Samuele Vannini

Investigation of the cytotoxic, genotoxic, and apoptosis-inducing effects of estragole isolated from fennel (Foeniculum vulgare).

The present study was undertaken to evaluate, in the HepG2 human hepatoma cell line, the in vitro cytotoxic, genotoxic, and apoptotic activities of estragole (1), contained in the essential oil of Foeniculum vulgare (fennel) and suspected to induce hepatic tumors in susceptible strains of mice. Toward this end, an MTT cytotoxicity assay, a trypan blue dye exclusion test, a double-staining (acridine orange and DAPI) fluorescence viability assay, a single-cell microgel-electrophoresis (comet) assay, a mitochondrial membrane potential (Δψm) assay, and a DNA fragmentation analysis were conducted. In terms of potential genotoxic effects, the comet assay indicated that estragole (1) was not able to induce DNA damage nor apoptosis under the experimental conditions used.

Occupational exposure to cytostatic/antineoplastic drugs and cytogenetic damage measured using the lymphocyte cytokinesis-block micronucleus assay: A systematic review of the literature and meta-analysis.

Many studies have reported the occurrence of work-environment contamination by antineoplastic drugs (ANPD), with significant incorporation of trace amounts of these hazardous drugs in hospital personnel. Given the ability of most ANPD to actively bind DNA, thus inducing genotoxic effects, it is of pivotal importance to assess the degree of genotoxic damage (i.e., residual genotoxic risk) in occupationally exposed subjects. The lymphocyte cytokinesis-block micronucleus (L-CBMN) assay is largely used for biological effect monitoring in subjects occupationally exposed to ANPD. In this study, we identified and analyzed the studies published reporting the use of the L-CBMN assay as biomarker of genotoxic risk in health care workers exposed to ANPD with the aim of performing meta-analysis and providing a meta-estimate of the genotoxic effect of exposure. We retrieved 24 studies, published from 1988 to 2015, measuring MN in peripheral blood lymphocytes in health care workers occupationally exposed to ANPD. In 15 out of the 24 studies (62.5%), increased MN frequencies were recognized in exposed subjects as compared to controls. The meta-analysis of MN frequency of the combined studies confirmed an association between occupational exposure to ANPD and cytogenetic effects with an overall meta-estimate of 1.67 [95% CI: 1.41-1.98]. In 16 out of the 24 studies (66.6%) at least one other genotoxicity biomarker, besides L-CBMN assay, was employed for biological effect monitoring. In several studies the effect of exposure to ANPD was evaluated also in terms of MN in exfoliated buccal cells. Other studies focused on genotoxicity endpoints, such as sister chromatid exchanges (3 studies), chromosome aberrations (6 studies), or primary DNA damage investigated by comet assay (7 studies). Overall, there was good agreement between other genotoxicity tests employed and L-CBMN assay outcomes.

Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the bodys response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFβ (transforming growth factor β) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

Buccal micronucleus cytome assay in primary school children: A descriptive analysis of the MAPEC_LIFE multicenter cohort study

BACKGROUND Recent data support the hypothesis that genetic damage occurring early in life during childhood can play an important role in the development of chronic diseases in adulthood, including cancer. OBJECTIVES The objective of this paper, part of the MAPEC_LIFE project, is to describe the frequency of micronuclei and meta-nuclear alterations in exfoliated buccal cells of 6-8year-old Italian children recruited in five Italian towns (i.e., Brescia, Torino, Pisa, Perugia and Lecce) with different air pollution levels. METHODS About 200 children per town were recruited from primary schools. Biological samples were collected twice from the same children, in two different seasons (winter 2014-15 and late spring 2015). Cytogenetic damage was evaluated by the buccal micronucleus cytome assay. RESULTS Overall,n = 1046 children represent the final cohort of the MAPEC_LIFE study. On the whole, the results showed a higher mean MN frequency in winter (0.42 ± 0.54‰) than late-spring (0.22 ± 0.34‰). MN frequency observed among the five Italian towns showed a trend that follows broadly the levels of air pollution in Italy: the highest MN frequency was observed in Brescia during both seasons, the lowest in Lecce (winter) and Perugia (late-spring). CONCLUSIONS To the best of our knowledge, the number of recruited children included in the analysis (n = 1046) is the highest compared to previous studies evaluating the frequency of MN in exfoliated buccal cells so far. MN frequency was associated with winter season and living in towns at various levels of air pollution, suggesting an important role of this exposure in determining early cytogenetic effects.

AIR POLLUTION BIOLOGICAL EFFECTS IN CHILDREN LIVING IN LECCE (ITALY) BY BUCCAL MICRONUCLEUS CYTOME ASSAY (THE MAPEC_LIFE STUDY)

The aim of the MAPEC_LIFE (Monitoring Air Pollution Effects on Children for Supporting Public Health Policy) study is to evaluate the associations between the concentrations of air pollutants and early biological effects in children living in five Italian towns (Brescia, Torino, Lecce, Perugia and Pisa) characterised by varying levels of air pollution. This paper presents the results of micronucleus cytome assays performed on the oral mucosa cells of subjects living in Lecce (Puglia, Italy) and their relationship to factors associated with indoor/outdoor exposure and lifestyles. The study was conducted on 6-8-year-old schoolchildren living in Lecce. The micronucleus cytome assay was performed on exfoliated buccal cells collected from the oral mucosa of children using a soft-bristled toothbrush. Micronuclei were evaluated only in normal differentiated cells. Overall, 43.0% of the samples tested were positive, with an average frequency of 0.28 MN/1000 differentiated cells. Data analysis shows positive associations between the frequency of MN in the children’s buccal mucosa cells and obesity, heavy traffic and smoking mothers, while outdoor sports seem to have the opposite effect. These data will be integrated with data from the other cities involved in the MAPEC_LIFE study and could be used

Sulforaphane and Epigallocatechin Gallate Restore Estrogen Receptor Expression by Modulating Epigenetic Events in the Breast Cancer Cell Line MDA-MB-231: A Systematic Review and Meta-Analysis

Background/Aims: Epigenetics refers to modifications in gene activity and expression without alteration at the DNA sequence. Environment and diet could influence gene expression. Diet modifications may be meaningful in preventing and treating chronic diseases, cancer included. Dietary bioactive compounds, such as polyphenols (e.g., curcumin, resveratrol, or epigallocatechin gallate [EGCG]) or isothiocyanate (e.g., sulforaphane [SFN]), can regulate histone acetylation. The aim of this systematic review and meta-analysis was to evaluate the effect of SFN and EGCG on breast cancer (BC) cells cultured in vitro. Methods: Due to the enormous variability observed in study protocols and the innumerable genes involved, only studies analyzing the number of apoptotic cells in the MDA-MB-231 cell line were evaluated. The effect size (ES) was computed as the ratio of means. Results: We identified 7 studies, 4 regarding the effect of 10 µM SFN on MDA-MB-231 cells (ES = 4.59, 95% confidence interval 4.05-5.20) and 3 focusing on the impact of 20 µM EGCG (ES = 2.84, 95% confidence interval 2.60-3.10). Conclusion: The findings suggest beneficial effects of dietary bioactive compounds such as SFN and EGCG and their effect on BC cells by restoring estrogen receptor gene expression, modulating epigenetic changes and events, and interfering with tumor growth rate. Publication bias limits the generalizability of the conclusions. High-quality studies are needed.

In Vitro Protective Effects of Lycium barbarum Berries Cultivated in Umbria (Italy) on Human Hepatocellular Carcinoma Cells

Lycium barbarum is a famous plant in the traditional Chinese medicine. The plant is known to have health-promoting bioactive components. The properties of Lycium barbarum berries cultivated in Umbria (Italy) and their effect on human hepatocellular carcinoma cells (HepG2) have been investigated in this work. The obtained results demonstrated that the Lycium barbarum berries from Umbria region display high antioxidant properties evaluated by total phenolic content and ORAC method, on hydrophilic and lipophilic fractions. Moreover, on HepG2 cell line Lycium barbarum berries extract did not change cell viability analyzed by MTT and Trypan blue exclusion assay and did not induce genotoxic effect analyzed by comet assay. Furthermore, it was demonstrated, for the first time, that the berries extract showed a protective effect on DNA damage, expressed as antigenotoxic activity in vitro. Finally, Lycium barbarum berries extract was able to modulate the expression of genes involved in oxidative stress, proliferation, apoptosis, and cancer. In particular, downexpression of genes involved in tumor migration and invasion (CCL5), in increased risk of metastasis and antiapoptotic signal (DUSP1), and in carcinogenesis (GPx-3 and PTGS1), together with overexpression of tumor suppressor gene (MT3), suggested that Umbrian Lycium barbarum berries could play a protective role against hepatocellular carcinoma.

Primary DNA damage in welders occupationally exposed to extremely-low-frequency magnetic fields (ELF-MF)

BACKGROUND Electric arc welding is known to involve considerable exposure to extremely-low-frequency magnetic fields (ELF-MF; 50 Hz). The aim of the present study was to evaluate individual exposure to ELF-MF during arc welding and to assess the eventually associated genotoxic hazard by evaluating primary DNA damage. METHODS The study group comprised 21 electric arc welders (exposed) and 21 non-exposed control subjects (healthy blood donors). Occupational exposure to ELF-MF was measured using personal dosimeters worn during one complete work-shift (7 am to 5 pm). The extent of primary DNA damage was measured in peripheral blood leukocytes with the standard procedure of the alkaline comet assay. RESULTS Tail length showed to have similar values in welders and controls. Whereas, the data showed a significant decrease for tail intensity (p = 0.01) and tail moment (p = 0.02) counts in exposed subjects compared to controls. CONCLUSIONS The different results of our present study and published investigations from other research groups reporting positive results in the comet assay might be a result of different chromium and/or nickel (or other metals) exposure levels, which lead to DNA-protein cross-links at lower concentrations and DNA single-strand breakages at higher concentrations. Since these results are derived from a small-scale pilot study, a larger scale study should be undertaken.

Effects of the "PreveDi" lifestyle modification trial on metabolic syndrome.

BACKGROUND Metabolic syndrome (MetS) is a cluster of metabolic disorders that includes central obesity, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia and high blood pressure (BP). Statistical reports suggest that the prevalence of MetS has dramatically increased during the recent years and is considered a worldwide epidemic. MetS has been found to be associated with increased risk of all-cause mortality, cardiovascular diseases (CVD), type 2 diabetes mellitus, chronic kidney disease and some types of cancer. MetS has a high socioeconomic cost and it is therefore extremely important that MetS is prevented and treated by simple and feasible methods. METHODS The PreveDi study is a pilot before/after preventive trial aimed at the evaluation of the impact of a brief lifestyle intervention on changes in metabolic syndrome (MetS) risk factors. Recruitment was carried out in two community (council-run) pharmacies in the province of Perugia, Italy and the sample population consisted of 186 adults aged 45 or more who volunteered to participate. At enrolment the participants received a booklet illustrating general recommendations for MetS. During the 6-months follow-up period, participants were invited (by brochures and text messaging on cellular phones) to attend five conferences, five cooking classes, and twelve physical activity sessions. The conferences and the kitchen course were aimed to disseminate a healthy diet strategy focused primarily on reducing glycemic and insulinemic response. At baseline and follow-up MetS parameters were evaluated using medical equipments available in community pharmacies. RESULTS At baseline, MetS was observed approximately in 52.2% of the PreveDi population, the MetS prevalence decreased with a higher education level in women, but not in men. Attendance to intervention programs was low and there was no significant difference between physical activity recorded at baseline and at follow-up. A slightly increased adherence to the healthy diet recommendations was observed for males. Waist circumference, systolic and diastolic blood pressure, fasting glucose and triglycerides did not change significantly, whereas weight, BMI and total cholesterol did. At follow-up the prevalence of MetS decreased, though not significantly, only in women (from 54,1 to 45.2%). Moreover, in women the negative correlation with education level was lost. CONCLUSIONS This pilot study, even if with some limitations, suggests that MetS can be prevented and/or treated by simple and sustainable methods.

In vitro testing of estragole in HepG2 cells: Cytokinesis-block micronucleus assay and cell-cycle analysis

The alkenylbenzene estragole (systematic name, 1-allyl-4-methoxybenzene) is a natural component of essential oils from various spices and herbs, including fennel, and it is used as a food and beverage flavouring agent. Estragole has been reported to be hepatocarcinogenic at high doses in rodents. However, in a previous in vitro study, we found that estragole did not exhibit cytotoxic effects after 4 hours of exposure, nor did it induce DNA damage or apoptosis in human HepG2 hepatoblastoma cells. As fennel tea is widely used for symptomatic treatment of spasmodic gastrointestinal conditions in infants, we aimed at further assessing its safety in a different experimental setting. We thus searched for possible cytogenetic effects and interference with cell-cycle progression in the same human hepatoblastoma cell line. Estragole did not show any clastogenic/aneugenic activities in the cytokinesis-block micronucleus assay, and no effects on cell-cycle checkpoints were observed.