Micronuclei and chromosome aberrations in subjects occupationally exposed to antineoplastic drugs: a multicentric approach.

OBJECTIVES: Recently published works showed that occupational exposure to antineoplastic drugs (ANPD) is still frequent in hospital settings, despite significant safety policy improvements. The aim of this study was to assess the current level of occupational exposure to ANPD and any potentially associated cytogenetic damages in hospital nurses routinely handling ANPD. METHODS: Occupationally ANPD-exposed (n = 71) and ANPD-unexposed (n = 77; control) nurses were recruited on a voluntary basis from five hospitals in Northern and Central Italy. Evaluation of surface contamination and dermal exposure to ANPD was assessed by determining cyclophosphamide (CP) on selected surfaces (wipes) and on exposed nurses' clothes (pads). The concentration of unmetabolized CP­as a biomarker of internal dose­was measured in end-shift urine samples. Biomonitoring of genotoxic effects (i.e., biological effect monitoring) was conducted by analyzing micronuclei (MN) and chromosome aberrations (CA) in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e., glutathione S-transferases) were analyzed as well. RESULTS: We observed a significant increase in MN frequency (5.30 ± 2.99 and 3.29 ± 1.97; mean values ± standard deviation; p < 0.0001) in exposed nurses versus controls, as well as in CA detection (3.30 ± 2.05 and 1.84 ± 1.67; p < 0.0001), exposed subjects versus controls. Our results provide evidence that, despite safety controlled conditions, ANPD handling still represents a considerable genotoxic risk for occupationally exposed personnel. CONCLUSIONS: Because both MN and CA have been described as being predictive of group-increased cancer risk, our findings point to a need for improving specific safety procedures in handling and administering ANPD.

Associations between fine particulate matter, gene expression, and promoter methylation in human bronchial epithelial cells exposed within a classroom under Air-Liquid Interface.

Associations between indoor air pollution from fine particulate matter (PM with aerodynamic diameter dp < 2.5 µm) and human health are poorly understood. Here, we analyse the concentration-response curves for fine and ultrafine PM, the gene expression, and the methylation patterns in human bronchial epithelial cells (BEAS-2B) exposed at the air-liquid interface (ALI) within a classroom in downtown Rome. Our results document the upregulation of aryl hydrocarbon receptor (AhR) and genes associated with xenobiotic metabolism (CYP1A1 and CYP1B1) in response to single exposure of cells to fresh urban aerosols at low fine PM mass concentrations within the classroom. This is evidenced by concentrations of ultrafine particles (UFPs, dp < 0.1 µm), polycyclic aromatic hydrocarbons (PAH), and ratios of black carbon (BC) to organic aerosol (OA). Additionally, an interleukin 18 (IL-18) down-regulation was found during periods of high human occupancy. Despite the observed gene expression dysregulation, no changes were detected in the methylation levels of the promoter regions of these genes, indicating that the altered gene expression is not linked to changes in DNA methylation and suggesting the involvement of another epigenetic mechanism in the gene regulation. Gene expression changes at low exposure doses have been previously reported. Here, we add the possibility that lung epithelial cells, when singly exposed to real environmental concentrations of fine PM that translate into ultra-low doses of treatment, may undergo epigenetic alteration in the expression of genes related to xenobiotic metabolism. Our findings provide a perspective for future indoor air quality regulations. We underscore the potential role of indoor UFPs as carriers of toxic molecules with low-pressure weather conditions, when rainfall and strong winds may favour low levels of fine PM.

Emission Factors of CO2 and Airborne Pollutants and Toxicological Potency of Biofuels for Airplane Transport: A Preliminary Assessment.

Aviation is one of the sectors affecting climate change, and concerns have been raised over the increase in the number of flights all over the world. To reduce the climate impact, efforts have been dedicated to introducing biofuel blends as alternatives to fossil fuels. Here, we report environmentally relevant data on the emission factors of biofuel/fossil fuel blends (from 13 to 17% v/v). Moreover, in vitro direct exposure of human bronchial epithelial cells to the emissions was studied to determine their potential intrinsic hazard and to outline relevant lung doses. The results show that the tested biofuel blends do not reduce the emissions of particles and other chemical species compared to the fossil fuel. The blends do reduce the elemental carbon (less than 40%) and total volatile organic compounds (less than 30%) compared to fossil fuel emissions. The toxicological outcomes show an increase in oxidative cellular response after only 40 min of exposure, with biofuels causing a lower response compared to fossil fuels, and lung-deposited doses show differences among the fuels tested. The data reported provide evidence of the possibility to reduce the climate impact of the aviation sector and contribute to the risk assessment of biofuels for aviation.

Kinetics of gamma-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation.

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.

Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species.

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Is it the time to study air pollution effects under environmental conditions? A case study to support the shift of in vitro toxicology from the bench to the field.

Air pollution and particulate matter are recognised cause of increased disease incidence in exposed population. The toxicological processes underlying air pollution associated effects have been investigated by in vivo and/or in vitro experimentation. The latter is usually performed by exposing cells cultured under submerged condition to particulate matter concentration quite far from environmental exposure expected in humans. Here we report for the first time the feasibility of a direct exposure of air liquid interface cultured cells to environmental concentration of particulate matter. Inflammatory proteins release was analysed in cell medium while differential expression of selected genes was analysed in cells. Significant association of anti-oxidant genes was observed with secondary and aged aerosol, while cytochrome activation with primary and PAHs enriched ultrafine particles. The results obtained clearly show the opportunity to move from the lab bench to the field for properly understanding the toxicological effects also of ultrafine particles on selected in vitro models.

Cytotoxicity, genotoxicity and gene expression changes elicited by exposure of human hepatic cells to Ginkgo biloba leaf extract.

The use of Ginkgo biloba leaf extract as nutraceutical is becoming increasingly common. As a consequence, the definition of a reliable toxicological profile is a priority for its safe utilization. Recently, contrasting data have been reported on the carcinogenic potential of Ginkgo biloba extract in rodent liver. We measured viability, Reactive Oxygen Species (ROS), apoptosis, colony-forming efficiency, genotoxicity by comet assay, and gene expression changes associated with hepato-carcinogenicity in human cells of hepatic origin (HepG2 and THLE-2) treated with different concentrations (0.0005-1.2 mg/mL) of Ginkgoselect®Plus. Our analyses highlighted a decrease of cell viability, not due to apoptosis, after treatment with high doses of the extract, which was likely due to ROS generation by a chemical reaction between extract polyphenols and some components of the culture medium. Comet assay did not detect genotoxic effect at any extract concentration. Finally, the array analysis detected a slight decrease in the expression of only one gene (IGFBP3) in Ginkgo-treated THLE-2 cells as opposed to changes in 28 genes in Aflatoxin B1 treated-cells. In conclusion, our results did not detect any significant genotoxic or biologically relevant cytotoxic effects and gross changes in gene expression using the Ginkgo extract in the hepatic cells tested.

Biodistribution and acute toxicity of a nanofluid containing manganese iron oxide nanoparticles produced by a mechanochemical process.

Superparamagnetic iron oxide nanoparticles are candidate contrast agents for magnetic resonance imaging and targeted drug delivery. Biodistribution and toxicity assessment are critical for the development of nanoparticle-based drugs, because of nanoparticle-enhanced biological reactivity. Here, we investigated the uptake, in vivo biodistribution, and in vitro and in vivo potential toxicity of manganese ferrite (MnFe2O4) nanoparticles, synthesized by an original high-yield, low-cost mechanochemical process. Cultures of murine Balb/3T3 fibroblasts were exposed for 24, 48, or 72 hours to increasing ferrofluid concentrations. Nanoparticle cellular uptake was assessed by flow-cytometry scatter-light measurements and microscopy imaging after Prussian blue staining; cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony-forming assays. After a single intravenous injection, in vivo nanoparticle biodistribution and clearance were evaluated in mice by Mn spectrophotometric determination and Prussian blue staining in the liver, kidneys, spleen, and brain at different posttreatment times up to 21 days. The same organs were analyzed for any possible histopathological change. The in vitro study demonstrated dose-dependent nanoparticle uptake and statistically significant cytotoxic effects from a concentration of 50 µg/mL for the MTT assay and 20 µg/mL for the colony-forming assay. Significant increases in Mn concentrations were detected in all analyzed organs, peaking at 6 hours after injection and then gradually declining. Clearance appeared complete at 7 days in the kidneys, spleen, and brain, whereas in the liver Mn levels remained statistically higher than in vehicle-treated mice up to 3 weeks postinjection. No evidence of irreversible histopathological damage to any of the tested organs was observed. A comparison of the lowest in vitro toxic concentration with the intravenously injected dose and the administered dose of other ferrofluid drugs currently in clinical practice suggests that there might be sufficient safety margins for further development of our formulation.

Direct and delayed X-ray-induced DNA damage in male mouse germ cells.

Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Various stages of spermatogenesis are characterized by large differences in radiosensitivity. Differentiating spermatogonia are susceptible to radiation-induced cell killing, but some of them can repair DNA damage and progress through differentiation. In this study, we applied the neutral comet assay, immunodetection of phosphorylated H2AX (γ-H2AX) and the Sperm Chromatin Structure Assay (SCSA) to detect DNA strand breaks in testicular cells and spermatozoa at different times following in vivo X-ray irradiation. Radiation produced DNA strand breaks in testicular cells that were repaired within the first few hours after exposure. Spermatozoa were resistant to the induction of DNA damage, but non-targeted DNA lesions were detected in spermatozoa derived from surviving irradiated spermatogonia. These lesions formed while round spermatids started to elongate within the testicular seminiferous tubules. The transcription of pro-apoptotic genes at this time was also enhanced, suggesting that an apoptotic-like process was involved in DNA break production. Our results suggest that proliferating spermatogonia retain a memory of the radiation insult that is recognized at a later developmental stage and activates a process leading to DNA fragmentation.