Carmela Marinaro

Mytilus galloprovincialis is a significant indicator species due to its ability to bioaccumulate environmental pollutants, such as lead (Pb), which can hinder essential reproductive molecular processes. This study aimed to examine the effect of exposure to lead (0.5, 1.5 and 5 μg/L PbCl₂) on the state of protamine-like (PL) proteins—the primary components of sperm nuclear basic proteins—and their interaction with DNA. PL proteins were analysed using acetic acid–urea PAGE and SDS-PAGE, after which their ability to bind and protect DNA from oxidative damage was also assessed. Exposure to lead resulted in SDS-PAGE-detectable alterations of the PL, particularly at levels of 1.5 µg/L and 5 µg/L of PbCl₂ and modified their capacity for DNA-binding at all doses of PbCl₂. Experiments testing the release of PLs from sperm nuclei further confirmed this, revealing a reduced release. In addition, the ability of PL proteins to protect DNA from oxidative damage was reduced at the highest exposure dose, suggesting improper condensation of sperm chromatin. Computational analyses of human protamines in the presence of lead indicated the formation of coordination complexes with Pb²⁺ in PLI-II and PL-III, potentially impairing DNA binding. Overall, our study demonstrates that exposure to lead alters the function of PL proteins and potentially destabilises the sperm chromatin of M. galloprovincialis. This provides valuable insights into the reproductive toxicity of this metal.

Background: Since the COVID-19 pandemic, managing acute infections in symptomatic individuals, regardless of vaccination status, has been widely debated and extensively studied. Even more concerning, however, is the impact of COVID-19 on pregnant women—especially its effects on fetuses and newborns. Several studies have documented complications in both expectant mothers and their infants following infection. Methods: In our previous works, we provided scientific evidence of the bacteriophage behavior of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). This demonstrated that a well-defined combination of two antibiotics, amoxicillin and rifaximin, is associated with the same statistics for subjects affected by severe cases of SARS-CoV-2, regardless of vaccination status. We considered the few cases in the literature regarding the management of pregnancies infected with SARS-CoV-2, as well as previous data published in our works. In this brief case series, we present two pregnancies from the same unvaccinated mother—one prior to the COVID-19 pandemic and the other during the spread of the Omicron variant—as well as one pregnancy from a mother vaccinated against COVID-19. We describe the management of acute maternal infection using a previously published protocol that addresses the bacteriophage and toxicological mechanisms associated with SARS-CoV-2. Results: The three pregnancies are compared based on fetal growth and ultrasound findings. This report highlights that, even in unvaccinated mothers, timely and well-guided management of symptomatic COVID-19 can result in positive outcomes. In all cases, intrauterine growth remained within excellent percentiles, and the births resulted in optimal APGAR scores. Conclusions: This demonstrates that a careful and strategic approach, guided by ultrasound controls, can support healthy pregnancies during SARS-CoV-2 infection, regardless of vaccination status.

In the last decades, there has been huge interest in Per- and Polyfluoroalkyl Substances (PFAS) worldwide because of the toxic effects on humans. In 2013, a large-scale contamination of PFASs in the Veneto region was caused by a fluorochemical plant in Vicenza. About 130,000 inhabitants were exposed to PFAS in their drinking water. To date, relatively few studies have investigated the associations between blood serum PFAS concentrations and oxidative stress in semen. This study compared the antioxidant activity, lipoperoxide levels and protection or induction of oxidative DNA damage by sperm nuclear basic proteins (SNBP) in subjects living in Veneto exposed to PFAS (VNT) with those living in a non-PFAS contaminated area (VSL). Although the semen parameters were within the WHO range, the VNT semen samples showed higher levels of lipoperoxides and lower antioxidant activity compared to the VSL samples. These differences were statistically significant. We also examined DNA damage following SNBP addition under pro-oxidative conditions, finding a significantly different distribution of DNA damage types between the two groups, where 0 means no damage and 1 to 3 means increasing damage with 3 indicating maximum damage. SNBP of VNT subjects showed a reduced ability to protect DNA from oxidative damage. In the VSL group, damage 0 was found in 56% of subjects, 35% of the VNT group show damage 1, 36% damage 2 and 18% damage 3, while only 11% of VNT subjects show damage 0. Additionally, VNT with 0-grade DNA oxidative damage also exhibited reduced antioxidant activity and higher levels of lipoperoxides, in contrast to VSL. The results of this study indicate that exposure to PFAS produces oxidative stress in the semen of VNT subjects, who were also found to have blood serum perfluorooctanoic acid (PFOA) levels above the threshold. This suggests the possibility of infertility issues and emphasises the necessity for additional research into the long-term consequences of oxidative stress on male fertility and the health of offspring.

Environmental pollutants have pervasive and far-reaching effects on both ecosystems and human health

In recent years, metabolomics has become a valuable new resource in environmental monitoring programs based on the use of bio-indicators such as Mytilus galloprovincialis. The reproductive system is extremely susceptible to the effects of environmental pollutants, and in a previous paper, we showed metabolomic alterations in mussel spermatozoa exposed to metal chlorides of copper, nickel, and cadmium, and the mixture with these metals. In order to obtain a better overview, in the present work, we evaluated the metabolic changes in the male gonad under the same experimental conditions used in the previous work, using a metabolomic approach based on GC-MS analysis. A total of 248 endogenous metabolites were identified in the male gonads of mussels. Statistical analyses of the data, including partial least squares discriminant analysis, enabled the identification of key metabolites through the use of variable importance in projection scores. Furthermore, a metabolite enrichment analysis revealed complex and significant interactions within different metabolic pathways and between different metabolites. Particularly significant were the results on pyruvate metabolism, glycolysis, and gluconeogenesis, and glyoxylate and dicarboxylate metabolism, which highlighted the complex and interconnected nature of these biochemical processes in mussel gonads. Overall, these results add new information to the understanding of how certain pollutants may affect specific physiological functions of mussel gonads.

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.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl₂, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram– bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.