl was required for the release from sperm nuclei of PL‐III, the main protamine‐like proteins component. Interestingly, protamine‐like proteins extracted from spermatozoa of exposed mussels promoted DNA oxidative damage in the presence of H2O2. These results demonstrate that also tolerable copper amount could affect the properties of protamine‐like proteins and determine negative effects on Mytilus galloprovincialis reproductive system. Finally, these analyses could be useful to develop quick and efficient chromatin‐based genotoxicity tests for pollution biomonitoring programs

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