A novel black poplar propolis extract with promising health-promoting properties: focus on its chemical composition, antioxidant, anti-inflammatory, and anti-genotoxic activities.

Propolis is a resinous mixture produced by honeybees which has been used since ancient times for its useful properties. However, its chemical composition and bioactivity may vary, depending on the geographical area of origin and the type of tree bees use for collecting pollen. In this context, this research aimed to investigate the total phenolic content (using the Folin-Ciocalteu assay) and the total antioxidant capacity (using the FRAP, DPPH, and ABTS assays) of three black poplar (Populus nigra L.) propolis (BPP) solutions (S1, S2, and S3), as well as the chemical composition (HPLC-ESI-MSn) and biological activities (effect on cell viability, genotoxic/antigenotoxic properties, and anti-inflammatory activity, and effect on ROS production) of the one which showed the highest antioxidant activity (S1). The hydroalcoholic BPP solution S1 was a prototype of an innovative, research-type product by an Italian nutraceutical manufacturer. In contrast, hydroalcoholic BPP solutions S2 and S3 were conventional products purchased from local pharmacy stores. For the three extracts, 50 phenolic compounds, encompassing phenolic acids and flavonoids, were identified. In summary, the results showed an interesting chemical profile and the remarkable antioxidant, antigenotoxic, anti-inflammatory and ROS-modulating activities of the innovative BPP extract S1, paving the way for future research. In vivo investigations will be a possible line to take, which may help corroborate the hypothesis of the potential health benefits of this product, and even stimulate further ameliorations of the new prototype.

Eating and lifestyle habits and primary school health-related pro-grams: a survey involving Italian children and teachers.

Background: Childhood overweight and obesity represent serious public health concerns in several countries worldwide, including Italy, where the highest prevalence in Europe of overweight and obesity among primary school children was recorded. Among others, primary schools represent suitable social environments for health education projects. In this perspective, to optimize resources and plan successful activities, it is necessary, first of all, to analyze the context of the intervention. Study design: A pilot survey involving children and teachers was conducted in a primary school in Jesi (Le Marche Region, Italy). Methods: A questionnaire was submitted by teachers between May and June 2023 to 104 pupils (aged 6-11) from the five different primary school system classes. A qualitative questionnaire was also filled out by the same teachers (n = 5) who were present while the children were filling the eating/lifestyle questionnaire. Results: Most of the children (96.1%) had breakfast before school, with milk and cereal (51.5%) representing the preferred com-bination. During recess, 59.2% of pupils usually have a sandwich, 23.3% usually have sweet snacks, whereas only 2.9% have a fruit. Over 55% of the sample preferred drinking high-sugar beverages to water, and 15.5% declared going to a fast-food restaurant more than once a week. Over 17% of children did sport only once a week or less often. Over 64% of children habitually played videogames, and 77.6% normally watched TV or played with a tablet/smartphone while eating. Finally, the teachers' interview highlighted that there were no active health-related projects addressed to the pupils, and the school did not normally organize me-etings with health professionals to increase children's and their parents' knowledge and awareness about healthy eating habits. Conclusions: This preliminary investigation analyzed the context for a future health and nutrition education project and will assist researchers in planning successful activities and increasing the efficiency of the intervention.

Genotoxicity assessment of 1,4-anhydro-4-seleno-D-talitol (SeTal) in human liver HepG2 and HepaRG cells.

1,4-Anhydro-4-seleno-D-talitol (SeTal) is a highly water-soluble selenosugar with interesting antioxidant and skin-tissue-repair properties; it is highly stable in simulated gastric and gastrointestinal fluids and is a potential pharmaceutical ingredient that may be administered orally. Hepatic toxicity is often a major problem with novel drugs and can result in drug withdrawal from the market. Predicting hepatotoxicity is therefore essential to minimize late failure in the drug-discovery process. Herein, we report in vitro studies to evaluate the cytotoxic and genotoxic potential of SeTal in HepG2 and hepatocyte-like differentiated HepaRG cells. Except for extremely high concentrations (10 mM, 68 h-treatment in HepG2), SeTal did not affect the viability of each cell type. While the highest examined concentrations (0.75 and 1 mM in HepG2; 1 mM in HepaRG) were observed to induce primary DNA damage, SeTal did not exhibit clastogenic or aneugenic activity toward either HepG2 or HepaRG cells. Moreover, no significant cytostasis variations were observed in any experiment. The clearly negative results observed in the CBMN test suggest that SeTal might be used as a potential active pharmaceutical ingredient. The present study will be useful for the selection of non-toxic concentrations of SeTal in future investigations.

The DianaWeb cohort during the first COVID-19 lockdown: changes in eating behaviour in women with breast cancer.

BACKGROUND AND AIM OF THE WORK: Home confinement during the first wave of COVID-19 subverted people's routines and generated a lot of stress among individuals. In addition to the 'pure' mental health consequences-a major public health concern, itself-this stressful situation led to a risk of worsening eating behaviours. The aim of this study was to investigate the potential deterioration of dietary habits in a cohort of women with breast cancer (BC). METHODS: We used data from 781 women with BC enrolled in the DianaWeb project. We used validated questionnaires to collect data concerning socio-demographic/anthropometric parameters, quality of life (QoL), lifestyle and the change in dietary habits before and during the lockdown period (December 2019 and June 2020). RESULTS: Data showed that psychiatric treatment, QoL and health perception significantly affected the food choices of the cohort (p = 0.048, p=0.002, and p=0.001, respectively), decisively contributing to a worsening in their eating behaviour. Moreover, sedentary behaviour during the lockdown also influenced food choice (p = 0.010): individuals who increased their sedentary behaviour were more likely to decrease their intake of recommended foods (p = 0.033). CONCLUSIONS: In summary, emotional state and mood-here investigated as QoL and health perception-definitely affected dietary habits in women with BC in the first COVID-19 lockdown. Given the centrality of correct eating behaviour in BC management, psychological aspects should not be contemplated merely as confined mental health matters but should be definitely taken into consideration also as factors that seriously affect an individual's healthy lifestyle.

Moderate red wine intake and cardiovascular health protection: a literature review.

Wine is a complex matrix consisting primarily of water (86%) and ethyl alcohol (12%), as well as other different molecules, such as polyphenols, organic acids, tannins, compound minerals, vitamins and biologically active compounds which play an important role in the specific characteristics of each wine. According to the Dietary Guidelines for Americans 2015-2020, moderate red wine consumption-defined as up to two units of alcohol per day for men and up to one unit of alcohol per day for women-significantly reduces the risk of cardiovascular disease which represents the major causes of mortality, and disability, in developed countries. We reviewed the available literature concerning the potential relationship between moderate red wine consumption and cardiovascular health. We searched Medline, Scopus and Web of Science (WOS) for randomized controlled studies and case-control studies published from 2002 to 2022. A total of 27 articles were selected for the review. According to epidemiological evidence, drinking red wine in moderation lowers the risk of developing cardiovascular disease and diabetes. Red wine contains both alcoholic and non-alcoholic ingredients; however, it is yet unclear which is to blame for these effects. Combining wine with the diet of healthy individuals may add additional benefits. New studies should focus more on the characterization of the individual components of wine, to allow the analysis and study of the impact of each of them on the prevention and treatment of certain diseases.

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels.

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

Effect of Cooking and Domestic Storage on the Antioxidant Activity of Lenticchia di Castelluccio di Norcia, an Italian PGI Lentil Landrace.

The aim of this work was to characterise Lenticchia di Castelluccio di Norcia (an Italian PGI lentil landrace) and assess the impact of cooking and storage on antioxidant activity. After opening the package (T0), samples were analysed using a set of chemical assays (i.e., total phenolic content, DPPH, ABTS, and ORAC assays). Analyses were also conducted on boiled, pressure-cooked, and 6-month-stored (T1) products. At both T0 and T1, raw Lenticchia di Castelluccio di Norcia PGI showed higher total phenolic content (T0: 9.08 mg GAE/g, T1: 7.76 mg GAE/g) and antioxidant activity (DPPH T0: 33.02 µmol TE/g, T1: 29.23 µmol TE/g; ABTS T0: 32.12 µmol CE/g, T1: 31.77 µmol CE/g; ORAC T0: 3.58 µmol TE/g, T1: 3.60 µmol TE/g) than boiled and pressure-cooked samples. Overall, pressure-cooking led to a smaller decline in total phenolic content and antioxidant activity than the common boiling procedure. Domestic storage led to a significant reduction in total phenolic content-both in raw and cooked products-but not in antioxidant activity. In summary, these results highlighted interesting amounts of phenols and antioxidant properties of this product, showing the impact of routine procedures. Given the relevance of pulses as sustainable plant-based meat alternatives and their importance in the prevention of non-communicable diseases, health professionals should consider these aspects in the context of correct nutrition education and scientific communication.

In vitro toxicological assessment of PhSeZnCl in human liver cells.

Phenylselenenylzinc chloride (PhSeZnCl) is an air-stable selenolate, easily synthesizable through oxidative insertion of elemental zinc into the Se-halogen bond of the commercially available phenylselenyl chloride. PhSeZnCl was shown to possess a marked GPx-like activity both in NMR and in vitro tests, and to effectively react with cellular thiols, and was supposed for a potential use in the chemotherapy of drug-resistant cancers. However, activity of PhSeZnCl in hepatic cells has never been tested before now. In this in vitro approach, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as the effects on cell cycle of PhSeZnCl in two preclinical hepatic models, namely HepG2 and HepaRG cells. Results showed that cell viability of HepG2 and HepaRG cells decreased in a dose-dependent manner, with a more marked effect in HepG2 tumour cells. Moreover, treatment with 50 µg/mL PhSeZnCl caused an increase of primary DNA damage (4 h) and a statistically significant increase of HepG2 cells arrested in G2/M phase. In addition, it altered mitochondrial membrane potential and induced chromosomal DNA fragmentation (24 h). In HepaRG cells, PhSeZnCl was able to determine a cell cycle-independent induction of apoptosis. Particularly, 50 µg/mL induced mitochondrial membrane depolarization after 24 h and apoptosis after 4 h treatment. Futhermore, all PhSeZnCl concentrations tested determined a significant increase of apoptotic cells after 24 h. Apoptosis was also highlighted by the detection of active Caspase-3 by Western Blot analysis after 24 h exposure. In conclusion, this first toxicological assessment provides new insights into the biological activity of PhSeZnCl in preclinical hepatic models that will be useful in future safety assessment investigation of this compound as a potential pharmaceutical. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00148-y.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

How the COVID-19 pandemic has affected eating habits and physical activity in breast cancer survivors: the DianaWeb study.

PURPOSE: Breast cancer (BC) survivors are increasingly interested in learning about healthy lifestyles to reduce the risk of BC recurrence and mortality. The DianaWeb study, a community-based participatory research, offers BC patients a specific interactive website to help them in adopting and maintain correct lifestyles, in line with World Cancer Research Fund recommendations. However, to limit the spread of the COVID-19 pandemic, most countries introduced restrictions which, inevitably, caused sudden lifestyle changes. The current study aimed at evaluating changes in lifestyle in BC survivors before, during, and after the first two waves of the COVID-19 pandemic. METHODS: We used data of 224 BC cancer survivors enrolled in the DianaWeb study. We evaluated the adherence to physical activity (PA) guidelines, to Mediterranean diet (MD), and WCRF recommendations, at baseline, during and after the first two waves of the COVID-19 pandemic. We estimated the association between PA, MD, and WCRF adherence with sociodemographic characteristics, years from enrolment in the DianaWeb study, and type of breast cancer. RESULTS: As expected, during confinement periods, we observed a significant decrease in walking activity and in the percentage of patients performing vigorous PA. In post-quarantine, total weekly energy expenditure increased significantly. BC patients participating in the DianaWeb study for more than 3 years were more likely to be more active. There were no changes in MD and WCRF adherence. CONCLUSIONS: Our results show that the proposed interactive website was useful in achieving durable lifestyle changes, that have not been undermined either during the COVID-19 pandemic. IMPLICATIONS FOR CANCER SURVIVORS: Patient education is essential for guiding BC survivors toward improving their health outcomes; therefore, an interactive website like the one proposed by the DianaWeb study may be useful to improve healthy lifestyles.

Cytotoxicity and Genotoxicity of Senecio vulgaris L. Extracts: An In Vitro Assessment in HepG2 Liver Cells.

Senecio vulgaris L. is a herbaceous species found worldwide. The demonstrated occurrence of pyrrolizidine alkaloids in this species and its ability to invade a great variety of habitats result in a serious risk of contamination of plant material batches addressed to the herbal teas market; this presents a potential health risk for consumers. In light of the above, this work aimed to assess the cytotoxic and genotoxic activity of S. vulgaris extracts in HepG2 cells. Dried plants were ground and extracted using two different methods, namely an organic solvent-based procedure (using methanol and chloroform), and an environmentally friendly extraction procedure (i.e., aqueous extraction), which mimicked the domestic preparation of herbal teas (5, 15, and 30 min of infusion). Extracts were then tested in HepG2 cells for their cytotoxic and genotoxic potentialities. Results were almost superimposable in both extracts, showing a slight loss in cell viability at the highest concentration tested, and a marked dose-dependent genotoxicity exerted by non-cytotoxic concentrations. It was found that the genotoxic effect is even more pronounced in aqueous extracts, which induced primary DNA damage after five minutes of infusion even at the lowest concentration tested. Given the broad intake of herbal infusions worldwide, this experimental approach might be proposed as a screening tool in the analysis of plant material lots addressed to the herbal infusion market.

Cytogenetic Effects in Children Exposed to Air Pollutants: A Systematic Review and Meta-Analysis.

The aim of this systematic review and meta-analysis was to assess the association between exposure to ambient air pollutants and micronuclei (MN) frequency in children. This work was performed according to the Cochrane Collaboration and the PRISMA guidelines and recommendations. Articles published before November 2021 were identified by an advanced search on PubMed/MEDLINE, Scopus and Web of Science databases. A critical appraisal using a specific tool was conducted to assess the quality of each included study. All analyses were carried out by using the Review Manager (RevMan) 5.4 software (The Cochrane Collaboration, London, UK). One hundred and forty-five references were firstly identified, and, at the end of selection process, 13 studies met the inclusion criteria. Six studies carried out a direct evaluation through the use of air samplers, whereas the other ones accessed environmental databases (n = 2) or used other tools (n = 3). In two cases, exposure was not directly investigated, with children sampled in two different areas with well-known different levels of pollution. The overall effect size (ES) was 1.57 ((95% CI = 1.39; 1.78), p-value < 0.00001) (total evaluated subjects: 4162), which highlighted a statistically significant association between outdoor air pollution and MN frequency in children. As a high MN frequency has been associated with a number of pathological states and a higher risk of developing chronic degenerative diseases, our results should be taken into consideration by policy makers to design and implement interventions aimed at reducing the introduction of pollutants in the atmosphere as well as at minimizing the exposure extent, particularly in children.

Rice-Husk Shredding as a Means of Increasing the Long-Term Mechanical Properties of Earthen Mixtures for 3D Printing.

This paper is part of a study on earthen mixtures for the 3D printing of buildings. To meet the ever increasing environmental needs, the focus of the paper is on a particular type of biocomposite for the stabilization of earthen mixtures-the rice-husk-lime biocomposite-and on how to enhance its effect on the long-term mechanical properties of the hardened product. Assuming that the shredding of the vegetable fiber is precisely one of the possible ways to improve the mechanical properties, we compared the results of uniaxial compression tests performed on cubic specimens, made with both shredded and unaltered vegetable fiber, for three curing periods. The results show that the hardened earthen mixture is not a brittle material, in the strict sense, because it exhibits some peculiar behaviors that are anomalous for a brittle material. However, being a "designable" material, its properties can be varied with a certain flexibility in order to become as close as possible to the desired ones. One of the peculiar properties of the hardened earthen mixture deserves further investigation, rather than corrections. This is the vulcanization that occurs (in a completely natural way) over the long term, thanks to the mineralization of the vegetable fiber by the carbonation of the lime.

Mechanical Properties of a 3D-Printed Wall Segment Made with an Earthen Mixture.

This study provides a contribution to the research field of 3D-printed earthen buildings, focusing, for the first time, on the load-bearing capacity of these structures. The study involves the entire production and testing process of the earthen elements, from the design, to the preparation of the mixture and the 3D printing, up to the uniaxial compression test on a wall segment. The results indicate that 3D-printed earthen elements have a compressive strength of 2.32 MPa, comparable to that of rammed earth structures. The experimental data also made it possible to draw conclusions on the action of the infill, which seems to have the function of stopping the propagation of cracks. This has a positive effect on the overall behavior of 3D-printed earthen elements, since it avoids the onset of dilative behavior in the final stages of the load test and maintains ultimate load values higher than 50% of the maximum load.

Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

COVID19 outbreak in Lombardy, Italy: An analysis on the short-term relationship between air pollution, climatic factors and the susceptibility to SARS-CoV-2 infection.

Short-term exposure to air pollution, as well as to climate variables have been linked to a higher incidence of respiratory viral diseases. The study aims to assess the short-term influence of air pollution and climate on COVID19 incidence in Lombardy (Italy), during the early stage of the outbreak, before the implementation of the lockdown measures. The daily number of COVID19 cases in Lombardy from February 25th to March 10th, 2020, and the daily average concentrations up to 15 days before the study period of particulate matter (PM10, PM2.5), O3, SO2, and NO2 together with climate variables (temperature, relative humidity - RH%, wind speed, precipitation), were analyzed. A univariable mixed model with a logarithm transformation as link function was applied for each day, from 15 days (lag15) to one day (lag1) before the day of detected cases, to evaluate the effect of each variable. Additionally, change points (Break Points-BP) in the relationship between incident cases and air pollution or climatic factors were estimated. The results did not show a univocal relationship between air quality or climate factors and COVID19 incidence. PM10, PM2.5 and O3 concentrations in the last lags seem to be related to an increased COVID19 incidence, probably due to an increased susceptibility of the host. In addition, low temperature and low wind speed in some lags resulted associated with increased daily COVID19 incidence. The findings observed suggest that these factors, in particular conditions and lags, may increase individual susceptibility to the development of viral infections such as SARS-CoV-2.

Effects of Titanium Dioxide Nanoparticles on Porcine Prepubertal Sertoli Cells: An "In Vitro" Study.

The increasing use of nanomaterials in a variety of industrial, commercial, medical products, and their environmental spreading has raised concerns regarding their potential toxicity on human health. Titanium dioxide nanoparticles (TiO2 NPs) represent one of the most commonly used nanoparticles. Emerging evidence suggested that exposure to TiO2 NPs induced reproductive toxicity in male animals. In this in vitro study, porcine prepubertal Sertoli cells (SCs) have undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposures at both subtoxic (5 µg/ml) and toxic (100 µg/ml) doses of TiO2 NPs. After performing synthesis and characterization of nanoparticles, we focused on SCs morphological/ultrastructural analysis, apoptosis, and functionality (AMH, inhibin B), ROS production and oxidative DNA damage, gene expression of antioxidant enzymes, proinflammatory/immunomodulatory cytokines, and MAPK kinase signaling pathway. We found that 5 µg/ml TiO2 NPs did not induce substantial morphological changes overtime, but ultrastructural alterations appeared at the third week. Conversely, SCs exposed to 100 µg/ml TiO2 NPs throughout the whole experiment showed morphological and ultrastructural modifications. TiO2 NPs exposure, at each concentration, induced the activation of caspase-3 at the first and second week. AMH and inhibin B gene expression significantly decreased up to the third week at both concentrations of nanoparticles. The toxic dose of TiO2 NPs induced a marked increase of intracellular ROS and DNA damage at all exposure times. At both concentrations, the increased gene expression of antioxidant enzymes such as SOD and HO-1 was observed whereas, at the toxic dose, a clear proinflammatory stress was evaluated along with the steady increase in the gene expression of IL-1α and IL-6. At both concentrations, an increased phosphorylation ratio of p-ERK1/2 was observed up to the second week followed by the increased phosphorylation ratio of p-NF-kB in the chronic exposure. Although in vitro, this pilot study highlights the adverse effects even of subtoxic dose of TiO2 NPs on porcine prepubertal SCs functionality and viability and, more importantly, set the basis for further in vivo studies, especially in chronic exposure at subtoxic dose of TiO2 NPs, a condition closer to the human exposure to this nanoagent.

B-Comet Assay (Comet Assay on Buccal Cells) for the Evaluation of Primary DNA Damage in Human Biomonitoring Studies.

Many subjects perceive venous blood collection as too invasive, and thus moving to better-accepted procedures for leukocytes collection might be crucial in human biomonitoring studies (e.g., biomonitoring of occupational or residential exposure to genotoxins) management. In this context, primary DNA damage was assessed in buccal lymphocytes (BLs), fresh whole venous, and capillary blood leukocytes, and compared with that in peripheral blood lymphocytes (PBLs)-the most frequently used cells-in 15 young subjects. Mouthwashes were collected after the volunteers rinsed their mouths with normal saline, and BLs were isolated by density gradient centrifugation. Blood samples were collected by venipuncture or by lancet. Anthropometric and lifestyle information was obtained by the administration of a structured questionnaire. As shown in the Bland-Altman plots, the level of agreement between BLs and PBLs lied within the accepted range, we thus enrolled a wider population (n = 54) to assess baseline DNA damage in BLs. In these cells, mean values of tail length (µm), tail intensity (%), and tail moment were 25.7 ± 0.9, 6.7 ± 0.4 and 1.0 ± 0.1, respectively. No significant association was observed between sex and smoking habit with any of the DNA damage parameters. Conversely, underweight subjects displayed significantly higher genomic instability compared with normal weight group (p < 0.05). In conclusion, we successfully managed to set up and update a non-invasive and well-accepted procedure for the isolation of BLs from saliva that could be useful in upcoming biomonitoring studies.

Imbalance in the antioxidant defence system and pro-genotoxic status induced by high glucose concentrations: In vitro testing in human liver cells.

It has been hypothesized that high glucose concentrations might contribute to the overall intracellular oxidative stress either by the direct generation of reactive oxygen species (ROS) or by altering the redox balance. Moreover, it has also been suggested that high glucose concentration can increase the susceptibility of DNA to genotoxic effects of xenobiotics. The aim of this approach was to test high glucose concentrations for pro-genotoxicity in human liver cells by setting up an in vitro model for hyperglycaemia. The experimental design included performing of tests on both human HepG2 tumour cells and HepaRG immortalized cells. Increased cell susceptibility to genotoxic xenobiotics was tested by challenging cell cultures with 4-nitroquinoline-N-oxide (4NQO) and evaluating the extent of primary DNA damage by comet assay. Moreover, we evaluated the relationship between glucose concentration and intracellular ROS, as well as the effects of glucose concentration on the induction of Nrf2-dependent genes such as Glutathione S-transferases, Heme­oxygenase-1, and Glutathione peroxidase-4. To investigate the involvement of ROS in the induced pro-genotoxic activity, parallel experimental sets were set up by considering co-treatment of cells with the model mutagen 4NQO and the antioxidant, glutathione precursor N-acetyl-L-cysteine. High glucose concentrations caused a significant increase in the levels of primary DNA damage, with a pro-genotoxic condition closely related to the concentration of glucose in the culture medium when cells were exposed to 4NQO. High glucose concentrations also stimulated the production of ROS and down-regulated genes involved in contrasting of the effects of oxidative stress. In conclusion, in the presence of high concentrations of glucose, the cells are in unfavourable conditions for the maintenance of genome integrity.

The Exposure to Osteoarthritic Synovial Fluid Enhances the Immunomodulatory Profile of Adipose Mesenchymal Stem Cell Secretome.

OBJECTIVE: Several clinical studies have proposed the infusion of adipose mesenchymal stem cells (AMSCs) as an alternative therapy for joint diseases with inflammatory components, such as osteoarthritis. Indeed, AMSCs are able to stimulate tissue repair through a paracrine activity and the interaction with the inflammatory microenvironment seems to have a critical role. DESIGN: To reproduce the inflammatory microenvironment, AMSCs were exposed to osteoarthritic synovial fluid (SF) for 48 h and the effect of their secretome on differentiation of monocytes (M0) into macrophages M1-like and mature dendritic cells (mDCs) was evaluated. Furthermore, the effect of the secretome of AMSCs exposed to SF was evaluated on the T cell population in terms of T cell proliferation and expansion of T regulatory cells (T reg). RESULTS: Our data show that the exposure of AMSCs to SF activates cells and promotes the release of immunosuppressive factors, which induce macrophage polarization of M0 into the M2-like phenotype and inhibit differentiation of monocytes into mature dendritic cells (mDCs). Only the secretome of exposed AMSCs was able to inhibit T cell proliferation and promote T reg expansion. CONCLUSIONS: Our results suggest that the microenvironment plays a fundamental role for the development of anti-inflammatory and immunomodulatory properties of AMSCs.