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1.  Comparative Study of Domoic Acid and Okadaic Acid Induced - Chromosomal Abnormalities in the CACO-2 Cell Line 
Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.
PMCID: PMC3785674  PMID: 16823071
Okadaic acid; Domoic acid; Micronuclei; Clastogenicity; Aneugenicity
2.  Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress 
Mutagenesis  2013;28(4):475-484.
Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally susceptible to DNA damage.
PMCID: PMC3681539  PMID: 23766106
3.  Micronuclei induced by reverse transcriptase inhibitors in mononucleated and binucleated cells as assessed by the cytokinesis-block micronucleus assay 
Genetics and Molecular Biology  2010;33(4):756-760.
This study evaluated the clastogenic and/or aneugenic potential of three nucleoside reverse transcriptase inhibitors (zidovudine - AZT, lamivudine - 3TC and stavudine - d4T) using the cytokinesis-block micronucleus (CBMN) assay in human lymphocyte cultures. All three inhibitors produced a positive response when tested in binucleated cells. The genotoxicity of AZT and 3TC was restricted to binucleated cells since there was no significant increase in the frequency of micronuclei in mononucleated cells. This finding indicated that AZT and 3TC caused chromosomal breakage and that their genotoxicity was related to a clastogenic action. In addition to the positive response observed with d4T in binucleated cells, this drug also increased the frequency of micronuclei in mononucleated cells, indicating clastogenic and aneugenic actions. Since the structural differences between AZT and 3TC and AZT and d4T involve the 3' position in the 2'-deoxyribonucleoside and in an unsaturated 2',3',dideoxyribose, respectively, we suggest that an unsaturated 2', 3', dideoxyribose is responsible for the clastogenic and aneugenic actions of d4T.
PMCID: PMC3036155  PMID: 21637587
lamivudine; micronucleus assay; stavudine; transcriptase inhibitors; zidovudine
4.  Assessment of the proliferative activity and radiosensitivity of human tumours using the cytokinesis-block micronucleus assay. 
British Journal of Cancer  1994;70(1):67-71.
We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.
PMCID: PMC2033295  PMID: 8018543
5.  Zinc deficiency or excess within the physiological range increases genome instability and cytotoxicity, respectively, in human oral keratinocyte cells 
Genes & Nutrition  2011;7(2):139-154.
Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO4) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO4 and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.
PMCID: PMC3316759  PMID: 21935692
Zinc; Cytotoxicity; DNA damage; Genomic stability; Human oral keratinocytes; Micronuclei
6.  The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability 
British Journal of Cancer  1999;80(10):1599-1607.
A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B–IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 × 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08–0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60–610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer. © 1999 Cancer Research Campaign
PMCID: PMC2363103  PMID: 10408406
cervical carcinoma; radiotherapy; micronucleus assay; predictive value
7.  Genetic Biomarkers for Neoplastic Colorectal Cancer in Peripheral Lymphocytes 
Mædica  2011;6(2):83-89.
Background: Loss of genomic stability appears as a key step in colorectal carcinogenesis. Micronucleus (MN) designates a chromosome fragment or an entire chromosme which lags behind mitosis. MN may be noticed as an additional nucleus within the cytoplasm cell during the intermediate mitosis phases. We tested the hypothesis that MN and its related anomalies may be associated with the presence of neoplastic colorectal lesions.
Method: Peripheral blood lymphocytes were cultured and microscopically examined. The frequency of micronuclei (FMN) and the presence of nucleoplasmic bridges (NPB) in binucleated cells were compared in patients with of without colorectal neoplastic lesions.
Results: We included 45 patients undergoing colonoscopy, 23 males and 22 females, with a median age of 59. 17 patients had polyps, 11 colorectal cancer (CRC) and 17 had a normal colonoscopy. The FMN was significantly higher in women than in men (8.14 vs 4.17, p=0.008); NPB were significantly less frequent in patients with advanced adenomas (>10mm or vilous) or CRC (p=0.044) when compared with patients with normal colonoscopy, hiperplastic polyps or non-advanced adenomas.
Conclusion: Micronuclei are more frequent in women, but its frequency was not significantly different in patients with advanced adenomas or CRC. Null or low frequency values for nucleoplasmic bridges presence in peripheral lymphocyte may be predictive for advanced adenomas and colorectal cancer.
PMCID: PMC3239403  PMID: 22205889
micronucleus; nucleoplasmic bridges; colon cancer
8.  Fast Image Analysis for the Micronucleus Assay in a Fully Automated High-Throughput Biodosimetry System 
Radiation research  2014;181(2):146-161.
The development of, and results from an image analysis system are presented for automated detection and scoring of micronuclei in human peripheral blood lymphocytes. The system is part of the Rapid Automated Biodosimetry Tool, which was developed at the Center for High-Throughput Minimally Invasive Radiation Biodosimetry for rapid radiation dose assessment of many individuals based on single fingerstick samples of blood. Blood lymphocytes were subjected to the cytokinesis-block micronucleus assay and the images of cell cytoplasm and nuclei are analyzed to estimate the frequency of micronuclei in binucleated cells. We describe an algorithm that is based on dual fluorescent labeling of lymphocytes with separate analysis of images of cytoplasm and nuclei. To evaluate the performance of the system, blood samples of seven healthy donors were irradiated in vitro with doses from 0–10 Gy and dose-response curves of micronuclei frequencies were generated. To establish the applicability of the system to the detection of high doses, the ratios of mononucleated cells to binucleated cells were determined for three of the donors. All of the dose-response curves generated automatically showed clear dose dependence and good correlation (R2 from 0.914–0.998) with the results of manual scoring.
PMCID: PMC4011502  PMID: 24502354
9.  Neutron Exposures in Human Cells: Bystander Effect and Relative Biological Effectiveness 
PLoS ONE  2014;9(6):e98947.
Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.
PMCID: PMC4045982  PMID: 24896095
10.  Genomic Instability in Human Lymphocytes from Male Users of Crack Cocaine 
Recent research suggests that crack cocaine use alters systemic biochemical markers, like oxidative damage and inflammation markers, but very few studies have assessed the potential effects of crack cocaine at the cellular level. We assessed genome instability by means of the comet assay and the cytokinesis-block micronucleus technique in crack cocaine users at the time of admission to a rehabilitation clinic and at two times after the beginning of withdrawal. Thirty one active users of crack cocaine and forty control subjects were evaluated. Comparison between controls and crack cocaine users at the first analysis showed significant differences in the rates of DNA damage (p = 0.037). The frequency of micronuclei (MN) (p < 0.001) and nuclear buds (NBUDs) (p < 0.001) was increased, but not the frequency of nucleoplasmic bridges (NPBs) (p = 0.089). DNA damage decreased only after the end of treatment (p < 0.001). Micronuclei frequency did not decrease after treatment, and nuclear buds increased substantially. The results of this study reveal the genotoxic and mutagenic effects of crack cocaine use in human lymphocytes and pave the way for further research on cellular responses and the possible consequences of DNA damage, such as induction of irreversible neurological disease and cancer.
PMCID: PMC4210963  PMID: 25264678
crack cocaine; drug withdrawal; DNA damage; comet assay; micronucleus
11.  Lack of a correlation between micronucleus formation and radiosensitivity in established and primary cultures of human tumours. 
British Journal of Cancer  1994;70(6):1112-1117.
The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.
PMCID: PMC2033705  PMID: 7981062
12.  Genomic instability in patients with type 2 diabetes mellitus on hemodialysis 
A previous study by our research group evaluated the levels of DNA damage using the comet assay in hemodialysis patients with type 2 diabetes mellitus. The same blood samples were also evaluated using the cytochalasin B micronucleus assay. A comparison of the results of the two assays is presented here.
Whole blood samples were collected from 22 type 2 diabetes mellitus patients on hemodialysis and from 22 control subjects. Samples were collected from patients early in the morning on Mondays, before the first weekly hemodialysis session. The cytokinesis-block micronucleus assay (CBMN) was used to evaluate genomic instability.
The frequencies of micronuclei and nuclear buds were higher in patients than in controls (p-value = 0.001 and p-value < 0.001, respectively). There was a correlation between the frequency of micronuclei and DNA damage with the results of the comet assay (p-value < 0.001). The difference in the frequency of micronuclei and nuclear buds between patients and controls was more pronounced in the group with higher median comet values than in the group with lower comet values.
Our results suggest that the increased rates of DNA damage as measured by the comet assay and influenced by the weekly routine therapy of these patients has a mutagenic effect, thereby increasing the risk of cancer in this group.
PMCID: PMC3459606  PMID: 23049381
Diabetes Mellitus, type 2; Micronucleus tests; Comet assay; Genomic instability; Renal dialysis
13.  Emergence of Micronuclei and Their Effects on the Fate of Cells under Replication Stress 
PLoS ONE  2010;5(4):e10089.
The presence of micronuclei in mammalian cells is related to several mutagenetic stresses. In order to understand how micronuclei emerge, behave in cells, and affect cell fate, we performed extensive time-lapse microscopy of HeLa H2B-GFP cells in the presence of hydroxyurea at low concentration. Micronuclei formed after mitosis from lagging chromatids or chromatin bridges between anaphase chromosomes and were stably maintained in the cells for up to one cell cycle. Nuclear buds also formed from chromatin bridges or during interphase. If the micronuclei-bearing cells entered mitosis, they either produced daughter cells without micronuclei or, more frequently, produced cells with additional micronuclei. Low concentrations of hydroxyurea efficiently induced multipolar mitosis, which generated lagging chromatids or chromatin bridges, and also generated multinuclear cells that were tightly linked to apoptosis. We found that the presence of micronuclei is related to apoptosis but not to multipolar mitosis. Furthermore, the structural heterogeneity among micronuclei, with respect to chromatin condensation or the presence of lamin B, derived from the mechanism of micronuclei formation. Our study reinforces the notion that micronucleation has important implications in the genomic plasticity of tumor cells.
PMCID: PMC2851613  PMID: 20386692
14.  Use of the Cytokinesis-Blocked Micronucleus Assay (CBMN) to Detect Gender Differences and Genetic Instability in a Lung Cancer Case-Control Study 
Although tobacco exposure is the predominant risk factor for lung cancer, other environmental agents are established lung carcinogens. Measuring the genotoxic effect of environmental exposures remains equivocal as increases in morbidity and mortality may be attributed to co-exposures such as smoking.
We evaluated genetic instability and risk of lung cancer associated with exposure to environmental agents (e.g., exhaust) and smoking among 500 lung cancer cases and 500 controls using the Cytokinesis-Blocked Micronucleus (CBMN) assay. Linear regression was applied to estimate the adjusted means of the CBMN endpoints (micronuclei and nucleoplasmic bridges). Logistic regression analyses were used to estimate lung cancer risk and to control for potential confounding by age, gender, and smoking.
Cases showed significantly higher levels of micronuclei and nucleoplasmic bridges as compared to controls (mean ± SEM=3.54±0.04 vs.1.81 ±0.04 and mean ± SEM=4.26±0.03 vs. 0.99±0.03, respectively; p <0.001) with no differences among participants with or without reported environmental exposure. No differences were observed when stratified by smoking or environmental exposure among cases or controls. A difference in lung cancer risk was observed between non-exposed male and female heavy smokers, although it was not statistically significant (I2=64.9%; P-value for Q statistic=0.09).
Our study confirms that the CBMN assay is an accurate predictor of lung cancer and supports the premise that heavy smoking may have an effect on DNA repair capacity and in turn modulate the risk of lung cancer.
Identifying factors that increase lung cancer risk may lead to more effective prevention measures.
PMCID: PMC3538922  PMID: 23195992
Lung cancer; CBMN assay; DNA damage; gender differences
15.  Sensitivity to NNKOAc is associated with renal cancer risk 
Carcinogenesis  2009;30(4):706-710.
Cigarette smoking has been investigated as a major risk factor for renal cell carcinoma (RCC). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant carcinogenic N-nitrosamines present in cigarette smoke. However, the association between repair capacity of NNK-induced DNA damage and RCC risk remains unknown. We used the comet assay to assess whether sensitivity to a NNK precursor 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) induced DNA damage, which partly reflects host sensitivity to NNK, was associated with increased risk of RCC in a population-based case-control study. The study included 95 RCC cases and 188 matched controls. Epidemiologic data were collected via in-person interview. Baseline and NNK-induced DNA damage in peripheral blood lymphocytes were measured using the comet assay and quantified by the Olive tail moment. The NNKOAc-induced median Olive tail moments were significantly higher in cases than in controls (2.27 versus 1.76, P = 0.002). Using the 75th percentile Olive tail moments of the controls as the cutoff point, we found that higher levels of NNKOAc-induced DNA damage were associated with a significantly increased risk of RCC [odds ratio, 2.06; 95% confidence interval, 1.17–3.61]. In quartile analysis, there was a dose–response association between NNKOAc-induced damage and risk of RCC (P for trend, 0.006). Our data strongly suggest that higher levels of NNKOAc-induced damage are associated with higher risks of RCC. Future studies with larger sample sizes are warranted to further investigate whether repair of NNKOAc-induced damage, as quantified by the comet assay, could be used as a predictive marker for RCC risk.
PMCID: PMC2722144  PMID: 19237609
16.  The application of the cytokinesis-block micronucleus assay on peripheral blood lymphocytes for the assessment of genome damage in long-term residents of areas with high radon concentration 
Journal of Radiation Research  2013;55(1):61-66.
Estimating the effects of small doses of ionising radiation on DNA is one of the most important problems in modern biology. Different cytogenetic methods exist to analyse DNA damage; the cytokinesis-block micronucleus assay (CBMN) for human peripheral blood lymphocytes is a simple, cheap and informative cytogenetic method that can be used to detect genotoxic-related markers. With respect to previous studies on radiation-induced genotoxicity, children are a poorly studied group, as evidenced by the few publications in this area. In this study, we assessed radon genotoxic effects by counting micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the lymphocytes of children who are long-term residents from areas with high radon concentrations. In the exposed group, radon was found to cause significant cytogenetic alterations. We propose that this method can be employed for biomonitoring to screen for a variety of measures.
PMCID: PMC3885124  PMID: 23908554
micronucleus assay; micronuclei; genotoxicity; cytochalasin B; ionising radiation; radon; children
17.  Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage in Workers: Results of a Multiple End Point Study 
Environmental Health Perspectives  2008;116(12):1689-1693.
Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory.
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 μg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
V2O5 causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.
PMCID: PMC2599764  PMID: 19079721
comet assay; cytokinesis-block micronucleus assay; DNA damage; occupational exposure; vanadium pentoxide
18.  Nuclear anomalies in the buccal cells of calcite factory workers 
Genetics and Molecular Biology  2010;33(2):374-378.
The micronucleus (MN) assay on exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. To determine the genotoxic effects of calcite dust that forms during processing, MN assay was carried out in exfoliated buccal cells of 50 (25 smokers and 25 non-smokers) calcite factory workers and 50 (25 smokers and 25 non-smokers) age- and sex-matched control subjects. Frequencies of nuclear abnormalities (NA) other than micronuclei, such as binucleates, karyorrhexis, karyolysis and ‘broken eggs', were also evaluated. Micronuclei and the other aforementioned anomalies were analysed by two way analysis of covariance. The linear correlations between the types of micronucleus and nuclear abnormalities were determined by Spearman's Rho. There was a positive correlation between micronuclei and other types of nuclear abnormalities in accordance with the Spearman's Rho test. Results showed statistically significant difference between calcite fabric workers and control groups. MN and NA frequencies in calcite fabric workers were significantly higher than those in control groups (p < 0.05). The results of this study indicate that calcite fabric workers are under risk of significant cytogenetic damage.
PMCID: PMC3036877  PMID: 21637497
calcite; exfoliated buccal cells; micronucleus (MN); genotoxicity
19.  Dynamics of Micronuclei in Rat Skin Fibroblasts after X Irradiation 
Radiation research  2009;172(1):106-113.
In a previous study, we demonstrated DNA damage, expressed as micronuclei, in binucleate dermal fibroblasts obtained from human skin 2-9 weeks after fractionated radiotherapy. Here we assessed micronuclei in X-irradiated skin fibroblasts from 9-14-week-old female Lewis rats as a function of time after a single dose of radiation to determine the lifetime of such damage in the skin. After irradiation with 5, 10, 15 and 18 Gy, formation of micronuclei at 1 day or 2 months postirradiation increased up to about 10 Gy, with evidence for a plateau at higher doses. The time course of micronuclei present in the skin fibroblasts demonstrated a plateau region (approximately 20 days after 18 Gy and about 2 months after 10 Gy) before the number of micronuclei started to decline. Residual micronuclei were observed for more than 1 year after irradiation. Monomicronucleated cells predominated in fibroblasts from nonirradiated skin, whereas in fibroblasts from irradiated skin, multimicronucleated cells predominated and persisted (together with monomicronucleated cells) in the residual levels of damage at late times. The results suggest that DNA damage in dermal fibroblasts can be assayed by the micronucleus assay in samples from irradiated skin up to 1 month after irradiation for doses up to at least 10 Gy. Further studies are needed to define the dose-response relationship in detail.
PMCID: PMC2747599  PMID: 19580512
20.  Investigation of genome instability in patients with non-alcoholic steatohepatitis 
AIM: To evaluate the occurrence of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in the mitogen-stimulated lymphocytes of patients with non-alcoholic steatohepatitis (NASH).
METHODS: The study was performed in 25 (9 females, 16 males) patients newly diagnosed with NASH, and 25 healthy subjects of similar ages and genders were used as a control group. None of the controls was known to be receiving any drugs for medical or other reasons or using alcohol. Hepatosteatosis was further excluded by abdominal ultrasound imaging in the control group. The numbers of MN, NPBs and NBUDs scored in binucleated (BN) cells were obtained from the mitogen-stimulated lymphocytes of patients and control subjects. Statistical comparisons of the numbers of BN cells with MN, NPBs and NBUDs and ages between the patients with NASH and control subjects were performed.
RESULTS: The mean ages of the patients and the control group were 41.92 ± 13.33 and 41.80 ± 13.09 years (P > 0.05), respectively. The values of the mean body mass index (BMI), HOMA-IR, hemoglobin, creatinin, aspartate aminotransferase, alanine aminotransferase, triglyceride, high density lipoprotein, and low density lipoprotein were 31.19 ± 4.62 kg/m2 vs 25.07 ± 4.14 kg/m2, 6.71 ± 4.68 vs 1.40 ± 0.53, 14.73 ± 1.49 g/dL vs 14.64 ± 1.30 g/dL, 0.74 ± 0.15 mg/dL vs 0.80 ± 0.13 mg/dL, 56.08 ± 29.11 U/L vs 16.88 ± 3.33 U/L, 92.2 ± 41.43 U/L vs 15.88 ± 5.88 U/L, 219.21 ± 141.68 mg/dL vs 102.56 ± 57.98 mg/dL, 16.37 ± 9.65 mg/dL vs 48.72 ± 15.31 mg/dL, and 136.75 ± 30.14 mg/dL vs 114.63 ± 34.13 mg/dL in the patients and control groups, respectively. The total numbers and frequencies of BN cells with MN, NPBs and NBUDs, which were scored using the CBMN cytome assay on PHA-stimulated lymphocytes, were evaluated in the patients with NASH and control group. We found significantly higher numbers of MN, NPBs and NBUDs in the BN cells of patients with NASH than in those of the control subjects (21.60 ± 9.32 vs 6.88 ± 3.91; 29.28 ± 13.31 vs 7.84 ± 3.96; 15.60 ± 5.55 vs 4.20 ± 1.63, respectively, P < 0.0001).
CONCLUSION: The increased numbers of MN, NPBs and NBUDs observed in the lymphocytes obtained from patients with NASH may reflect genomic instability.
PMCID: PMC3752563  PMID: 23983432
Non-alcoholic steatohepatitis; Micronucleus; Nucleoplasmic bridges; Nuclear buds
21.  Suppression of p53 and p21CIP1/WAF1 reduces arsenite-induced aneuploidy 
Chemical research in toxicology  2010;23(2):357-364.
Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21CIP1/WAF1 in aneugenesis and clastogenesis was investigated in telomerase immortalized diploid human fibroblasts using siRNA suppression of p53 and p21CIP1/WAF1. Cells were exposed to the environmental carcinogen sodium arsenite (15 and 20 µM), and the induction of micronuclei (MN) was evaluated in binucleated cells using the cytokinesis-block assay. To determine whether MN resulted from missegregation of chromosomes or from chromosomal fragments, we used a fluorescent in situ hybridization with a centromeric DNA probe. Micronuclei were predominantly of clastogenic origin in control cells regardless of p53 or p21CIP1/WAF1 expression. MN with centromere signals in cells transfected with NSC siRNA or Mock increased 30% after arsenite exposure, indicating that arsenite induced aneuploidy in the tGM24 cells. Although suppression of p53 increased the fraction of arsenite-treated cells with MN, it caused a decrease in the fraction of with centeromeric DNA. Suppression of p21CIP1/WAF1 like p53 suppression decreased the fraction of with centromeric DNA. Our results suggest that cells lacking normal p53 function cannot become aneuploid because they die by mitotic arrest-associated apoptosis, whereas cells with normal p53 function that are able to exit from mitotic arrest can become aneuploid. Furthermore our current results support this role for p21CIP1/WAF1. Since suppression of p21CIP1/WAF1 caused a decrease in aneuploidy induced by arsenite suggesting that p21CIP1/WAF1 plays a role in mitotic exit.
PMCID: PMC2825144  PMID: 20000476
aneuploidy; arsenite; p21CIP1/WAF1; p53
22.  Cytotoxic and genotoxic effects of sodium hypochlorite on human peripheral lymphocytes in vitro 
Cytotechnology  2009;59(2):113-119.
Chlorination is widely used method in the disinfection of drinking and utility water worldwide. In this study, cytotoxic and genotoxic effects of sodium hypochlorite were investigated by the cytokinesis-block micronucleus assay and chromosomal aberration analysis on human peripheral lymphocytes in vitro. A significant increase in chromosomal aberration frequency was observed in all treatments of NaOCl (0.030, 0.065, 0.100, 0.25, 0.5, 1, 2, 4 μg/mL) at 24 and 48 h compared with the negative control and mitomycin C (MMC, 0.3 μg/mL), which was used as a positive control. NaOCl significantly increased the frequency of micronuclei in a dose dependent manner. The results showed that there was a significant correlation between NaOCl concentration and chromosomal aberration, micronuclei frequency, necrotic cells, apoptotic cells and binucleated cells.
PMCID: PMC2698443  PMID: 19484372
Sodium hypochlorite; Chromosomal aberrations (CA); Micronucleus (MN) assay; Necrotic cell; Apoptotic cell
23.  Clinical Application of Micronucleus Test: A Case-Control Study on the Prediction of Breast Cancer Risk/Susceptibility 
PLoS ONE  2014;9(11):e112354.
The micronucleus test is a well-established DNA damage assay in human monitoring. The test was proposed as a promising marker of cancer risk/susceptibility mainly on the basis of studies on breast cancer. Our recent meta-analysis showed that the association between micronuclei frequency, either at baseline or after irradiation, and breast cancer risk or susceptibility, has been evaluated in few studies of small size, with inconsistent results. The aim of the present study is to investigate the role of micronucleus assay in evaluating individual breast cancer susceptibility. Two-hundred and twenty untreated breast cancer patients and 295 female controls were enrolled in the study. All women were characterized for cancer family history and 155 subjects were evaluated for the presence of BRCA mutations. Micronuclei frequency was evaluated at baseline and after irradiation with 1-Gy gamma rays from a 137Cs source. The results show a non significant increase of frequency of micronucleated binucleated lymphocytes in cancer patients compared with the controls at baseline (Mean (S.E.): 16.8 (0.7) vs 15.7 (0.5), but not after irradiation (Mean (S.E.): 145.8 (3.0) vs 154.0 (2.6)). Neither a family history of breast cancer nor the presence of a pathogenic mutation in BRCA1/2 genes were associated with an increased micronuclei frequency. Our results do not support a significant role of micronucleus frequency as a biomarker of breast cancer risk/susceptibility.
PMCID: PMC4240584  PMID: 25415331
24.  Study of Radioprotective Effect of Green Tea against Gamma Irradiation Using Micronucleus Assay on Binucleated Human Lymphocytes 
The aim of this study was to investigate the radioprotective effect of green tea against genotoxicity induced by gamma irradiation in cultured blood lymphocytes from 5 human volunteers.
Materials and Methods
Peripheral blood samples were collected from volunteers before and 1, 3 and 5 hr after drinking a decoction 4 g green tea in 280 ml boiling water for 5 constitutive days with the same quantity. At each time point, the whole blood samples were exposed to 200 cGy of 60 Co gamma irradiation and then were cultured with mitogenic stimulation to determine the chromosomal aberration in micronucleus assay on cytokinesis-blocked binucleated cells.
As expected, for each volunteer, the results showed a significant increase in the incidence of micronuclei after exposure to gamma irradiation as compared to non-irradiated control samples. Only lymphocytes blood sample collected 3 hr after drinking green tea exhibited a significant decrease in incidence of micronuclei compared to non-treated irradiated samples.
These results suggest the radioprotective ability of green tea against ionizing radiation in human lymphocytes, at specified time after consumptior.
PMCID: PMC3586922  PMID: 23493517
Gamma irradiation; Green tea; Lymphocytes; Micronucleus; Natural radioprotective
25.  Genome Damage in Oropharyngeal Cancer Patients Treated by Radiotherapy 
Croatian medical journal  2008;49(4):515-527.
To estimate genome damage in oropharyngeal cancer patients before, during, and after radiotherapy and to measure the persistence of caused genome damage relevant in the evaluation of secondary cancer risk.
DNA damage was evaluated in peripheral blood lymphocytes of 10 oropharyngeal cancer patients using alkaline comet assay, analysis of structural chromosome aberrations, and micronucleus assay. Blood samples were taken 2 hours before irradiation on day 1 of the first radiotherapy cycle, 2 hours after the application of the first dose, in the middle of the radiotherapy cycle, within 2 hours after the last received radiotherapy dose, and after 6 and 12 months after radiotherapy.
In most participants, the highest level of primary DNA damage was recorded in blood samples collected after the administration of first radiation dose (mean tail length 25.04 ± 6.23 μm). Most patients also had increased frequency of comets with long tail-nucleus (LTN comets) after the administration of the first radiation dose (mean, 10.50 ± 7.71 per 100 comets), which remained increased in the middle of radiotherapy (mean, 18.30 ± 27.62 per 100 comets). Later on, the levels of primary DNA damage as recorded by the comet assay, slightly diminished. The frequency of structural chromosome aberrations in lymphocytes gradually increased during the radiation cycle (26.50 ± 27.72 per 100 metaphases at the end of the therapy), as well as the frequency of micronuclei (mean total number of micronuclei 167.20 ± 35.69; per 1000 binuclear cells).
Oropharyngeal cancer patients had relatively high levels of primary DNA damage in their peripheral blood lymphocytes even before therapy. The frequency of complex structural chromosome aberrations and the frequency of micronuclei increased with the progression of the radiation cycle and the doses delivered. As the frequency of chromosomal aberrations a year after radiotherapy mostly did not return to pre-therapy values, it represents an important risk factor related to the onset of second cancer.
PMCID: PMC2525833  PMID: 18716999

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