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1.  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.
doi:10.1590/S1415-47572010005000084
PMCID: PMC3036155  PMID: 21637587
lamivudine; micronucleus assay; stavudine; transcriptase inhibitors; zidovudine
2.  Use of the Cytokinesis-Blocked Micronucleus Assay (CBMN) to Detect Gender Differences and Genetic Instability in a Lung Cancer Case-Control Study 
Background
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.
Methods
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.
Results
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).
Conclusions
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.
Impact
Identifying factors that increase lung cancer risk may lead to more effective prevention measures.
doi:10.1158/1055-9965.EPI-12-0435
PMCID: PMC3538922  PMID: 23195992
Lung cancer; CBMN assay; DNA damage; gender differences
3.  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.
doi:10.1093/carcin/bgp045
PMCID: PMC2722144  PMID: 19237609
4.  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
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
doi:10.1038/sj.bjc.6690569
PMCID: PMC2363103  PMID: 10408406
cervical carcinoma; radiotherapy; micronucleus assay; predictive value
6.  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.
Images
PMCID: PMC2033705  PMID: 7981062
7.  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
8.  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.
doi:10.1590/S1415-47572010005000021
PMCID: PMC3036877  PMID: 21637497
calcite; exfoliated buccal cells; micronucleus (MN); genotoxicity
9.  Micronuclei in genotoxicity assessment: from genetics to epigenetics and beyond 
Frontiers in Genetics  2013;4:131.
Micronuclei (MN) are extra-nuclear bodies that contain damaged chromosome fragments and/or whole chromosomes that were not incorporated into the nucleus after cell division. MN can be induced by defects in the cell repair machinery and accumulation of DNA damages and chromosomal aberrations. A variety of genotoxic agents may induce MN formation leading to cell death, genomic instability, or cancer development. In this review, the genetic and epigenetic mechanisms of MN formation after various clastogenic and aneugenic effects on cell division and cell cycle are described. The knowledge accumulated in literature on cytotoxicity of various genotoxins is precisely reflected and individual sensitivity to MN formation due to single gene polymorphisms is discussed. The importance of rapid MN scoring with respect to the cytokinesis-block micronucleus assay is also evaluated.
doi:10.3389/fgene.2013.00131
PMCID: PMC3708156  PMID: 23874352
micronuclei; chromosome-defective; epigenetics; genotoxicity; DNA methylation; histones; small RNAs
10.  Genomic instability in patients with type 2 diabetes mellitus on hemodialysis 
Objective
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.5581/1516-8484.20120011
PMCID: PMC3459606  PMID: 23049381
Diabetes Mellitus, type 2; Micronucleus tests; Comet assay; Genomic instability; Renal dialysis
11.  Study of Radioprotective Effect of Green Tea against Gamma Irradiation Using Micronucleus Assay on Binucleated Human Lymphocytes 
Objective(s)
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.
Results
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.
Conclusion
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
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.
doi:10.1093/jrr/rrt091
PMCID: PMC3885124  PMID: 23908554
micronucleus assay; micronuclei; genotoxicity; cytochalasin B; ionising radiation; radon; children
13.  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.
doi:10.1371/journal.pone.0064532
PMCID: PMC3652818  PMID: 23675539
14.  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.
doi:10.1021/tx900353v
PMCID: PMC2825144  PMID: 20000476
aneuploidy; arsenite; p21CIP1/WAF1; p53
15.  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.
Background
Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
Objective
The aim of the study was to investigate the impact of V2O5 on DNA stability in workers from a V2O5 factory.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1289/ehp.11438
PMCID: PMC2599764  PMID: 19079721
comet assay; cytokinesis-block micronucleus assay; DNA damage; occupational exposure; vanadium pentoxide
16.  Protective effect of ascorbic acid against oxidative damage induced by hydrogen peroxide in cultured human peripheral blood lymphocytes 
Hydrogen peroxide is one of the reactive oxygen species for cellular injury. It is overproduced during oxidative stress and is known to damage proteins, nucleic acids and cell membranes. The present study was aimed to study the protective effect of ascorbic acid against the toxic doses of hydrogen peroxide using lipid peroxidation and cytokinesis blocked micronucleus assay. Hydrogen peroxide was studied at 50, 100 and 200μM and was found to increase a dose dependent increase in lipid peroxidation and micronuclei frequency. The treatment of 100 and 200μM of hydrogen peroxide separately along with 20, 40 and 80μM of ascorbic acid showed a dose dependent decrease in the lipid peroxidation and micronuclei frequency. The results suggest a protective effect of ascorbic acid against the hydrogen peroxide induced oxidative damage in cultured human peripheral blood lymphocytes.
doi:10.1007/s12291-009-0055-5
PMCID: PMC3453308  PMID: 23105852
Hydrogen peroxide; Ascorbic acid; Lipid peroxidation; Micronucleus; Reactive oxygen species
17.  4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) regulates CTL activation and memory programming 
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the major carcinogens in tobacco. NNK has been associated with various cancers in tobacco users, especially lung cancer. However, the effects of NNK on cytotoxic T lymphocytes (CTLs), the cells responsible for destrcution of maligant and pathogen-infected cells, has not been elucidated. Using transgenic CTLs in vitro and in vivo, we show that NNK can directly affect CTL activation. NNK can enhance the expression of adhesion molecule CD62L in CTLs during their activation in vitro, but has no effects on their expansion and production of effector molecules such as IFN and granzyme B. After transferred into recipient mice, however, the NNK pretreated CTLs suffer an early loss in expansion. The percentage of memory precursors is higher in NNK pretreated CTLs, but the total amount of memory precursors is similar to controls. The final memory CTL population from NNK pretreated CTLs is reduced, but sustains a more central memory phenotype. In conclusion, NNK can affect CTL activation by modulating adhension molecule expression and reducing memory programming.
doi:10.1016/j.bbrc.2013.05.011
PMCID: PMC3787869  PMID: 23673295
Carcinogen; CTLs; NNK; CD62L; Cytokines; Memory; Activation
18.  The effect of aging on micronuclei frequency and proliferation in human peripheral blood lymphocytes 
Indian Journal of Human Genetics  2012;18(1):95-100.
INTRODUCTION:
Increase in the instability of cellular genome with an increasing age is the result of an accumulation of cellular damage and mutations. This instability which might be observed as chromosome damage or chromosome losses can be measured by the micronucleus technique.
AIM:
The aim of this study was to investigate the effect of aging and oxidative stress induced by non-toxic levels of H2O2 on micronuclei induction and their relationship to cell proliferation in human peripheral blood lymphocytes.
MATERIALS AND METHODS:
Healthy volunteers with different ages were choosen. Spontaneous and H2O2 induced micronuclei frequencies were measured in peripheral blood lymphocytes of 30 volunteers by the micronucleus method.
RESULTS:
Spontaneous micronuclei frequencies increased first then started to decrease after 50 years of age. This biphasic response was significantly higher than micronucleus (MN) frequencies induced by H2O2 (P < 0.05), which followed the similar shape of response to increasing ages with lower frequencies. Proliferative capacity of cells either treated with H2O2 or not did not differ with an increasing age giving similar responses.
CONCLUSION:
These results indicate biphasic character of chromosome damage; first increase and decrease after 50 years with an increasing age. But this change pattern was not correlated with the steady state of proliferation capacity of cells through an increasing age. Decreases in H2O2-induced MN frequencies compared to spontaneous MN frequencies may be inducing an apoptosis by H2O2 treatment leading to underscoring damaged cells.
doi:10.4103/0971-6866.96671
PMCID: PMC3385189  PMID: 22754230
Aging; cell kinetics; lymphocytes; micronucleus
19.  DNA repair and mutagen sensitivity of epithelial cells and lymphocytes in oropharyngeal cancer 
Oncology Letters  2011;3(1):100-106.
Tobacco-associated nitrosamines are known carcinogens causing DNA damage in epithelial cells of the head and neck. A matched case-control study was performed to evaluate the sensitivity of patients with squamous cell cancer (SCC) of the oropharynx, and controls to tobacco-associated nitrosamines. Quantitative DNA repair was evaluated following a period of 15 and 30 min. Fresh biopsies from 100 male donors of macroscopically healthy oropharyngeal cells and lymphocytes (50 SCC patients and 50 controls) were incubated with N-nitrosodiethylamine (NDEA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or N-nitrosonornicotine (NNN). DNA damage in epithelial cells and lymphocytes was assessed using the comet assay. Following incubation with NDEA, cells underwent a period of DNA repair. All of the nitrosamines caused equivalent genotoxic damage in mucosal cells and lymphocytes of the two groups. Lymphocyte DNA repair capacity in the control group (26.8 and 37.1% after 15 and 30 min) was comparable to the tumor group (23.6 and 40.6%). However, epithelial cell DNA repair capacity of carcinoma patients was significantly reduced to 17.1% (15 min) and 23% (30 min) compared to the DNA repair of the control group (36.2%, 15 min and 46.0%, 30 min). Mutagen sensitivity was comparable in patients and controls. Thus, reduced epithelial cell DNA repair capacity of tumor patients is a possible endogenous risk factor for the development of head and neck squamous cell cancer.
doi:10.3892/ol.2011.417
PMCID: PMC3362385  PMID: 22740863
mutagen sensitivity; DNA repair; lymphocytes; oropharynx; comet assay; repair capacity
20.  Maternal and Gestational Factors and Micronucleus Frequencies in Umbilical Blood: The NewGeneris Rhea Cohort in Crete 
Environmental Health Perspectives  2011;119(10):1460-1465.
Background: The use of cancer-related biomarkers in newborns has been very limited.
Objective: We investigated the formation of micronuclei (MN) in full-term and preterm newborns and their mothers from the Rhea cohort (Crete), applying for the first time in cord blood a validated semiautomated analysis system, in both mono- and binucleated T lymphocytes.
Methods: We assessed MN frequencies in peripheral blood samples from the mothers and in umbilical cord blood samples. We calculated MN in mononucleated (MNMONO) and binucleated (MNBN) T lymphocytes and the cytokinesis block proliferation index (CBPI) in 251 newborns (224 full term) and 223 mothers, including 182 mother–child pairs. Demographic and lifestyle characteristics were collected.
Results: We observed significantly higher MNBN and CBPI levels in mothers than in newborns. In newborns, MNMONO and MNBN were correlated (r = 0.35, p < 0.001), and we found a moderate correlation between MNMONO in mothers and newborns (r = 0.26, p < 0.001). MNMONO frequencies in newborns were positively associated with the mother’s body mass index and inversely associated with gestational age and mother’s age, but we found no significant predictors of MNBN or CBPI in newborns.
Conclusions: Although confirmation is needed by a larger study population, the results indicate the importance of taking into account both mono- and binucleated T lymphocytes for biomonitoring of newborns, because the first reflects damage expressed during in vivo cell division and accumulated in utero, and the latter includes additional damage expressed as MN during the in vitro culture step.
doi:10.1289/ehp.1003246
PMCID: PMC3230441  PMID: 21622084
folate; gestational age; micronuclei; mononucleated cells; newborns; vitamin B12
21.  The tobacco carcinogen nitrosamine induces a differential gene expression response in tumour susceptible A/J and resistant C3H mouse lungs 
The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), an important carcinogen found in tobacco products, causes lung cancer in genetically susceptible animals. In addition to mutations of the K-Ras gene, NNK has non-mutagenic effects that include alterations in gene expression and immunomodulation in the lung. Here we report the identification of two gene sets associated with NNK-induced pulmonary tumourigenesis. First, to identify genes involved in the susceptibility to NNK, we compared the lung transcriptomes of NNK-resistant C3H mice with that of the NNK-susceptible A/J mice, identifying differential expression of genes related to innate immunity and inflammation. Second, to identify gene expression induced by NNK, we compared the lung transcriptomes of C3H and A/J mice post-treatment. The Resistin-like alpha (Retnla) gene was highly upregulated in response to NNK only in susceptible mice. This gene product is known to recruit immune cells to the lung, and accumulation of CD45 positive cells in A/J lungs correlated with increased Retnla expression. Genetic susceptibility to NNK-induced lung tumourigenesis may relate in part to gene expression changes and alterations in the immune response to create a protumourigenic environment, acting in concert with NNK’s mutagenic effects.
doi:10.1016/j.ejca.2012.08.027
PMCID: PMC3587785  PMID: 23010150
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone; Nitrosamine; Susceptibility; Lung cancer; Resistin-like alpha; Inflammation; Retnla
22.  Modulation of Radiation-Induced Genetic Damage by HCMV in Peripheral Blood Lymphocytes from a Brain Tumor Case-Control Study 
Cancers  2010;2(2):420-435.
Human cytomegalovirus (HCMV) infection occurs early in life and viral persistence remains through life. An association between HCMV infection and malignant gliomas has been reported, suggesting that HCMV may play a role in glioma pathogenesis and could facilitate an accrual of genotoxic damage in the presence of γ-radiation; an established risk factor for gliomas. We tested the hypothesis that HCMV infection modifies the sensitivity of cells to γ-radiation-induced genetic damage. We used peripheral blood lymphocytes (PBLs) from 110 glioma patients and 100 controls to measure the level of chromosome damage and cell death. We evaluated baseline, HCMV-, γ-radiation and HCMV + γ-radiation induced genetic instability with the comprehensive Cytokinesis-Blocked Micronucleus Cytome (CBMN-CYT). HCMV, similar to radiation, induced a significant increase in aberration frequency among cases and controls. PBLs infected with HCMV prior to challenge with γ-radiation led to a significant increase in aberrations as compared to baseline, γ-radiation and HCMV alone. With regards to apoptosis, glioma cases showed a lower percentage of induction following in vitro exposure to γ-radiation and HCMV infection as compared to controls. This strongly suggests that, HCMV infection enhances the sensitivity of PBLs to γ-radiation-induced genetic damage possibly through an increase in chromosome damage and decrease in apoptosis.
doi:10.3390/cancers2020420
PMCID: PMC3835085  PMID: 24281077
HCMV; brain tumors; CBMN-CYT assay; chromosome aberrations
23.  Urinary levels of the tobacco-specific carcinogen N′-nitrosonornicotine and its glucuronide are strongly associated with esophageal cancer risk in smokers 
Carcinogenesis  2011;32(9):1366-1371.
N′-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are tobacco-specific nitrosamines. NNN and NNK can induce cancers of the esophagus and lung, respectively, in laboratory animals, but data on human esophageal cancer are lacking. The association between levels of NNN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), an NNK metabolite, in urine samples collected before diagnosis and risk of esophageal cancer was examined in 77 patients with esophageal cancer and 223 individually matched controls, all current smokers, from a cohort of 18244 Chinese men in Shanghai, China, followed from 1986 to 2008. Urinary total NNN (free NNN plus NNN-N-glucuronide) was significantly higher, whereas the percentage of its detoxification product NNN-N-glucuronide was significantly lower in cases than controls. Odds ratios (95% confidence intervals) of esophageal cancer for the second and third tertiles of total NNN were 3.99 (1.25–12.7) and 17.0 (3.99–72.8), respectively, compared with the first tertile after adjustment for urinary total NNAL and total cotinine and smoking intensity and duration (Ptrend < 0.001). The corresponding figures for the percentage of NNN-N-glucuronides were 0.37 (0.17–0.80) and 0.27 (0.11–0.62) (Ptrend = 0.001). Urinary total NNN and the percentage of NNN-N-glucuronides almost completely accounted for the observed association for urinary total NNAL (free NNAL plus its glucuronides), urinary total cotinine and smoking intensity with esophageal cancer risk. These findings along with results of previous studies in laboratory animals support a significant and unique role of NNN in esophageal carcinogenesis in humans.
doi:10.1093/carcin/bgr125
PMCID: PMC3202311  PMID: 21734256
24.  DNA breaks and chromosome pulverization from errors in mitosis 
Nature  2012;482(7383):53-58.
Whether whole-chromosome aneuploidy promotes tumorigenesis has been controversial, in large part because of the paucity of insight into underlying mechanisms. Here we identify a mechanism by which mitotic chromosome segregation errors generate DNA breaks via the formation of structures called micronuclei. Whole chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and frequently pulverization of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for “chromothripsis” in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.
doi:10.1038/nature10802
PMCID: PMC3271137  PMID: 22258507
25.  Genetic Biomarkers for Neoplastic Colorectal Cancer in Peripheral Lymphocytes 
Mædica  2011;6(2):83-89.
ABSTRACT
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

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