Every year, thousands of donors are exposed to granulocyte-colony stimulating factor (G-CSF) for stem cell mobilization in hematopoietic stem cell transplantations (HSCT). Previous studies about the genotoxicity of G-CSF were inconclusive. In this study, the genotoxic effects of G-CSF in peripheral blood stem cell (PBSC) donors were evaluated prospectively by using three different validated and reliable methods for the first time in the literature to the best of our knowledge.
Donors of PBSC transplantation (n=36), who received G-CSF were evaluated for genotoxicity by micronucleus test (MNT), nuclear division index (NDI), and comet assay (CA). Genotoxic effects are expected to cause an increase in MNT and CA values and decrease in NDI. Blood samples were collected at three timepoints (TP): before starting G-CSF (TP1), after G-CSF for five days (TP2), and one month after the last dose (TP3). Sixteen controls were included for baseline comparison of genotoxicity tests. CD34 cell counts and hemograms were also analyzed.
MNT and CA parameters; comet and tail length, tail DNA%, and tail moment, showed no change in time whereas another CA parameter, Olive`s tail moment (OTM) was increased significantly at TP3 compared to both baseline and TP2 (p=0.002 and p=0.017, respectively). Nuclear division index decreased significantly at TP2 (p < 0.001), then increased above baseline at TP3 (p=0.004). Baseline comparison with controls showed higher MN frequency in donors without statistical significance (p=0.059). Whereas, CA results were significantly higher in controls. CD34 cell count showed moderate positive correlation with white blood cell count at TP2 (Pearson R=0.495, p=0.004).
Our results showed the genotoxic effect of G-CSF in healthy donors, in two of the three tests performed, short-term effect in NDI, and long-lasting effect in OTM. So, this study provides novel information for the debate about the genotoxicity of G-CSF and supports the need for further studies with a larger sample size and longer follow-up.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

Environmental studies which aim to assess the ecological impact of chemical and other types of pollution should employ a complex weight-of-evidence approach with multiple lines of evidence (LoEs). This study focused on in situ genotoxicological methods such as the comet and micronucleus assays and randomly amplified polymorphic DNA analysis as one of the multiple LoEs (LoE3) on the fish species Alburnus alburnus (bleak) as a bioindicator. The study was carried out within the Joint Danube Survey 4 (JDS4) at nine sites in the Danube River Basin in the Republic of Serbia. Out of nine sampling sites, two were situated at the Tisa, Sava, and Velika Morava rivers, and three sites were at the Danube River. The three additionally employed LoEs were: SumTUwater calculated based on the monitoring data in the database of the Serbian Environmental Protection Agency (SEPA) (LoE1); in vitro analyses of JDS4 water extracts employing genotoxicological methods (LoE2); assessment of the ecological status/potential by SEPA and indication of the ecological status for the sites performed within the JDS4 (LoE4). The analyzed biomarker responses in the bleak were integrated into the unique integrated biomarker response index which was used to rank the sites. The highest pollution pressure was recorded at JDS4 39 and JDS4 36, while the lowest was at JDS4 35. The impact of pollution was confirmed at three sites, JDS4 33, 40, and 41, by all four LoEs. At other sampling sites, a difference was observed regarding the pollution depending on the employed LoEs. This indicates the importance of implementing a comprehensive weight-of-evidence approach to ensure the impact of pollution is not overlooked when using only one LoE as is often the case in environmental studies.

The in vivo comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point-of-contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal- and cell-specific considerations may compromise applicability to the comet assay. In the present study, 1,1\'-methylenebis(4-isocyanatobenzene) (4,4\'-MDI) was administered to rats for 6 h by nose-only inhalation, and the comet assay was conducted to evaluate genotoxicity in the site-of-contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site-of-contact- including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress- were used to set appropriate exposure concentrations, in addition to the standard systemic measures of toxicity. In the range-finding study, a concentration of 4 mg/m3 was considered the maximum noninflammatory concentration; hence target concentrations of 2, 5, and 11 mg/m3 were selected for the comet study. In the lung lavage, MDI exposure substantially increased total protein and β-glucuronidase, along with cellular apoptosis. Although MDI did not increase the comet assay response (% tail DNA) in any of the tissues examined, the positive control (ethyl methanesulfonate, EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the comet assay.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods\' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

In case of mass radiological emergencies, new strategies involving biological and clinical endpoints are requested for an efficient triage classification of casualties. For this purpose, we developed a novel protocol combining the two most established cytogenetic methods used in biological dosimetry (dicentric and micronucleus assays) into a single one, in order to have a time-saving, inexpensive and potentially automatable instrument to be used for triage purposes in case of large-scale radiological events. This method could be considered as a \'three in one\' assay allowing the simultaneous scoring of chromosome aberrations and micronuclei on a single slide, and also enabling to discriminate between metaphases in first and second cell division without the Fluorescence plus Giemsa staining. This method needs further validation through inter-comparisons involving biological dosimetry laboratories, to verify its reproducibility. Moreover, the possibility to apply the already existing software for automation for dicentric and micronucleus assays could be also verified.

Vitamin B deficiency in patients with inflammatory bowel disease (IBD) is well-documented; however, few studies have explored genomic damage in patients with IBD using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. This study investigated the frequency of micronuclei (MNi) using the CBMN-Cyt assay and the level of vitamin B in patients with IBD. This prospective study was conducted in 15 patients with ulcerative colitis, 15 patients with Crohn\'s disease, and 30 healthy controls from one tertiary hospital. Serum vitamin B and homocysteine levels were measured, and the MNi status was analyzed using the CBMN-Cyt assay. The patients with IBD showed significantly lower serum pyridoxine levels and significantly higher homocysteine levels than controls. The frequencies of binucleated cells (BNCs) with MNi, nucleoplasmic bridges (NPBs), and nuclear buds (Nbuds) were 8.5 [5.8-13.5], 1.0 [0.0-1.9], and 5.4 [4.3-7.4] for the IBD group, and 5.9 [4.8-7.7], 0.2 [0.0-1.0], and 3.5 [2.9-5.4] for the control group (P =  0.011, P =  0.010, and P =  0.002), respectively. This study suggests that patients with IBD have increased frequencies of MNi and decreased levels of pyridoxine than healthy controls.

The present study investigated the effects of two expired commercial medicines, like Buscopan Plus and Mesulid, commonly classified as household medical wastes, on hemocytes of mussel Mytilus galloprovincialis. Mussel hemocytes\' lysosomal membrane stability (in terms of neutral red retention assay), superoxide anions (O2·-) and nitric oxides (NO, in terms of nitrites) production, lipid peroxidation (in terms of malondialdehyde/MDA content) and the formation of nuclear abnormalities (using the micronucleus/MN assay) were assessed in hemocytes of mussels treated for 7 days with appropriate amounts of each drug (the concentrations of active substances were considered in each case, due to the absence of data related with the excipients) as well as in hemocytes of post-treated/recovered mussels (7 days post-treatment/recovery period). According to the results, treated mussels showed significantly decreased NRRT values, enhanced O2·-, NO and MDA levels, as well as high frequencies of nuclear abnormalities in both cases. Thοse effects showed a drastic reduction in almost all cases, after the post-treatment/recovery period. Moreover, the \"stress on stress\" method, commonly performed for estimating mussels\' ability to survive in air, showed significantly reduced LT50 values in challenged mussels, compared to values observed in control mussels. The current findings revealed for the first time that both expired commercial drugs could affect mussels, probably via the formation of active substances bioactivated metabolites, as well as excipients, such as TiO2 and SiO2, at least in case of Buscopan plus. Although further research is needed, the current findings indicate the environmental impact of expired commercial drugs, thus revealing the need for the proper disposal of household medical wastes.

Exposure to certain pollutants induces a series of alterations in deoxyribonucleic acid (DNA) that may result in genotoxic/mutagenic effects in exposed individuals. The present study aimed to monitor genotoxic, mutagenic, and recombinogenic potential and consequently water quality in two streams in the Paranaíba River basin in the state of Minas Gerais, Brazil, using two bioindicator fish (Rhamdia quelen and Geophagus brasiliensis). The micronucleus (MN) test and somatic recombination and mutation test (SMART) were employed to assess DNA damage. The water quality index (WQI) at the reference site control (S1) due to its proximity to the river source was compared to Córrego do Óleo (S2) with respect to chemical parameter levels of biochemical oxygen demand (BOD), dissolved-oxygen rates (DO), and total solid and fecal coliform counts. These chemical parameters were above the permitted limits at Córrego do Óleo (S2). At a third site, Córrego Liso (S3), a poor WQI was detected, attributed to the influence of domestic and industrial activities where BOD, DO, total solid, fecal coliform, total phosphorus, and turbidity rates exceeded premissible limits. The MN frequencies and the numbers of MN per cell (CMN) at sites S2 and S3 were significantly higher than those at S1 in both species. It is of interest that the increased frequency of MN was similar to the positive control cyclophosphamide only at S3, suggesting that the effects of water contaminants were most severe at this site. At sites assessed (S2 and S3), there was a significant rise in somatic mutation and recombination in the wings of Drosophila melanogaster, indicating the presence of trace elements, mainly lead (Pb) and cadmium (Cd), in the effluents in the Paranaíba River basin sites.

In this work, the model plant for genotoxicity studies Vicia faba L. was used to investigate the relation between Boron (B) content and bioavailability in soil and plant genotoxic/phytotoxic response. A total of nine soil samples were investigated: two soil samples were collected from a B-polluted industrial area in Cecina (Tuscany, Italy), the other samples were obtained by spiking control soil (from a not polluted area of the basin) with seven increased doses of B, from about 20 to 100 mg B kg-1. As expected, B availability, evaluated by chemical extraction, was higher (twofold) in spiked soils when compared with collected polluted soils with the same B total content. To analyze the phytotoxic effects of B, seed germination, root elongation, biomass production, and B accumulation in plant tissues were considered in V. faba plants grown in the various soils. Moreover, the cytotoxic/genotoxic effects of B were investigated in root meristems by mitotic index (MI) and micronuclei frequency (MCN) analysis. The results highlighted that V. faba was a B-sensitive plant and the appearance of phytotoxic effects, which altered plant growth parameters, were linearly correlated to the bioavailable B concentration in soils. Concerning the occurrence of cytotoxic/genotoxic effects induced by B, no linear correlation was observed even if MCN frequency was logarithmic correlated with the concentration of B bioavailable in soils.