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International journal of radiation biology, 93, 36--47
2017

RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay).

Depuydt, Julie, Baeyens, Ans, Barnard, Stephen, Beinke, Christina, Benedek, Anett, Beukes, Philip, Buraczewska, Iwona, Darroudi, Firouz, De Sanctis, Stefania, Dominguez, Inmaculada, Monteiro Gil, Octávia, Hadjidekova, Valeria, Kis, Enikő, Kulka, Ulrike, Lista, Florigio, Lumniczky, Katalin, M'kacher, Radhia, Moquet, Jayne, Obreja, Doina, Oestreicher, Ursula, Pajic, Jelena, Pastor, Nuria, Popova, Ljubomira, Regalbuto, Elisa, Ricoul, Michelle, Sabatier, Laure, Slabbert, Jacobus, Sommer, Sylwester, Testa, Antonella, Thierens, Hubert, Wojcik, Andrzej, Vral, Anne

In the framework of the 'Realizing the European Network of Biodosimetry' (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1206231

Mol Med Rep, 14(1), 103–110
July, 2016

A semi‑automated FISH‑based micronucleus‑centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation.

Vral, Anne, Decorte, Veerle, Depuydt, Julie, Wambersie, André, Thierens, Hubert

The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.

Digital object identifier (DOI): 10.3892/mmr.2016.5265

Mol Med Rep, 13(1), 130–136
January, 2016

Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa.

Herd, Olivia, Francies, Flavia, Kotzen, Jeffrey, Smith, Trudy, Nxumalo, Zwide, Muller, Xanthene, Slabbert, Jacobus, Vral, Anne, Baeyens, Ans

Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation‑induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)‑positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV‑negative and 15 HIV‑positive) and 20 healthy controls were exposed to X‑rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV‑positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients.

Digital object identifier (DOI): 10.3892/mmr.2015.4504

Journal of applied toxicology : JAT
2016

Genotoxic risk of ethyl-paraben could be related to telomere shortening.

Finot, F, Kaddour, A, Morat, L, Mouche, I, Zaguia, N, Cuceu, C, Souverville, D, Négrault, S, Cariou, O, Essahli, A, Prigent, N, Saul, J, Paillard, F, Heidingsfelder, L, Lafouge, P, Al Jawhari, M, Hempel, W M, El May, M, Colicchio, B, Dieterlen, A, Jeandidier, E, Sabatier, L, Clements, J, M'Kacher, R

The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley & Sons, Ltd.

Digital object identifier (DOI): 10.1002/jat.3425

Mutagenesis, 29(3), 165--175
May, 2014

Automation and validation of micronucleus detection in the 3D EpiDerm?human reconstructed skin assay and correlation with 2D dose responses.

K. E. Chapman, A. D. Thomas, J. W. Wills, S. Pfuhler, S. H. Doak, G J S. Jenkins

Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay's fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 ?g/ml and methyl methanesulfonate (MMS) at 1750 ?g/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 ?g/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm?. Our preliminary validation here demonstrates that the RSMN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives.

Int J Radiat Biol, 90(4), 318--324
April, 2014

Effect of hypothermia on radiation-induced micronuclei and delayof cell cycle progression in TK6 cells.

Halina Lisowska, Karl Brehwens, Friedo Z{\"o}lzer, Aneta Wegierek-Ciuk, Joanna Czub, Anna Lankoff, Siamak Haghdoost, Andrzej Wojcik

Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis.Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8°C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8°C or 37.0°C.The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected.The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.

Int J Radiat Biol, 90(2), 193--202
February, 2014

Inter- and intra-laboratory comparison of a multibiodosimetric approachto triage in a simulated, large scale radiation emergency.

Elizabeth A. Ainsbury, Jenna Al-Hafidh, Ainars Bajinskis, Stephen Barnard, Joan Francesc Barquinero, Christina Beinke, Virginie {de Gelder}, Eric Gregoire, Alicja Jaworska, Carita Lindholm, David Lloyd, Jayne Moquet, Reetta Nylund, Ursula Oestreicher, Sandrine Roch-Lef{\'e}vre, Kai Rothkamm, Horst Romm, Harry Scherthan, Sylwester Sommer, Hubert Thierens, Charlot Vandevoorde, Anne Vral, Andrzej Wojcik

The European Union's Seventh Framework Programme-funded project 'Multi-disciplinary biodosimetric tools to manage high scale radiological casualties' (MULTIBIODOSE) has developed a multiparametric approach to radiation biodosimetry, with a particular emphasis on triage of large numbers of potentially exposed individuals following accidental exposures. In November 2012, an emergency exercise took place which tested the capabilities of the MULTIBIODOSE project partners. The exercise described here had a dual purpose: Intercomparison of (i) three biodosimetric assays, and (ii) the capabilities of the seven laboratories, with regards to provision of triage status for suspected radiation exposed individuals.Three biological dosimetry tools - the dicentric, micronucleus and gamma-H2AX (the phosphorylated form of member X of histone H2A, in response to DNA double-strand breaks) foci assays - were tested, in addition to provision of the triage status results (low exposure: 2 Gy) by the MULTIBIODOSE software. The exercise was run in two modes: An initial triage categorisation of samples (based on the first dose estimates for each assay received from each laboratory) followed by collation of the full set of estimated doses (all the results from all modes of each assay carried out by the participating laboratories) calculated using as many modes of operation as possible of the different assays developed during the project. Simulated acute whole body and partial body exposures were included.The results of the initial triage categorisation and the full comparison of assays and methods within and between laboratories are presented here.The data demonstrate that the MULTIBIODOSE approach of applying multiparametric tools to radiation emergencies is valid and effective.

Int J Radiat Biol, 90(2), 149--158
February, 2014

Induction and disappearance of gammaH2AX foci and formation of micronuclei after exposure of human lymphocytes to (60)Co gamma-rays and p(66)+ Be(40) neutrons.

Veerle Vandersickel, Philip Beukes, Bram {Van Bockstaele}, Julie Depuydt, Anne Vral, Jacobus Slabbert

To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (gammaH2AX foci) after in vitro exposure of human lymphocytes to either (60)Co gamma-rays or p(66)+ Be(40) neutrons.MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0-4 Gy gamma-rays or 0-2 Gy neutrons. Also, gammaH2AX foci DR curves were obtained following exposure to doses ranging from 0-0.5 Gy of either gamma-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy gamma-rays or neutrons were studied up to 24 hours post-irradiation.Micronuclei yields following neutron exposure were consistently higher compared to that from (60)Co gamma-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced gammaH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from gamma-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from gamma-rays. Foci formations were more likely to be over-dispersed for neutron irradiations.Although neutrons are more effective to induce MN, the absolute number of induced gammaH2AX foci are less at first compared to gamma-rays. With time neutron-induced foci are more persistent. These findings are helpful for using £^H2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

Radiat Res
July, 2013

NATO BIODOSIMETRY STUDY: Laboratory Intercomparison of the Cytokinesis-BlockMicronucleus Assay.

H. Romm, S. Barnard, H. Boulay-Greene, A. {De Amicis}, S. {De Sanctis}, M. Franco, F. Herodin, A. Jones, U. Kulka, F. Lista, P. Martigne, J. Moquet, U. Oestreicher, K. Rothkamm, H. Thierens, M. Valente, V. Vandersickel, A. Vral, H. Braselmann, V. Meineke, M. Abend, C. Beinke

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83\% accuracy and up to 100\% specificity).

Toxicol Sci
July, 2013

Genotoxicity Profile of Azidothymidine In Vitro.

Andreas Zeller, Julie Koenig, Georg Schmitt, Thomas Singer, Melanie Gu{\'e}rard

Azidothymidine (Zidovudine, AZT) is part of the standard care of treatment for acquired immunodeficiency syndrome since many years. A great number of studies on the genotoxic potential of AZT have been published, but no comprehensive hypothesis yet explains all observations. We investigated a multitude of genotoxic endpoints, both in vitro and in vivo, with the goal to complete the picture. The mutagenic potential of AZT in bacteria was found to be restricted to strains with an #ochre# target sequence and could be abrogated both by thymidine supplementation and rat liver S9 mix. Single-strand breaks in mammalian cells were detected in the comet assay after short-term treatment (3h) with AZT, which did not induce micronuclei. The latter were mainly seen after prolonged exposure (24 and 48h) and are probably not directly related to AZT incorporation into DNA. Our data demonstrate that short-term exposure to low AZT concentrations does not induce biologically relevant micronucleation. Only treatment with high concentrations of AZT for prolonged time periods manifests in substantial micronucleus induction. Furthermore, we found that high concentrations of thymidine have no effect in the comet assay but increase micronucleus frequency in a manner very similar to AZT. These results lead us to the following hypothesis: AZT is triphosphorylated and then incorporated into DNA strands, leading to mutations and cytotoxicity. Cellular attempts to repair these DNA lesions as well as stalled replication forks due to chain termination are detectable with the comet assay. Increased micronucleus frequency is likely related to nucleotide pool imbalance.

Toxicol Sci
July, 2013

Assessment of the Genotoxic Potential of Azidothymidine in the Comet, Micronucleus, and Pig-a Assay.

Melanie Guerard, Julie Koenig, Matthias Festag, Stephen D. Dertinger, Thomas Singer, Georg Schmitt, Andreas Zeller

The genotoxic potential of azidothymidine (Zidovudine, AZT), chosen as a model compound for nucleotide analogs, was comprehensively assessed in vivo for gene mutation, clastogenicity, and DNA breakage endpoints. Male Wistar rats were treated by oral gavage over 7 days with AZT at dose levels of 2×0 (control), 2×250, 2×500, and 2×1000mg/kg/day with a final single dose given on day 8. DNA damage was then evaluated with the comet assay in liver, stomach, and peripheral blood and with the micronucleus test in bone marrow and peripheral blood (by flow cytometry) in the same animals. After a treatment-free period of upto 42 days, the Pig-a gene mutation assay was performed in peripheral blood of the high-dose animals. In the comet assay as well as the micronucleus test, AZT caused a considerable dose-dependent increase in DNA damage in all tissues evaluated and was highly cytotoxic to bone marrow and peripheral blood cells. These data are well in line with published results. Surprisingly, AZT did not significantly increase the number of Pig-a mutant cells. We speculate that two factors likely contributed to this negative result: a predominance of large deletions caused by AZT, and the relatively low statistical power of the first-generation scoring method used for this study.

Mutat Res, 726(1), 36--41
November, 2011

A rapid, semi-automated method for scoring micronuclei in mononucleatedmouse lymphoma cells.

Ann T Doherty, Julie Hayes, Mick Fellows, Sarah Kirk, Mike O'Donovan

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.

Mutagenesis, 26(1), 11--17
January, 2011

The micronucleus assay as a biological dosimeter of in vivo ionisingradiation exposure.

Anne Vral, Michael Fenech, Hubert Thierens

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus (MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected. In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation.

Mutagenesis, 26(1), 169--175
January, 2011

Automated scoring of lymphocyte micronuclei by the MetaSystems Metaferimage cytometry system and its application in studies of human mutagensensitivity and biodosimetry of genotoxin exposure.

Andrea Rossnerova, Milada Spatova, Christian Schunck, Radim J Sram

Automated image analysis scoring of micronuclei (MN) in cells can facilitate the objective and rapid measurement of genetic damage in mammalian and human cells. This approach was repeatedly developed and tested over the past two decades but none of the systems were sufficiently robust for routine analysis of MN until recently. New methodological, hardware and software developments have now allowed more advanced systems to become available. This mini-review presents the current stage of development and validation of the Metasystems Metafer MNScore system for automated image analysis scoring of MN in cytokinesis-blocked binucleated lymphocytes, which is the best-established method for studying MN formation in humans. The results and experience of users of this system from 2004 until today are reviewed in this paper. Significant achievements in the application of this method in research related to mutagen sensitivity phenotype in cancer risk, radiation biodosimetry and biomonitoring studies of air pollution (enriched by new data) are described. Advantages as well as limitations of automated image analysis in comparison with traditional visual analysis are discussed. The current increased use of the Metasystems Metafer MNScore system in various studies and the growing number of publications based on automated image analysis scoring of MN is promising for the ongoing and future application of this approach.

Mutagenesis, 26(1), 111--117
January, 2011

Heritability of baseline and induced micronucleus frequencies.

Harald Surowy, Antje Rinckleb, Manuel Luedeke, Madeleine Stuber, Anna Wecker, Dominic Varga, Christiane Maier, Josef Hoegel, Walther Vogel

The scoring of micronuclei (MN) is widely used in biomonitoring and mutagenicity testing as a surrogate marker of chromosomal damage inflicted by clastogenic agents or by aneugens. Individual differences in the response to a mutagenic challenge are known from studies on cancer patients and carriers of mutations in DNA repair genes. However, it has not been studied to which extent genetic factors contribute to the observed variability of individual MN frequencies. Our aim was to quantify this heritable genetic component of both baseline and radiation-induced MN frequencies. We performed a twin study comprising 39 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs. Due to the small number of DZ pairs, we had to recruit controls from which 38 age- and gender-matched random control pairs (CPs) were generated. For heritability estimates, we used biometrical modelling of additive genetic, common environmental, and unique environmental components (ACE model) of variance and direct comparison of variance between the sample groups. While heritability estimates from MZ to DZ comparisons produced inconclusive results, both estimation methods revealed a high degree of heritability (h(2)) for baseline MN frequency (h(2) = 0.68 and h(2) = 0.72) as well as for the induced frequency (h(2) = 0.68 and h(2) = 0.57) when MZ were compared to CP. The result was supported by the different intraclass correlation coefficients of MZ, DZ and CP for baseline (r = 0.63, r = 0.31 and r = 0.0, respectively) as well as for induced MN frequencies (r = 0.79, r = 0.74 and r = 0.0, respectively). This study clearly demonstrates that MN frequencies are determined by genetic factors to a major part. The strong reflection of the genetic background supports the idea that MN frequencies represent an intermediate phenotype between molecular DNA repair mechanisms and the cancer phenotype and affirms the approaches that are made to utilise them as predictors of, for example, cancer risk.

Radiation Measurements, 46(2), 169 - 175
2011

Micronucleus test for radiation biodosimetry in mass casualty events: Evaluation of visual and automated scoring

Claudia Bolognesi, Cristina Balia, Paola Roggieri, Francesco Cardinale, Paolo Bruzzi, Francesca Sorcinelli, Florigio Lista, Raffaele D'Amelio, Enzo Righi

In the case of a large-scale nuclear or radiological incidents a reliable estimate of dose is an essential tool for providing timely assessment of radiation exposure and for making life-saving medical decisions. Cytogenetics is considered as the #gold##standard# for biodosimetry. The dicentric analysis (DA) represents the most specific cytogenetic bioassay. The micronucleus test (MN) applied in interphase in peripheral lymphocytes is an alternative and simpler approach. A dose-effect calibration curve for the MN frequency in peripheral lymphocytes from 27 adult donors was established after in vitro irradiation at a dose range 0.15-8 Gy of 137Cs gamma rays (dose rate 6 Gy min-1). Dose prediction by visual scoring in a dose-blinded study (0.15-4.0 Gy) revealed a high level of accuracy (R = 0.89). The scoring of MN is time consuming and requires adequate skills and expertise. Automated image analysis is a feasible approach allowing to reduce the time and to increase the accuracy of the dose estimation decreasing the variability due to subjective evaluation. A good correlation (R = 0.705) between visual and automated scoring with visual correction was observed over the dose range 0-2 Gy. Almost perfect discrimination power for exposure to 1-2 Gy, and a satisfactory power for 0.6 Gy were detected. This threshold level can be considered sufficient for identification of sub lethally exposed individuals by automated CBMN assay.

Mutat Res
April, 2010

Cytosine arabinoside, vinblastine, 5-fluorouracil and 2-aminoanthracenetesting in the in vitro micronucleus assay with L5178Y mouse lymphomacells at Sanofi Aventis, with different cytotoxicity measurements,in support of the draft OECD Test Guidel

Olivier Cariou, Nathalie Laroche-Prigent, Sandrine Ledieu, Isabelle Guizon, Fran{\c{c}}oise Paillard, V{\'e}ronique Thybaud

Cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair), vinblastine (an aneugen that inhibits tubulin polymerisation), 5-fluorouracil (a nucleoside analogue with a steep response profile), and 2-aminoanthracene (a metabolism-dependent reference genotoxin) were tested in the in vitro micronucleus assay with L5178Y mouse lymphoma cells, without cytokinesis block. The four chemicals were independently evaluated in two Sanofi Aventis laboratories, one of which used an image analyser to score micronuclei, while the other scored micronucleated cells manually. Very similar results were obtained in the two laboratories, highlighting the robustness of the assay. The four test chemicals induced significant increases in the incidence of micronucleated cells at concentrations that produced no more than a 55+/-5\% reduction in survival growth, as measured with the three parameters recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) for chemical testing, namely the relative increase in cell counts, relative population doubling, and the relative cell count. These results support the premise that the relative increase in cell counts and relative population doubling, that take into account both cell death and cytostasis, are appropriate measures of survival growth reduction in the in vitro micronucleus test conducted in the absence of cytokinesis block, as recommended in MNvit.

Int J Radiat Biol, 86(1), 2--11
January, 2010

Automated micronucleus (MN) scoring for population triage in caseof large scale radiation events.

Petra Willems, Liezel August, Jacobus Slabbert, Horst Romm, Ursula Oestreicher, Hubert Thierens, Anne Vral

PURPOSE: In case of a large-scale radiation accident when hundreds of people may be exposed, it is important to distinguish the severely exposed individuals (> or =1 gray), who require early medical treatment, from those less exposed. The aim of our study was to develop a quick population triage method based on automated micronucleus (MN) scoring. MATERIALS AND METHODS: Using the MN software module developed by MetaSystems specifically for the Metafer4 platform, about 60 blood samples can be scored in one day. Standard dose response curves were determined for manual and automated MN scoring. RESULTS: The automated MN assay results were closely correlated with MN yields obtained with the manual procedure. A dose of 1 Gy can be estimated with an uncertainty of 0.2 Gy. Corrections for false positives and false negatives by visual inspection of the image gallery did not result in an improved accuracy or reproducibility. To test the automated MN assay in a multicenter setting, an inter-laboratory comparison was performed whereby irradiated blood samples were processed in Ghent University (Belgium) and BfS (Bundesamt fuer Strahlenschutz; Germany). Both laboratories obtained comparable results. CONCLUSIONS: These results confirm the efficacy of the automated MN assay for fast population triage in a multicenter setting, in the case of large radiation accidents.

Digital object identifier (DOI): 10.3109/09553000903264481

Mutation Research, 669(2), 42-47
2009

The impact of air pollution on the levels of micronuclei measured by automated image analysis.

A. Rossnerova, M. Spatova, P. Rossner, I. Solansky, R.J. Sram

The measurement of micronuclei (MN) in human peripheral blood lymphocytes is frequently used in molecular epidemiology as one of the preferred methods for assessing chromosomal damage resulting from environmental mutagen exposure. In the present study, we evaluated the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), volatile organic compounds (VOC) and smoking on the frequency of MN in a group of 56 city policemen living and working in Prague. The average age of the participants was 34+/-6 years. The study was conducted on the same subjects in February and May 2007. The concentrations of air pollutants were obtained from personal and stationary monitoring. A statistically significant decrease in the levels of pollutants was observed in May when compared with February, with the exception of toluene levels measured by stationary monitoring. The frequency of MN was determined by the automatic image scoring (MetaSystems Metafer 4, version 3.2.1) of DAPI-stained slides. The results of the image analysis indicated a significant difference in the frequency of MN (mean levels 7.32+/-3.42 and 4.67+/-2.92, for February and May, respectively). Our study suggests that automatic image analysis of MN is a highly sensitive method for evaluating the effect of c-PAHs and confirms that there are no differences between smokers and nonsmokers. These results demonstrate the ability of c-PAHs to increase MN frequency, even if the exposure to c-PAHs occurred up to 60 days before the collection of biological material. Our work is the first human biomonitoring study focused on the measurement of MN by automated image analysis for assessing chromosomal damage as a result of environmental mutagen exposure.