Nucleoplasmic Bridges Research Articles

ASSESSMENT OF CYTOGENETIC DAMAGE TO EXFOLIATED GINGIVAL CELLS IN PATIENTS WITH CHRONIC GENERALIZED PERIODONTITIS.

SUMMARYThe aim of this cross-sectional study was to investigate the occurrence of chromosomal abnormalities through the frequency of micronuclei and other genomic damage markers in patients with chronic generalized periodontitis and without periodontal disease. Micronucleus assay was performed in exfoliated gingival epithelial cells of 35 patients with generalized chronic periodontitis and 30 control subjects with healthy periodontium. Full mouth clinical examination was performed to define periodontal condition. The mean number of cells with micronuclei observed in chronic periodontitis and control groups was 1.8 (±1.49) and 2.0 (±1.34), respectively. Differences between the groups were not significant (p=0.574). Compared to control subjects, patients with chronic periodontitis showed a significant increase in the number of binucleated cells (p≤0.001) and number of cells with nucleoplasmic bridges (p=0.042). Study results indicated that chronic periodontitis was not associated with higher occurrence of chromosomal damage in gingival cells compared to individuals with healthy periodontium.

DETERMINATION OF THE OCCURENCE OF NUCLEAR ANOMALIES IN DANIO RERIO PERIPHERAL BLOOD ERYTH- ROCYTES DEPENDING ON THE DOSE OF IONIZING RADIATION

Ionizing radiation induces double-stranded breaks in the DNA structure, following by the formation of dicentric chromosomes. With the passage of subsequent postradiation mitoses, dicentric chromosomes are unevenly distributed to the poles of the cell, as a result of which nuclear anomalies of various types are formed, distinguishable at the light-optical level. The aim of this study was to assess the occurrence of nuclear anomalies in the peripheral blood erythrocytes of freshwater fish Danio rerio after exposure to ionizing radiation, as well as to assess the possibility of using these organisms as model animals in radiobiological studies. To determine the frequency of occurrence of nuclear anomalies in peripheral blood erythrocytes of freshwater fish Danio rerio, fish were exposed to X-ray radiation at doses of 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0 Gy. 48 hours after irradiation in fish peripheral blood erythrocytes by light microscopy, four types of nuclear anomalies were identified: micronuclei, nuclear protrusions, nucleoplasmic bridges, dumbbell-shaped nuclei. The frequency of detection of micronuclei and nuclear protrusions was found to be dose-dependent (r = 0.9245, p < 0.05 and r = 0.9062, p < 0.05 respectively), while the appearance of nucleoplasmic bridges and dumbbell-shaped nuclei did not correlate with the dose. More than that, the frequencies of micronuclei and nuclear protrusions detected after irradiation of fish at doses of more than 4 Gy (4.0, 6.0 and 8.0 Gy) significantly differed from the control values. Thus, Danio rerio can be used as a laboratory test system for radiobiological research, for example, to determine the effect of the developed radioprotectors and radiosensitizers, despite the extreme radioresistance of these organisms. It is advisable to use micronuclei or nuclear protrusions as cell markers, given, however, that the identification of these markers will allow detecting radiation exposure only in doses above 4 Gy.

Micronucleus, nucleoplasmic bridge and nuclear bud frequencies in patients with laryngeal carcinoma

SUMMARYThe aim of the study is to determine and compare micronucleus (MN), nucleoplasmic bridge (NPB) and nuclear bud (NBUD) frequencies in patients with laryngeal carcinoma and healthy controls. The study was conducted in the School of Medicine of Onsekiz Mart University. A total of 102 volunteers, 51 of whom had laryngeal carcinoma and 51 of whom were healthy control subjects, participated in this study. The Cytokinesis-Block Micronucleus Assay (CBMN) was applied to peripheral blood lymphocytes taken from patients and controls. We evaluated MN, NPBs and NBUDs frequencies in patients with laryngeal carcinoma and compared the results with those in the control group. The frequencies of MN, NPBs and NBUDs of patients with laryngeal carcinoma were found significantly higher than those in the control group (P = 0.01, P = 0.004, P = 0.01, respectively). MN, NPB and NBUD frequencies were also compared in the patients with and without pesticide exposure, and the means of all frequencies was higher in patients with pesticide exposure (P = 0.001, P = 0.02 respectively). The MN, NPBs and NBUDs frequencies of the patients with laryngeal cancer were significantly higher than those of the control group, and pesticide exposure might be a risk factor that increases genomic instability and risk of laryngeal cancer.

Biomonitoring findings for occupational lead exposure in battery and ceramic tile workers using biochemical markers, alkaline comet assay, and micronucleus test coupled with fluorescence in situ hybridisation.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints.

Dietary exposure to mercury and its relation to cytogenetic instability in populations from “La Mojana” region, northern Colombia

Fish consumption and chronic exposure to low doses of mercury (Hg) seems to activate several molecular mechanisms leading to carcinogenic and/or teratogenic processes. However, Hg genotoxic effects on humans are not completely described. In the present study, we assessed cytogenetic damage in isolated human peripheral lymphocytes using the cytokinesis-block micronucleus cytome assay (CBMN-Cyt), micronucleus formation with anti-kinetochore antibody (CREST staining), levels of total Hg in hair (T-Hg), fish consumption, and estimated Hg dose. The study comprised 39 non-exposed, and 73 residents from La Mojana region, an area with a well-documented Hg contamination. Data showed a significant increase in micronuclei (MNBN), nucleoplasmic bridges (NPB), and necrotic and apoptotic cell frequencies in residents of “La Mojana.” The overall mean T-Hg level in hair for exposed residents was 1.12 ± 0.94 mg kg−1 and 0.15 ± 0.05 in individuals from the reference area. Approximately 40% of analyzed individuals showed T-Hg levels that exceeded US Environmental Protection Agency (USEPA) reference dose. Increased T-Hg levels in hair were related to increased MNBN frequencies and high fish consumption. Other cellular markers, such as necrotic and apoptotic cell frequencies, were also correlated with high fish intake and T-Hg contents. Results of the CREST staining demonstrated that in vivo exposure to Hg induces genetic instability by chromosome fragment loss (clastogenic). Additionally, a high average intake of some fish species, particularly with carnivorous habits like Caquetaia kraussii, Hoplias malabaricus, and Sorubin cuspicaudus, seems to increase MNBN frequencies significantly.

Targeting DNA Repair to Overcome Drug Resistance in Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by rare tumor-initiating Hodgkin and Reed-Sternberg cells (HRS) surrounded by a microenvironment with a reactional immune cells infiltrate. cHL accounts for 15% to 25% of all lymphomas. This neoplasm is curable in the majority of cases with chemotherapy including ABVD (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) or BEACOPP (Bleomycin, etoposide, Doxorubicin, cyclophosphamide, Vincristine, procarbazine and Prednisone) and/or radiation. However, 15-20% of high-risk patients ultimately relapse and high dose chemotherapy in combination with autologous stem-cell transplantation is successful in only half of the patients with relapsed/refractory cHL (Eichenaueret al., Eur. Soc. Med. Oncol,2018). HRS cells are characterized by genetic instability, abnormal DNA damage response and repair that may play a role in drug resistance in high-risk cHL (Reichelet al., Blood,2015, Monroyet al. Mol. Carcinog,2011). In this work, we hypothesized that inhibiting DNA repair mechanisms using small molecules might represent a promising strategy to overcome drug resistance to genotoxic agents in cHL, such as cyclophosphamide and topoisomerase II inhibitors (O'Connor,Mol. Cell, 2015; Shaheenet al., Blood 2011). We characterized the drug-response of 4 cHL cell lines to DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1 inhibitor), AZD6738 (ATR inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor) and genotoxic agents used in standard chemotherapy (cyclophosphamide, doxorubicin and etoposide). We showed that DNA repair inhibitors targeting ATR, CHK1 or Wee1 significantly induced apoptosis of cHL cell lines (AnnexinV staining, p&amp;lt;0.05), significant inhibition of proliferation (BrdU incorporation). ATR inhibitor induces a significant accumulation of cHL cells in G0/G1 phase (p&amp;lt;0.05) whereas CHK1 and Wee1 inhibitors induced a significant accumulation in G2/M phase (p&amp;lt;0.01). Moreover, ATR, CHK1 and Wee1 inhibitors induced double strand breaks monitored by 53BP1 foci formation, γH2A.X staining (p&amp;lt;0.01) and chromosomic instability characterized by a higher frequency of micronucleation (p&amp;lt;0.05) and nucleoplasmic bridges formation (p&amp;lt;0.01). Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 DNA repair targeted treatments with conventional genotoxic agents in cHL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone (Srivaset al., Mol. Cell, 2016), a total of two synthetic lethal combinations in cHL cells have been identified: CHK1 inhibitor (PF477736) combined with a Wee1 inhibitor (AZD1775) (IC50 reduced from 5.00 µM to 1.75 µM for L-428 and from 4.81 µM to 1.86 µM for KMH2) and cyclophosphamide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 3.26 µM to 1.12 µM for L-428 and from 1.27µM to 0.10µM for KMH2). In addition, we identified 3 synergistic/additive combinations (combination index &amp;lt; 1): doxorubicin combined with a DNAPK inhibitor (NU7441) (IC50 reduced from 44 nM to 22 nM for L-428 and from 27 nM to 8 nM for KMH2), cyclophosphamide combined with CHK1 inhibitor (PF477736) (IC50 reduced from 2.9 µM to 2.4 µM for L-428 and from 1.2µM to 0.9µM for KMH2) and etoposide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 420 nM to 195 nM for KMH2). The treatment of Hodgkin's lymphoma is successful for a majority of patients. However, patients with advanced-stage or high-risk disease are only cured in ∼70% of cases. Alternative treatment options for the subsets of patients for whom first- or second-line therapies fail are needed. These results open new perspectives to improve the treatment of relasped/refractory cHL patients and provide new strategies to overcome drug resistance. Disclosures Moreaux: Diag2Tec:Consultancy.

Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability.

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

Short-term in vitro glutamine restriction differentially impacts the chromosomal stability of transformed and non-transformed cells.

Glutamine (Gln) is a non-essential amino acid central for generating building blocks and cellular energy in tumours and rapidly proliferating non-transformed cells. However, the influence of Gln on regulating chromosomal stability of transformed and non-transformed cells remain poorly understand. We hypothesised that Gln is required for maintaining a homeostatic level of chromosomal stability. To this end, transformed cells HeLa and A375 and non-transformed cells NCM460 and HUVEC cells were intervened with varying concentrations of Gln (10, 1, 0.1 and 0.01 mM), with or without cisplatin (0.1 µg/ml), for 24 h. The cytokinesis-block micronucleus (MN) assay was used to determine chromosomal instability (CIN), the extent of which is reflected by the frequency of MN, nucleoplasmic bridge (NPB) and nuclear bud (NB). We demonstrated an unexpected decrease in the spontaneous rate of MN, but not NPB and NB, after Gln restriction in HeLa and A375 cells. Gln restriction reduced cisplatin-induced MN, but not NPB and NB, in HeLa and A375 cells. We further revealed that Gln restriction suppressed the proliferation of HeLa cells with high CIN induced by nocodazole, partially explaining why Gln restriction decreased the frequency of spontaneous and cisplatin-induced MN in transformed cells. In contrast, Gln restriction increased MN and NB, but not NPB, in NCM460 cells. In HUVEC cells, Gln restriction increased MN, NPB and NB. Meanwhile, Gln restriction sensitised NCM460 cells to cisplatin-induced genotoxicity. A similar but more pronounced pattern was observed in HUVEC cells. Collectively, these results suggest that the in vitro influences of Gln metabolism on CIN depend on cellular contexts: Transformed cells require high Gln to fine tune their CIN in an optimal rate to maximise genomic heterogeneity and fitness, whereas non-transformed cells need high Gln to prevent CIN.

The secretome of non-tumorigenic mammary cells MCF-10A elicits DNA damage in MCF-7 and MDA-MB-231 breast cancer cells

Radiotherapy is used in breast cancer to destroy tumor cells lingering after surgery. It is accepted that lethal effects of ionizing radiation occur as a result of damage to DNA in irradiated (IR) cells. However, response mechanisms may promote cell survival with efficient DNA repair or genomic alterations. Chromosomal aberrations are frequent in surviving cells and may enhance chromosomal instability (CIN) which is associated with increased risk of recurrence and metastasis. Intercellular communication can affect the response in IR cells and cause damage in non-irradiated (N-IR) cells. We evaluated the effect of the secretome of non-tumorigenic mammary cells (MCF-10A) on proliferation and DNA damage in breast cancer cells (MCF-7 and MDA-MB-231). Results showed that conditioned media from IR and N-IR MCF-10A cells produced cycles of DNA double-strand breaks in N-IR and IR tumor cells leaving them with residual damage. CIN markers (micronuclei, nucleoplasmic bridges, nuclear buds) were also increased in IR and N-IR tumor cells, being the effect of conditioned media from IR MCF-10A greater in many cases. The inhibition of phosphorylation/activation of Src kinase in cancer cells hindered CIN markers' increment. Besides, clonogenic survival of tumor cells was differentially modulated by conditioned media from MCF-10A: decreased in MCF-7 and enhanced in MDA-MB-231 cells. These results signal the relevance of tumor-host interaction in tumor behavior and the response to radiotherapy.

Mitomycin C induced genotoxic stress in endothelial cells is associated with differential expression of proinflammatory cytokines

Mitomycin C (MMC) is an alkylating chemotherapy drug that causes DNA crosslinking resulting in transcription arrest and apoptosis. DNA crosslinking is a critical damage to DNA that can be caused not only by MMC and other antitumor drugs, but also by various environmental and anthropogenic endo- and exogenous agents. Mammalian cells exposed to alkylating mutagens are characterized by severe genotoxic stress. Somatic mutations and genotoxic stress may lead to endothelial dysfunction, which is the initial stage of atherosclerosis, a leading cause of morbidity and mortality worldwide. Here we studied DNA damage, protein secretion and gene expression of IL6 and IL8 in primary human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) in vitro exposed to 500 ng/mL MMC. We observed an increase in levels of cytogenetic damage (micronuclei, nucleoplasmic bridges and nuclear buds) in MMC-treated cells compared to control cells. After 6 h incubation with MMC, both HCAEC and HITAEC displayed a decrease in IL8 concentration and the mRNA level of IL6 and IL8 compared to control cells. Removal of MMC from cultures after 6 h followed by 24 h incubation of cells in complete growth media led to a sharp increase in secretion and gene expression of the studied cytokines in both HCAEC and HITAEC. Moreover, HCAEC were more susceptible to mutagenic exposure compared to HITAEC. These findings suggest that the MMC-induced genotoxic stress in endothelial cells derived from different arteries is associated with differential secretion and gene expression of proinflammatory cytokines IL6 and IL8.

Positive association of a Sirt1 variant and parameters of oxidative stress on Alzheimer’s disease

Alzheimer's disease (AD) is a complex neurodegenerative disorder and the most common type of dementia in the elderly. Although its cause is not completely known, several studies suggest that oxidative stress plays an important role in the etiology of this disease. The SIRT1 and SOD2 proteins are linked to pathways that may impair oxidative stress. In this study, we analyzed the association between polymorphisms in these genes and in the APOE gene, through RT-PCR, as well as between environmental factors and the risk of AD. Additionally, the thiobarbituric acid reactive substance assay was performed to estimate the plasma level of malondialdehyde (MDA), a biomarker of lipid peroxidation. Furthermore, some cytogenetic studies indicate that cells of AD patients show increased chromosomal damage; thus, we performed the micronucleus cytome assay to assess cytogenetic damage in AD patients. As expected, the APOE polymorphisms were found to be highly associated with AD. Additionally, the CT genotype of the SIRT1 gene showed a positive association with the disease. The frequencies of genomic damage (micronucleus, buds, nucleoplasmic bridges and binucleated cells), the presence of cell death biomarkers (condensed chromatin, karyorrhexis and pyknosis), and the plasma level of MDA were significantly greater in AD patients than in controls. Our results support the hypothesis that AD is a condition with increased oxidative stress and genomic instability, which may contribute to the neurodegeneration in AD.

Women rag pickers at a dump in Ahmedabad: Genotoxicity and oxidative stress

Municipal solid waste (MSW) generated in Ahmedabad, India, and the surrounding area is dumped at the Pirana site; rag pickers collect materials for re-sale. We have compared genotoxicity and oxidative stress in samples from women rag pickers working at this site, with women involved only in door-to-door waste picking (in residential areas near the university campus) as “controls”. The buccal Cytokinesis-Block Micronucleus (CBMN) assay showed significantly higher frequencies of Micronucleus (MN), Nucleoplasmic Bridges (NPB), and Nuclear Buds (NB) in the rag pickers than in the “controls”. The buccal Micronuclei Cytome (BMCyt) assay showed significantly higher prevalence of nuclear anomalies, such as micronucleus, karyorrhexis, karyolytic cells, and nuclear buds. Blood samples from the rag pickers showed lower levels of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase), lower total serum protein concentrations, and greater lipid peroxidation compared to the “control” group. Exposure to hazardous solid waste may lead to increased oxidative damage and genotoxicity; improved safety procedures and the use of personal protective equipment are recommended.

Biomarkers of susceptibility and effect in car painters exposed to organic solvents.

Introduction: Car painters are routinely exposed to organic solvents classified as carcinogenic and mutagenic substances. Objective: To characterize the population susceptibility and evaluate the genotoxic effects of exposure to organic solvents. Methods: A cross-sectional study comparing a group of car painters exposed to organic solvents with a non-exposed group. CYP2E1 polymorphisms and the presence of micronuclei in lymphocytes were determined. Results: One hundred twenty-two workers participated in the study: 62 who worked in car paint shops and were exposed to solvents, and 60 who were not exposed. There were statistically significant differences between the two groups regarding micronucleated cells and nucleoplasmic bridges frequencies (p= 0.042 and p= 0.046, respectively; exact likelihood ratio). Significant differences were found at the interaction between the CYP2E1 genotype c1c1 and occupational exposure to solvents, with higher frequencies of micronuclei (p= 0.013) and micronucleated cells (p= 0.015). However, when the frequencies of micronuclei, micronucleated cells and nucleoplasmic bridges in the exposure group were compared between the c1c1 and c2c2/c1c2 allele groups of the CYP2E1 polymorphism, statistically significant differences were found. Conclusions: This study confirms that when workers with CYP2E1 polymorphisms, specifically the c1c1 genotype, are exposed to organic solvents, they are more likely to have somatic cell mutations, a condition associated with increased susceptibility to diseases such as cancer

Genotoxicity of the organophosphate pesticide malathion and its metabolite dimethylthiophosphate in human cells in vitro

Organophosphate (OP) pesticides are biotransformed into metabolites such as dialkylphosphates (DAPs). We have evaluated the genotoxicity of malathion and its metabolite dimethylthiophosphate (DMTP) in the human hepatic cell lines HepG2 and WRL-68 and in peripheral blood mononuclear cells (PBMC). In the Cytokinesis-Block Micronucleus assay (CBMN), malathion and DMTP increased the frequencies of micronuclei (MN) and nucleoplasmic bridges (NPB). Malathion was primarily clastogenic whereas DMTP was aneuploidogenic. When HepG2 or WRL-68 cells were treated with DMTP in the presence of sulconazole, a non-specific cytochrome P450 inhibitor, MN frequency was reduced, indicating that DMTP genotoxicity requires P450-cataliyzed metabolism.

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

The genotoxicity of an organic solvent mixture: A human biomonitoring study and translation of a real-scenario exposure to in vitro

This study aimed to evaluate occupational exposure to a styrene and xylene mixture through environmental exposure assessment and identify the potential genotoxic effects through biological monitoring. Secondly, we also exposed human peripheral blood cells in vitro to both xylene and styrene either alone or in mixture at concentrations found in occupational settings in order to understand their mechanism of action. The results obtained by air monitoring were below the occupational exposure limits for both substances. All biomarkers of effect, except for nucleoplasmic bridges, had higher mean values in workers (N = 17) compared to the corresponding controls (N = 17). There were statistically significant associations between exposed individuals and the presence of nuclear buds and oxidative damage. As for in vitro results, there was no significant influence on primary DNA damage in blood cells as evaluated by the comet assay. On the contrary, we did observe a significant increase of micronuclei and nuclear buds, but not nucleoplasmic bridges upon in vitro exposure. Taken together, both styrene and xylene have the potential to induce genomic instability either alone or in combination, showing higher effects when combined. The obtained data suggested that thresholds for individual chemicals might be insufficient for ensuring the protection of human health.

Radioprotective efficacy of plastic polymer against the toxicogenomic effects of radiopharmaceutical 18F-FDG on human lymphocytes

BackgroundHealthcare workers occupationally exposed to 18F-FDG cannot wear protective equipment, such as lead aprons, since the interaction between high energy radiation (511 keV) and metal increases the dose of radiation absorption. The objective of this study was to evaluate the shielding efficacy of a plastic polymer against the toxicogenomic effects of ionizing radiation in human lymphocytes, using cytokinesis-block micronucleus assays.MethodsHuman peripheral blood lymphocytes were isolated from three subjects and cultured under standard conditions. The cultures were exposed to 300 mCi of 18F-FDG at a distance of 10 cm for 10 min, in the absence of shielding or with lead, polymer, and lead + polymer shields.ResultsLead shielding was found to increase the number of counts detected by Geiger-Müller radiation monitors as a consequence of the photoelectron effect. Conversely, the lead + polymer shield reduced the number of counts. The lead, polymer, and lead + polymer shields significantly reduced the frequency of micronuclei, nucleoplasmic bridges, and nuclear buds induced by ionizing radiation. Regarding cytotoxicity, only the lead + polymer shield re-established the cell cycle at the level observed for the negative control.ConclusionsLead aprons that are internally coated with polymer increased the radiological protection of individuals occupationally exposed to 18F-FDG PET/CT, especially during examinations.

Potential biological damage of human peripheral blood lymphocytes induced by computed tomography examination of the oromaxillofacial region

The aim of this study was to examine whether oromaxillofacial computed tomography (CT) examination causes biologic damage in lymphocytes and whether the biologic damage is related to radiation dose, patient age, or gender. Peripheral blood was taken from 51 individuals and divided into control, in vivo, and in vitro irradiation groups. Biologic damage was assessed by comparing rates of chromosomal aberrations (CAs), including dicentric chromosomes (dics), centric rings, and acentric fragments; and nuclear aberrations, including micronuclei (MN), nuclear buds (NBUDs), and nucleoplasmic bridges (NPBs) in the peripheral blood before and after CT examination. Absorbed and effective doses were calculated with the software VirtualDose, and the blood dose was estimated accordingly. The rates of acentric fragments, MN, NBUDs, and NPBs in the in vivo (P ≤ .008) and in vitro (P ≤ .003) irradiation groups were significantly higher than those in the control groups. The acentric fragment rate (P=.013) and MN rate (P=.002) were higher in the in vitro group than in the in vivo group. There was no correlation between change rates of CAs and nuclear aberrations with radiation dose. Positive correlations of MN rates with age were found in all groups (ρ ≥ 0.590). Certain doses of radiation in oromaxillofacial CT examination may induce CAs and nuclear aberrations in lymphocytes.

Different working conditions shift the genetic damage levels of pesticide-exposed agriculture workers.

In the current study, we had two main purposes. Firstly, we aimed to compare genetic damages in the agricultural workers of two different types of environmental conditions including the greenhouse and open fields. Secondly, we aimed to compare genetic damages in the total agricultural workers as the exposed group (greenhouse and open field workers) (n = 114) and the non-exposed control group (n = 98) living in the same area in Canakkale, Turkey. For these purposes, we investigated the incidence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in peripheral blood lymphocytes. We observed that the frequencies of MN, NPB, and NBUD obtained for the greenhouse workers were statistically significantly higher than those obtained for the open field workers. When the results of the control group were compared with those of the total workers, there were statistically significant differences in terms of MN and NBUD frequencies. We found that age and MN were correlated at a significant level in both the agricultural workers and the control group. The MN frequency of the female workers was 1.5 times greater than that of the male workers, and it was a significant level in the agricultural workers.

In vitro genomic damage induced by urban fine particulate matter on human lymphocytes

Urban air pollution represents a global problem, since everyday many mutagenic and carcinogens compounds are emitted into the atmosphere, with consequent adverse health effects on humans and biota. Specifically, particulate matter air pollution was associated with increased risks in human mortality and morbidity. In this paper, we analyse the genomic effects on human lymphocytes of different concentrations of annual Turin PM2.5 extract by an in vitro micronuclei assay. Samplings were collected from an urban meteorological-chemical station positioned in Turin (Italy), one of the most polluted cities in Europe. PM2.5 sampled on filters was used for organic extraction in monthly pools and successively aggregated to produce a mixture representative for a full year PM2.5 collection. Lymphocytes were exposed to four concentrations of PM2.5: 5, 10, 15 and 20 μg/mL and micronuclei, nucleoplasmic bridges and nuclear buds were scored. With respect to controls, PM2.5 significantly increased the frequencies of all analysed biomarkers at all tested concentrations, whereas the CBPI index was significantly reduced only at the concentration of 20 μg/mL. Such in vitro effects can both to stimulate local authorities to adopt efficient measures for air pollution mitigation and to improve human monitoring to detect early precancer lesions.