Sara Levorato

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.

Socio-economic and environmental factors associated with overweight and obesity in children aged 6–8 years living in five Italian cities (The MAPEC_LIFE cohort)

The prevalence of obesity among Italian children has reached such alarming levels as to require detailed studies of the causes of the phenomenon. A cross-sectional study was carried out in order to assess the weight status of 1164 Italian children aged 6–8 years (the Monitoring Air Pollution Effects on Children for Supporting Public Health Policy (MAPEC_LIFE) cohort) and to identify any associations between selected socio-economic and environmental factors and overweight/obesity. The data were obtained by means of a questionnaire given to parents, and any associations were examined by binomial logistic regression analyses. Overweight was found to be positively associated with male gender, parents of non-Italian origin, and parents who smoke, and negatively associated with the parents’ level of education and employment. In addition, the frequency of overweight varied in relation to the geographical area of residence, with a greater prevalence of overweight children in the cities of central-southern Italy. This study highlights the need to implement appropriate obesity prevention programs in Italy, which should include educational measures concerning lifestyle for parents from the earliest stages of their child’s life.

Feasibility and reliability of a questionnaire for evaluation of the exposure to indoor and outdoor air pollutants, diet and physical activity in 6-8-year-old children

INTRODUCTION The MAPEC-Life project aims to study the biological effects of early exposure to air pollutants on the oral mucosa cells of school-age children in five Italian cities. A questionnaire was created to evaluate the association between outdoor and indoor airborne pollutants, lifestyle, diet and biomarker effects. The feasibility and reliability of the questionnaire were evaluated. METHODS A questionnaire was drawn up to be filled in by the parents of 6-8-year-old children. It consisted of 148 questions on the childrens health, physical activity, environmental exposures and the frequency of food consumption at the main meals. First we conducted a questionnaire feasibility study involving 53 volunteer parents. We then performed a reliability study by administering the questionnaire to a further 156 parents and again one month later (test/retest method). The correlations between answers at the first and second administration of the questionnaire were evaluated using the Kappa statistic and Spearmans coefficient. RESULTS After verifying the feasibility of the questionnaire, we conducted a reliability analysis on 132 completed questionnaires. The percentage of agreement between the first and the second responses given was over 70%, all K values being greater than 0.6. The analysis of calories and macronutrients also showed good agreement. CONCLUSIONS The questionnaire drawn up for the study proved to be sufficiently reliable for gathering information about the factors of interest in our study of the relationship between air pollution and early biological effects in children.

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.

Are 6-8 year old Italian children moving away from the Mediterranean diet?

BACKGROUND The Mediterranean diet (MD) is considered one of the healthiest dietary models, as it decreases the risk of chronic diseases and may modulate the organisms early response to environmental pollution. In recent decades, Mediterranean countries have been replacing their traditional diet with other less healthy eating habits, especially among children and teenagers. OBJECTIVE The aim of this study was to evaluate the MD and the level of adherence to it in 6-8 year old Italian children, in relation to residence, lifestyle, and social and family contexts. METHODS A questionnaire was administered to the childrens parents in two seasons in 5 Italian towns. The diet section contained 116 questions investigating the frequency of consumption of different types of food. The Italian Mediterranean Index (IMI) was calculated according to the intake of 6 typical Mediterranean and 4 non-Mediterranean foods. On the basis of IMI score, MD adherence was classified as low (≤ 3 IMI score), medium (4-5) and high (≥ 6). Total energy load and diet composition in micro- and macronutrients were calculated from consumption frequency. RESULTS Diet analysis was computed on 1164 subjects with two complete questionnaires. Body mass index, calculated for each subject, showed that 28.9% of the children were overweight, the figure varying slightly with area of residence. Our findings showed that 59.0% of the children had a low score for MD adherence. CONCLUSIONS The results of this study showed that most Italian children did not follow the MD and socio-economic characteristics appeared not to be associated with type of diet.

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.

In vitro toxicity evaluation of estragole-containing preparations derived from Foeniculum vulgare Mill. (fennel) on HepG2 cells

Estragole, a common component of herbs and spices, is a wellknown genotoxic hepatocarcinogen in rodents, whereas its potential toxic effect in humans is still debated. In the European contest, one of the major sources of human exposure to this phytochemical is Foeniculum vulgare Mill. (fennel). Therefore, the aim of this study was to evaluate the in vitro toxicity of estragole in the context of two complex phytochemical mixtures derived from fennel: fennel seed powder (FSPw) and fennel seed essential oil (FSEO). The estragole-containing preparations were analysed for their ability to cause cytotoxicity, genotoxicity, apoptosis and cell cycle perturbation in the human hepatoma (HepG2) cell line. None of the tested concentrations of FSPw induced DNA damage, nor apoptosis or cell cycle perturbation. Although FSEO did not induce any genetic damage as well, it exerted marked dose-dependent apoptotic effects on HepG2 cells with a concurrent cell cycle arrest in G2/M at the highest tested dose. Although prospective analyses are required to clarify the observed toxic effects of FSEO, our results support the hypothesis that the genotoxicity of estragole may be significantly reduced or null in the context of botanical mixtures.

No evidence of DNA damage by co-exposure to extremely low frequency magnetic fields and aluminum on neuroblastoma cell lines.

Whether exposure to 50-60Hz extremely low frequency magnetic fields (ELF-MF) exerts neurotoxic effects is a debated issue. Analogously, the potential role of Aluminum (Al) in neurodegeneration is a matter of controversial debate. As all living organisms are exposed to ELF-MF and/or Al daily, we found investigating the early effects of co-exposure to ELF-MF and Al in SH-SY5Y and SK-N-BE-2 human neuroblastoma (NB) cells intriguing. SH-SY5Y5 and SK-N-BE-2 cells underwent exposure to 50Hz ELF-MF (0.01, 0.1 or 1mT) or AlCl3 (4 or 40μM) or co-exposure to 50Hz ELF-MF and AlCl3 for 1h continuously or 5h intermittently. The effects of the treatment were evaluated in terms of DNA damage, redox status changes and Hsp70 expression. The DNA damage was assessed by Comet assay; the cellular redox status was investigated by measuring the amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) while the inducible Hsp70 expression was evaluated by western blot analysis and real-time RT-PCR. Neither exposure to ELF-MF or AlCl3 alone induced DNA damage, changes in GSH/GSSG ratio or variations in Hsp70 expression with respect to the controls in both NB cell lines. Similarly, co-exposure to ELF-MF and AlCl3 did not have any synergic toxic effects. The results of this in vitro study, which deals with the effects of co-exposure to 50Hz MF and Aluminum, seem to exclude that short-term exposure to ELF-MF in combination with Al can have harmful effects on human SH-SY5Y and SK-N-BE-2 cells.

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.