Production and Characterization of New Biosurfactants/Bioemulsifiers from Pantoea alhagi and Their Antioxidant, Antimicrobial and Anti-Biofilm Potentiality Evaluations.

The present work aimed to develop rapid approach monitoring using a simple selective method based on a positive hemolysis test, oil spreading activity and emulsification index determinations. It is the first to describe production of biosurfactants (BS) by the endophytic Pantoea alhagi species. Results indicated that the new BS evidenced an E24 emulsification index of 82%. Fourier-transform infrared (FTIR) results mentioned that the described BS belong to the glycolipid family. Fatty acid profiles showed the predominance of methyl 2-hyroxydodecanoate in the cell membrane (67.00%) and methyl 14-methylhexadecanoate (12.05%). The major fatty acid in the BS was oleic acid (76.26%), followed by methyl 12-methyltetradecanoate (10.93%). Markedly, the BS produced by the Pantoea alhagi species exhibited antimicrobial and anti-biofilm activities against tested human pathogens. With superior antibacterial activity against Escherchia coli and Staphylococcus aureus, a high antifungal effect was given against Fusarium sp. with a diameter of zone of inhibition of 29.5 mm, 36 mm and 31 mm, obtained by BS dissolved in methanol extract. The DPPH assay indicated that the BS (2 mg/mL) showed a higher antioxidant activity (78.07 inhibition percentage). The new BS exhibited specific characteristics, encouraging their use in various industrial applications.

In Vitro Combination of Ascorbic and Ellagic Acids in Sperm Oxidative Damage Inhibition.

It is known that an altered redox balance interferes with normal spermatic functions. Exposure to genotoxic substances capable of producing oxidative stress (OS) can cause infertility in humans. The use of antioxidants to reduce oxidative stress contributes to the improvement in reproductive function. This study focused on an antigenotoxic evaluation of ellagic acid (EA) and ascorbic acid (AA) in combination against benzene genotoxic action on human spermatozoa in vitro. In addition to the evaluation of sperm parameters, damage in sperm genetic material and intracellular ROS quantification were assessed after AA, EA and benzene co-exposure using the TUNEL technique and DCF assay. The results showed that the combination of the two antioxidants generates a greater time-dependent antigenotoxic action, reducing both the sperm DNA fragmentation index and the oxidative stress. The genoprotective effect of AA and EA association in sperm cells lays the foundations for a more in-depth clinical study on the use of antioxidants as a therapy for male infertility.

In vitro ameliorative effects of ellagic acid on vitality, motility and DNA quality in human spermatozoa.

Oxidative stress (OS) plays a significant role in the etiology of male infertility, resulting in the impairment of male reproduction. This condition, characterized by an imbalance in the levels of oxidizing and antioxidant species in the seminal fluid, has a harmful impact on sperm functions and DNA integrity. The present study aimed to evaluate the anti-genotoxic action of ellagic acid, a polyphenolic molecule of natural origin having a powerful antigenotoxic, anti-inflammatory and antiproliferative role. An OS condition was induced in vitro by incubating normozoospermic human semen samples in benzene for 45, 60 and 90 min. DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, RAPD-PCR was performed to calculate the genome template stability, while the percentage of intracellular reactive oxygen species (ROS) was assessed by the 2', 7'-dichlorofluorescein assay. Our results showed that ellagic acid has a consistent protective effect on DNA integrity, as well as on sperm vitality and motility, by counteracting generation of intracellular ROS. The results of this study suggest ellagic acid as a suitable molecule to protect sperm DNA from oxidative stress, with a potentially significant translational impact on the management of the male infertility.

Protective activity of ellagic acid in counteract oxidative stress damage in zebrafish embryonic development.

During its development, embryo is easily susceptible to reactive oxygen species (ROS). Evidence demonstrate protective role of the antioxidants, improving both cellular growth and embryonic development. Among these, ellagic acid (EA) is a natural antioxidant with anti-inflammatory and anti-carcinogen properties. The aim of this work was to assess in vitro the protective and anti-genotoxic role of EA during Danio rerio (zebrafish) embryonic development. For the study, zebrafish embryos were treated with H2O2 (15 µM, 30 µM and 45 µM) to simulate an oxidative damage, and with EA (2.5 mM, 5 mM and 10 mM) for 8, 20, 24, 48, 96 hpf (hours post fertilization). Vitality rate, alterations in the morphology and behavior of the larvae and the genomic stability were analyzed. The exposure to H2O2 caused genotoxicity for all exposure times. The incubation in 45 µM H2O2 and 30 µM H2O2 resulted in increased mortality rate of the larvae, as well as 10 mM EA. The co-exposure was performed using to 15 µM H2O2 and 2.5 mM and 5 mM EA and it demonstrated the EA capacity to protect the embryo DNA and development from the oxidative insult. Particularly, the co-exposure to 15 mM H2O2 and 5 mM EA showed an increase in the embryo survival rate and absence of alterations in morphology and behavior at 96 hpf. Interestingly, we observed a higher genomic stability at 8h and 20h co-exposure (15 mM H2O2 and 5 mM EA) time. The decline observed in ROS concentration for both exposure times confirmed the observation. In conclusion, EA protects the zebrafish embryonic development from DNA oxidative damage increasing the embryo survival rate and improving morphological parameters of the larvae.

In vitro genotoxic effects of titanium dioxide nanoparticles (n-TiO2 ) in human sperm cells.

Titanium dioxide nanoparticles (TiO2 -NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO 2 -NPs genotoxic effects on human spermatozoa in vitro. TiO 2 -NPs are able to cross the blood-testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n-TiO 2 concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n-TiO 2 genotoxicity. Primary n-TiO 2 characterization was performed by transmission electron microscopy. The dispersed state of the n-TiO 2 in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD-PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD-PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n-TiO 2 . The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n-TiO 2 exposure. This research provides the evaluation of n-TiO 2 potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.

A prolonged microscopic observation improves detection of underpopulated cells in peripheral blood smears.

We evaluated an extended time in the microscopic review in samples in which the potential clinical information could be increased with respect to those that could be achieved with the usual laboratory methodologies. We used samples containing nucleated red blood cells in a small amount and cytopenic samples. For these purposes for each peripheral blood smear, the timing of eye-count differential was increased up to 20 min, regardless of the final number of cells which could be counted. In addition, an automated system for digital analysis of peripheral blood smears was employed and the number of cells counted was brought up to 1000 leukocytes. In both manual and automatic light microscopy extended observation, we obtained more diagnostic information in respect to those with routine or standard methods. Both automated and manual increase systems of the timing for microscopic review are useful tools to find diagnostic information that otherwise would be lost using normal and standard procedures. So, these methods should be used especially when there is a higher pre-test probability for discovery of pathological cells.

Genotoxicity assessment of TiO2 nanoparticles in the teleost Danio rerio.

Titanium dioxide nanoparticles (TiO2 NPs), widely used in paints, pharmaceutical preparations and in many consumer products, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses both in vitro and in vivo. Numerous studies have shown the potential impact of nanoparticles on a series of aquatic organisms and their toxicity has been linked to their dissolution, surface properties and size. In vitro studies have raised concerns about the toxicity of TiO2 NPs, but there are very limited data on ecotoxicity to aquatic life. This in vivo study aimed to describe the genotoxicity of TiO2 NPs in the zebrafish Danio rerio. After 2 weeks of adaptation, groups of zebrafish were exposed to TiO2 NPs (1 and 10µg/L) for 5, 7, 14, 21 and 28 days. The genotoxic potential of TiO2 NPs was assessed by the Comet assay, the Diffusion assay and RAPD-PCR technique. The use of multi-biomarkers has become an important aspect of ecotoxicology to evaluate environmental quality through a wide panel of biological responses triggered by contaminants. The highest genotoxic effect was observed at the maximum concentrations of nanoparticles (10µg/L) with all three tests at 14 and 21 days of exposure. The results suggests the presence of mechanisms that can reduce the n-TiO2 genotoxicity. Future studies are necessary to analyze the DNA repairing capacity in zebrafish cells and so verify the role of the antioxidant defence system in modulating the response to exposure to n-TiO2 in fish.

Anti-genotoxic ability of α-tocopherol and Anthocyanin to counteract fish DNA damage induced by musk xylene.

Many compounds released into the environment are able to interact with genetic material. The main purpose of genetic toxicology is to investigate the adverse effects of genotoxic molecules such as reduced fitness, changes in gene frequencies and their impact on genetic diversity in populations following genotoxic exposure. However, the ecological effects of many genotoxic compounds remain poorly understood. The aim of this research was to evaluate the genotoxic activity of an artificial musk (musk xylene, MX) and the potential anti-genotoxicity against this chemical compound of two antioxidant substances (α-tocopherol and an anthocyanins enriched extract). The studies were performed both in vivo and in vitro, using the teleost Danio rerio and the DLEC (Dicentrarchus labrax embryonic cells) cell line. We carried out the exposure to these substances at different times. DNA and cell damage and their possible repair were detected by various experimental approaches: DNA strand breaks (Comet Assay), degree of apoptosis (Diffusion Assay) and molecular alterations at the genomic level (RAPD-PCR technique). Data were collected and analyzed for statistical significance using the Student's t test. The results of this study showed that MX exhibited a genotoxic activity even after short exposure times. The anti-genotoxicity experiments evidenced that both α-tocopherol and Anthocyanin were able to contrast the genotoxic effects induced by MX, both in vivo and in vitro.

Reproductive cytotoxic and genotoxic impact of polystyrene microplastic on Paracentrotus lividus spermatozoa.

In recent decades, industrialization, intensive agriculture, and urban development have severely impacted marine environments, compromising the health of aquatic and terrestrial organisms. Inadequate disposal results in hundreds of tons of plastic products released annually into the environment, which degrade into microplastics (MPs), posing health risks due to their ability to biomagnify and bioaccumulate. Among these, polystyrene MPs (PS-MPs) are significant pollutants in marine ecosystems, widely studied for their reproductive toxicological effects. This research aimed to evaluate the reproductive cytotoxic and genotoxic effects of PS-MPs on sea urchin (Paracentrotus lividus) spermatozoa in vitro. Results showed that PS-MPs significantly reduced sperm viability and motility without altering morphology, and induced sperm DNA fragmentation mediated by reactive oxygen species production. Furthermore, head-to-head agglutination of the spermatozoa was observed exclusively in the sample treated with the plastic agents, indicating the ability of microplastics to adhere to the surface of sperm cells and form aggregates with microplastics on other sperm cells, thereby impeding movement and reducing reproductive potential. These findings suggest that PS-MPs can adversely affect the quality of sea urchin sperm, potentially impacting reproductive events.

Oxidative Stress Biomarkers in Male Infertility: Established Methodologies and Future Perspectives.

Male fertility can be affected by oxidative stress (OS), which occurs when an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them arises. OS can damage cells and influence sperm production. High levels of lipid peroxidation have been linked to reduced sperm motility and decreased fertilization ability. This literature review discusses the most commonly used biomarkers to measure sperm damage caused by ROS, such as the high level of OS in seminal plasma as an indicator of imbalance in antioxidant activity. The investigated biomarkers include 8-hydroxy-2-deoxyguanosine acid (8-OHdG), a marker of DNA damage caused by ROS, and F2 isoprostanoids (8-isoprostanes) produced by lipid peroxidation. Furthermore, this review focuses on recent methodologies including the NGS polymorphisms and differentially expressed gene (DEG) analysis, as well as the epigenetic mechanisms linked to ROS during spermatogenesis along with new methodologies developed to evaluate OS biomarkers. Finally, this review addresses a valuable insight into the mechanisms of male infertility provided by these advances and how they have led to new treatment possibilities. Overall, the use of biomarkers to evaluate OS in male infertility has supplied innovative diagnostic and therapeutic approaches, enhancing our understanding of male infertility mechanisms.

Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

BACKGROUND: Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. METHODS: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. RESULTS: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. CONCLUSIONS: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

Artificial Intelligence in Andrology: From Semen Analysis to Image Diagnostics.

Artificial intelligence (AI) in medicine has gained a lot of momentum in the last decades and has been applied to various fields of medicine. Advances in computer science, medical informatics, robotics, and the need for personalized medicine have facilitated the role of AI in modern healthcare. Similarly, as in other fields, AI applications, such as machine learning, artificial neural networks, and deep learning, have shown great potential in andrology and reproductive medicine. AI-based tools are poised to become valuable assets with abilities to support and aid in diagnosing and treating male infertility, and in improving the accuracy of patient care. These automated, AI-based predictions may offer consistency and efficiency in terms of time and cost in infertility research and clinical management. In andrology and reproductive medicine, AI has been used for objective sperm, oocyte, and embryo selection, prediction of surgical outcomes, cost-effective assessment, development of robotic surgery, and clinical decision-making systems. In the future, better integration and implementation of AI into medicine will undoubtedly lead to pioneering evidence-based breakthroughs and the reshaping of andrology and reproductive medicine.

Green Synthesis and Characterization of Novel Silver Nanoparticles Using Achillea maritima subsp. maritima Aqueous Extract: Antioxidant and Antidiabetic Potential and Effect on Virulence Mechanisms of Bacterial and Fungal Pathogens.

Novel silver nanoparticles were synthesized based on a simple and non-toxic method by applying the green synthesis technique, using, for the first time, the aqueous extract of an extremophile plant belonging to the Achillea maritima subsp. maritima species. AgNP characterization was performed via UV-Visible, front-face fluorescence spectroscopy, and FTIR and XRD analyses. AgNP formation was immediately confirmed by a color change from yellow to brown and by a surface plasmon resonance peak using UV-Vis spectroscopy at 420 nm. The biosynthesized AgNPs were spherical in shape with a size ranging from approximatively 14.13 to 21.26 nm. The presented silver nanoparticles exhibited strong antioxidant activity following a DPPH assay compared to ascorbic acid, with IC50 values of about 0.089 µg/mL and 22.54 µg/mL, respectively. The AgNPs showed higher antidiabetic capacities than acarbose, by inhibiting both alpha amylase and alpha glucosidase. The silver nanoparticles could affect various bacterial mechanisms of virulence, such as EPS production, biofilm formation and DNA damage. The silver nanoparticles showed no lysozyme activity on the cell walls of Gram-positive bacteria. The AgNPs also had a strong inhibitory effect on the Candida albicans virulence factor (extracellular enzymes, biofilm formation). The microscopic observation showed abnormal morphogenesis and agglomeration of Candida albicans exposed to AgNPs. The AgNPs showed no cytotoxic effect on human cells in an MTT assay. The use of novel silver nanoparticles is encouraged in the formulation of natural antimicrobial and antidiabetic agents.

Polymorphic Rearrangements of Human Chromosome 9 and Male Infertility: New Evidence and Impact on Spermatogenesis.

Chromosomal polymorphisms are structural variations in chromosomes that define the genomic variance of a species. These alterations are recurrent in the general population, and some of them appear to be more recurrent in the infertile population. Human chromosome 9 is highly heteromorphic, and how its rearrangement affects male fertility remains to be fully investigated. In this study, we aimed to investigate the association between the polymorphic rearrangements of chromosome 9 and male infertility via an Italian cohort of male infertile patients. Cytogenetic analysis was carried out, along with Y microdeletion screening, semen analysis, fluorescence in situ hybridization, and TUNEL assays using spermatic cells. Chromosome 9 rearrangements were observed in six patients: three of them showed a pericentric inversion, while the others showed a polymorphic heterochromatin variant 9qh. Of these, four patients exhibited oligozoospermia associated with teratozoospermia, along with a percentage of aneuploidy in the sperm of above 9%, in particular, an increase in XY disomy. Additionally, high values for sperm DNA fragmentation (≥30%) were observed in two patients. None of them had microdeletions to the AZF loci on chromosome Y. Our results suggest that polymorphic rearrangements of chromosome 9 might be associated with abnormalities in sperm quality due to incorrect spermatogenesis regulation.

Environmental and Genetic Traffic in the Journey from Sperm to Offspring.

Recent advancements in the understanding of how sperm develop into offspring have shown complex interactions between environmental influences and genetic factors. The past decade, marked by a research surge, has not only highlighted the profound impact of paternal contributions on fertility and reproductive outcomes but also revolutionized our comprehension by unveiling how parental factors sculpt traits in successive generations through mechanisms that extend beyond traditional inheritance patterns. Studies have shown that offspring are more susceptible to environmental factors, especially during critical phases of growth. While these factors are broadly detrimental to health, their effects are especially acute during these periods. Moving beyond the immutable nature of the genome, the epigenetic profile of cells emerges as a dynamic architecture. This flexibility renders it susceptible to environmental disruptions. The primary objective of this review is to shed light on the diverse processes through which environmental agents affect male reproductive capacity. Additionally, it explores the consequences of paternal environmental interactions, demonstrating how interactions can reverberate in the offspring. It encompasses direct genetic changes as well as a broad spectrum of epigenetic adaptations. By consolidating current empirically supported research, it offers an exhaustive perspective on the interwoven trajectories of the environment, genetics, and epigenetics in the elaborate transition from sperm to offspring.

Development of a risk score to predict portal vein tumor thrombosis in patients with hepatocellular carcinoma.

BACKGROUND: Portal vein tumor thrombosis (PVTT) is a common complication of hepatocellular carcinoma and is one of the most negative prognostic factors. The management of patients with PVTT is challenging. The aim of the study was to develop a score predictive of tumor thrombosis. METHODS: Data from a large cohort of 2243 hepatocellular carcinoma patients (all stages) recorded in the Progetto Epatocarcinoma Campania (January 2013-April 2021) database were analyzed. To construct the score, univariate generalized estimated equation models, the bootstrap approach for internal validation, and a regression coefficient-based scoring system were used. RESULTS: PVTT (any location) was found in 14.4% of cases and was related to shorter survival. Males, younger patients, and symptomatic cases were more prevalent among the PVTT group. At multivariate analysis, size ≥5 cm, massive or infiltrative hepatocellular carcinoma growth, and alpha-fetoprotein ≥400 ng/mL were significantly associated with PVTT. A risk prediction score of PVTT based on eight variables was developed. Using a continuous score, the risk was associated with an odds ratio (OR) of 1.30 (1.27-1.34; P  < 0.001). Considering a dichotomous score >8 versus a score ≤8 the OR for PVTT was 11.33 (8.55-15.00; P  < 0.001). CONCLUSION: The risk score for PVTT might be useful for clinicians to optimize hepatocellular carcinoma management by picking out patients with more aggressive cancers and higher mortality rates. Prospective validation of the score is needed before its application in daily clinical practice.

Controversy and Consensus on the Management of Elevated Sperm DNA Fragmentation in Male Infertility: A Global Survey, Current Guidelines, and Expert Recommendations.

PURPOSE: Sperm DNA fragmentation (SDF) has been associated with male infertility and poor outcomes of assisted reproductive technology (ART). The purpose of this study was to investigate global practices related to the management of elevated SDF in infertile men, summarize the relevant professional society recommendations, and provide expert recommendations for managing this condition. MATERIALS AND METHODS: An online global survey on clinical practices related to SDF was disseminated to reproductive clinicians, according to the CHERRIES checklist criteria. Management protocols for various conditions associated with SDF were captured and compared to the relevant recommendations in professional society guidelines and the appropriate available evidence. Expert recommendations and consensus on the management of infertile men with elevated SDF were then formulated and adapted using the Delphi method. RESULTS: A total of 436 experts from 55 different countries submitted responses. As an initial approach, 79.1% of reproductive experts recommend lifestyle modifications for infertile men with elevated SDF, and 76.9% prescribe empiric antioxidants. Regarding antioxidant duration, 39.3% recommend 4-6 months and 38.1% recommend 3 months. For men with unexplained or idiopathic infertility, and couples experiencing recurrent miscarriages associated with elevated SDF, most respondents refer to ART 6 months after failure of conservative and empiric medical management. Infertile men with clinical varicocele, normal conventional semen parameters, and elevated SDF are offered varicocele repair immediately after diagnosis by 31.4%, and after failure of antioxidants and conservative measures by 40.9%. Sperm selection techniques and testicular sperm extraction are also management options for couples undergoing ART. For most questions, heterogenous practices were demonstrated. CONCLUSIONS: This paper presents the results of a large global survey on the management of infertile men with elevated SDF and reveals a lack of consensus among clinicians. Furthermore, it demonstrates the scarcity of professional society guidelines in this regard and attempts to highlight the relevant evidence. Expert recommendations are proposed to help guide clinicians.

Micronucleus test and comet assay for the evaluation of zebrafish genomic damage induced by erythromycin and lincomycin.

An enormous quantity of pharmacologically active principles are currently being introduced into the environment, with consequent escalation of environmental problems, but only a small number of studies are focusing on an assessment of their genotoxic effects. The aim of this article is to assess the genotoxic effects of erythromycin, lincomycin, and of a combination of these two antibiotics on the genome of the zebrafish. The genotoxicity of the two antibiotics was assessed by applying the micronucleus test to erythrocytes and performing a Comet assay on erythrocytes and hepatocytes. The fish were exposed to antibiotics at different concentrations and times of exposure, under standard laboratory conditions. Depending on the different experimental conditions, erythromycin and lincomycin induced a significant increase in DNA migration (tail moment) and a significant increase in micronuleus frequency. We also conducted an analysis on the activation of repair mechanisms when the genotoxic agent was removed. Only a few of the cells displayed a decrease in damage under these test conditions.

Genotoxic effects in fish induced by pharmacological agents present in the sewage of some Italian water-treatment plants.

The presence of pharmaceutical substances in the municipal effluents is currently considered the principal source of bio-active molecule emissions into aquatic environments. This study analyzes the genotoxic damage caused by gemfibrozil and atorvastatin, two regulators of the hematic level of lipids, and sildenafil citrate, a vasodilator, on the teleost Danio rerio. The genotoxicity of these three compounds was evaluated using the comet assay, diffusion assay, and RAPD-PCR. The alkaline version (pH 12.1) of the comet assay was used for the erythrocytes of the zebrafish to evaluate the presence of single strand DNA breaks. Furthermore, the diffusion assay was used to estimate the number of apoptotic cells. The fish were treated with the three pharmacological agents at the average concentrations previously found at some Italian treatment plants and were then sacrificed from 5 to 35 days after exposure. The data of the comet assay showed a statistically significant loss of DNA integrity after 5 days of exposure to atorvastatin and after one week of exposure to gemfibrozil. This damage was, however, repaired after 14 days. Sildenafil citrate produced, instead, a statistically significant loss of DNA integrity at the concentrations found only after 35 days of exposure. The genotoxicity at the molecular level was tested by RAPD-PCR. The results from this investigation are in agreement with those from two other tests, confirming the efficacy of the use of the three experimental approaches for the complete evaluation of genotoxic damage.

Evaluation of Zebrafish DNA Integrity after Individual and Combined Exposure to TiO2 Nanoparticles and Lincomycin.

Environmental contamination by nanoparticles (NPs) and drugs represents one of the most debated issues of the last years. The aquatic biome and, indirectly, human health are strongly influenced by the negative effects induced by the widespread presence of pharmaceutical products in wastewater, mainly due to the massive use of antibiotics and inefficient treatment of the waters. The present study aimed to evaluate the harmful consequences due to exposure to antibiotics and NPs, alone and in combination, in the aquatic environment. By exploiting some of their peculiar characteristics, such as small size and ability to bind different types of substances, NPs can carry drugs into the body, showing potential genotoxic effects. The research was conducted on zebrafish (Danio rerio) exposed in vivo to lincomycin (100 mg/L) and titanium dioxide nanoparticles (TiO2 NPs) (10 µg/L) for 7 and 14 exposure days. The effects on zebrafish were evaluated in terms of cell viability, DNA fragmentation, and genomic template stability (GTS%) investigated using Trypan blue staining, TUNEL assay, and the random amplification of polymorphic DNA PCR (RAPD PCR) technique, respectively. Our results show that after TiO2 NPs exposure, as well as after TiO2 NPs and lincomycin co-exposure, the percentage of damaged DNA significantly increased and cell viability decreased. On the contrary, exposure to lincomycin alone caused only a GTS% reduction after 14 exposure days. Therefore, the results allow us to assert that genotoxic effect in target cells could be through a synergistic effect, also potentially mediated by the establishment of intermolecular interactions between lincomycin and TiO2 NPs.