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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.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  A Mode-of-Action Approach for the Identification of Genotoxic Carcinogens 
PLoS ONE  2013;8(5):e64532.
Distinguishing between clastogens and aneugens is vital in cancer risk assessment because the default assumption is that clastogens and aneugens have linear and non-linear dose-response curves, respectively. Any observed non-linearity must be supported by mode of action (MOA) analyses where biological mechanisms are linked with dose-response evaluations. For aneugens, the MOA has been well characterised as disruptors of mitotic machinery where chromosome loss via micronuclei (MN) formation is an accepted endpoint used in risk assessment. In this study we performed the cytokinesis-block micronucleus assay and immunofluorescence mitotic machinery visualisation in human lymphoblastoid (AHH-1) and Chinese Hamster fibroblast (V79) cell lines after treatment with the aneugen 17-β-oestradiol (E2). Results were compared to previously published data on bisphenol-A (BPA) and Rotenone data. Two concentration-response approaches (the threshold-[Td] and benchmark-dose [BMD] approaches) were applied to derive a point of departure (POD) for in vitro MN induction. BMDs were also derived from the most sensitive carcinogenic endpoint. Ranking comparisons of the PODs from the in vitro MN and the carcinogenicity studies demonstrated a link between these two endpoints for BPA, E2 and Rotenone. This analysis was extended to include 5 additional aneugens, 5 clastogens and 3 mutagens and further concentration and dose-response correlations were observed between PODs from the in vitro MN and carcinogenicity. This approach is promising and may be further extended to other genotoxic carcinogens, where MOA and quantitative information from the in vitro MN studies could be used in a quantitative manner to further inform cancer risk assessment.
PMCID: PMC3652818  PMID: 23675539
19.  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
20.  Evaluation of DNA damage in COPD patients and its correlation with polymorphisms in repair genes 
BMC Medical Genetics  2013;14:93.
We investigated a potential link between genetic polymorphisms in genes XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr) with the level of DNA damage and repair, accessed by comet and micronucleus test, in 51 COPD patients and 51 controls.
Peripheral blood was used to perform the alkaline and neutral comet assay; and genetic polymorphisms by PCR/RFLP. To assess the susceptibility to exogenous DNA damage, the cells were treated with methyl methanesulphonate for 1-h or 3-h. After 3-h treatment the % residual damage was calculated assuming the value of 1-h treatment as 100%. The cytogenetic damage was evaluated by buccal micronucleus cytome assay (BMCyt).
COPD patients with the risk allele XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) showed higher DNA damage by comet assay. The residual damage was higher for COPD with risk allele in the four genes. In COPD patients was showed negative correlation between BMCyt (binucleated, nuclear bud, condensed chromatin and karyorrhexic cells) with pulmonary function and some variant genotypes.
Our results suggest a possible association between variant genotypes in XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr), DNA damage and progression of COPD.
PMCID: PMC3848611  PMID: 24053728
COPD; DNA damage; DNA repair; Genetic polymorphisms; BMCyt
21.  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
22.  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
23.  Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients 
Pelvic organs morbidity after irradiation of cancer patients remains a major problem although new technologies have been developed and implemented. A relatively simple and suitable method for routine clinical practice is needed for preliminary assessment of normal tissue intrinsic radiosensitivity. The micronucleus test (MNT) determines the frequency of the radiation induced micronuclei (MN) in peripheral blood lymphocytes, which could serve as an indicator of intrinsic cell radiosensitivity.
To investigate a possible use of the micronucleus test (MNT) for acute radiation morbidity prediction in gynecological cancer patients.
Materials and methods
Forty gynecological cancer patients received 50 Gy conventional external pelvic irradiation after radical surgery. A four-field “box” technique was applied with 2D planning. The control group included 10 healthy females.
Acute normal tissue reactions were graded according to NCI CTCAE v.3.0. From all reaction scores, the highest score named “summarized clinical radiosensitivity” was selected for a statistical analysis.
MNT was performed before and after in vitro irradiation with 1.5 Gy. The mean radiation induced frequency of micronuclei per 1000 binucleated cells (MN/1000) and lymphocytes containing micronuclei per 1000 binucleated cells (cells with MN/1000) were evaluated for both patients and controls.
An arbitrary cut off value was created to pick up a radiosensitive individual: the mean value of spontaneous frequency of cells with MN/1000 ± 2SD, found in the control group.
Both mean spontaneous frequency of cells with MN/1000 and MN/1000 were registered to be significantly higher in cancer patients compared to the control group (t = 2.46, p = 0.02 and t = 2.51, p = 0.02). No statistical difference was registered when comparing radiation induced MN frequencies between those groups.
Eighty percent (32) of patients developed grade 2 summarized clinical radiosensitivity, with great variations in MNT parameters. Only three patients with grade 2 “summarized clinical radiosensitivity” had values of cells with MN/1000 above the chosen radiosensitivity threshold.
The present study was not able to confirm in vitro MNT applicability for radiosensitivity prediction in pelvic irradiation.
PMCID: PMC3863151  PMID: 24376993
Pelvic irradiation; Normal tissue radiosensitivity; Micronucleus test; Predictive methods
24.  Baseline Micronuclei Frequency in Children: Estimates from Meta- and Pooled Analyses 
Environmental Health Perspectives  2005;113(9):1226-1229.
The number of studies evaluating the effect of environmental exposure to genotoxic agents in children has rapidly increased in the last few years. The frequency of micronuclei (MN) in peripheral blood lymphocytes determined with the cytokinesis block assay is among the most popular biomarkers used for this purpose, although large inter- and intralaboratory variability of this end point has been observed in population studies. The availability of reference measures is therefore necessary for laboratories to validate protocols and analytical procedures, and for molecular epidemiologists, as well, to estimate the statistical power of studies and to assess the quality of data. In this article, we provide estimates of the baseline frequency of MN in children, conducting a meta-analysis of MN frequency reported by field studies in children and a pooled analysis of individual data [available from published studies and from the Human Micronucleus International Collaborative Study (HUMN) database]. Thirteen articles were selected for meta-analysis, and individual data included in the pooled analysis were retrieved from the databases of 12 laboratories. Overall means of 4.48 [95% confidence interval (CI), 3.35–5.98] and 5.70 (95% CI, 4.29–7.56) MN per 1,000 binucleated cells were estimated by the meta- and pooled analysis, respectively. A clear effect of age was detected, even within the restricted range of pediatric age considered, with significantly lower frequency values in newborns. No influence of sex was found. The study showed the advantage of using data from large collaborative studies and suggested a synergistic use of meta- and pooled analysis.
PMCID: PMC1280406  PMID: 16140632
biomarker; children; environmental exposure; genetic damage; meta-analysis; micronucleus assay; molecular epidemiology; pooled analysis
25.  Interlaboratory Evaluation of a Flow Cytometric, High Content In Vitro Micronucleus Assay 
Mutation research  2007;650(2):181-195.
An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) 56–66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei—the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.
PMCID: PMC2705921  PMID: 18182318
Micronuclei; Automation; Flow cytometry; Genotoxicity; Cytotoxicity; L5178Y

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