<p>The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ?15 \% was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication.</p>

Ionizing radiation is a known human carcinogen that can induce a variety of biological effects depending on the physical nature, duration, doses and dose-rates of exposure. However, the magnitude of health risks at low doses and dose-rates (below 100mSv and/or 0.1mSvmin(-1)) remains controversial due to a lack of direct human evidence. It is anticipated that significant insights will emerge from the integration of epidemiological and biological research, made possible by molecular epidemiology studies incorporating biomarkers and bioassays. A number of these have been used to investigate exposure, effects and susceptibility to ionizing radiation, albeit often at higher doses and dose rates, with each reflecting time-limited cellular or physiological alterations. This review summarises the multidisciplinary work undertaken in the framework of the European project DoReMi (Low Dose Research towards Multidisciplinary Integration) to identify the most appropriate biomarkers for use in population studies. In addition to logistical and ethical considerations for conducting large-scale epidemiological studies, we discuss the relevance of their use for assessing the effects of low dose ionizing radiation exposure at the cellular and physiological level. We also propose a temporal classification of biomarkers that may be relevant for molecular epidemiology studies which need to take into account the time elapsed since exposure. Finally, the integration of biology with epidemiology requires careful planning and enhanced discussions between the epidemiology, biology and dosimetry communities in order to determine the most important questions to be addressed in light of pragmatic considerations including the appropriate population to be investigated (occupationally, environmentally or medically exposed), and study design. The consideration of the logistics of biological sample collection, processing and storing and the choice of biomarker or bioassay, as well as awareness of potential confounding factors, are also essential.

<p>Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), -7/7q (17.2%), and 11q- (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.</p>

Evidence suggests that de novo, therapy-related and benzene-induced acute myeloid leukemias (AML) occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes has been detected in benzene-related leukemia patients as well as in healthy benzene-exposed workers, suggesting that aneuploidy precedes and may be a potential mechanism underlying benzene-induced leukemia. Here, we analyzed the peripheral blood lymphocytes of 47 exposed workers and 27 unexposed controls using a novel OctoChrome fluorescence in situ hybridization (FISH) technique that simultaneously detects aneuploidy in all 24 chromosomes. Through this chromosome-wide aneuploidy study (CWAS) approach, we found heterogeneity in the monosomy and trisomy rates of the 22 autosomes when plotted against continuous benzene exposure. In addition, statistically significant, chromosome-specific increases in the rates of monosomy [5, 6, 7, 10, 16 and 19] and trisomy [5, 6, 7, 8, 10, 14, 16, 21 and 22] were found to be dose dependently associated with benzene exposure. Furthermore, significantly higher rates of monosomy and trisomy were observed in a priori defined 'susceptible' chromosome sets compared with all other chromosomes. Together, these findings confirm that benzene exposure is associated with specific chromosomal aneuploidies in hematopoietic cells, which suggests that such aneuploidies may play roles in benzene-induced leukemogenesis.

The capital city of Prague is one of the most polluted areas of the Czech Republic. The impact of air pollution on the level of chromosomal aberrations was systematically studied: analyses were performed using fluorescence in situ hybridization (FISH) with whole-chromosome painting for chromosomes #1 and #4. In the present study, we analyzed the levels of stable (one-way and two-way translocations) and unstable (acentric fragments) chromosomal aberrations in 42 mothers living in Prague and in their newborns. The average age of the mothers was 29 years (range, 20-40 years). Blood samples were collected from October 2007 to February 2008. The average levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and benzo[a]pyrene (B[a]P) in respirable particles (PM2.5), as determined by stationary monitoring, were 21.0+/-12.3ng/m(3) and 2.9+/-1.8ng/m(3), respectively. We did not observe any effect of either c-PAH or B[a]P exposure on the genomic frequency of translocations (per 100 cells, F(G)/100) in either group due to their similar exposure during the winter months. The mean values of F(G)/100 representing stable aberrations were 0.09+/-0.13 vs 0.80+/-0.79 (p

Both sporadic and familial Alzheimer's disease (AD) patients exhibit increased chromosome aneuploidy, particularly trisomy 21, in neurons and other cells. Significantly, trisomy 21/Down syndrome patients develop early onset AD pathology. We investigated the mechanism underlying mosaic chromosome aneuploidy in AD and report that FAD mutations in the Alzheimer Amyloid Precursor Protein gene, APP, induce chromosome mis-segregation and aneuploidy in transgenic mice and in transfected cells. Furthermore, adding synthetic Abeta peptide, the pathogenic product of APP, to cultured cells causes rapid and robust chromosome mis-segregation leading to aneuploid, including trisomy 21, daughters, which is prevented by LiCl addition or Ca(2+) chelation and is replicated in tau KO cells, implicating GSK-3beta, calpain, and Tau-dependent microtubule transport in the aneugenic activity of Abeta. Furthermore, APP KO cells are resistant to the aneugenic activity of Abeta, as they have been shown previously to be resistant to Abeta-induced tau phosphorylation and cell toxicity. These results indicate that Abeta-induced microtubule dysfunction leads to aneuploid neurons and may thereby contribute to the pathogenesis of AD.

The international radiation biodosimetry community has recently been engaged in activities focused on establishing cooperative networks for biodosimetric triage for radiation emergency scenarios involving mass casualties. To this end, there have been several recent publications in the literature regarding the potential for shared scoring in such an accident or incident. We present details from a medical irradiation case where two independently validated laboratories found very different yields of dicentric chromosome aberrations. The potential reasons for this disparity are discussed, and the actual reason is identified as being the partial-body nature of the radiation exposure combined with differing criteria for metaphase selection. In the context of the recent networking activity, this report is intended to highlight the fact that shared scoring may produce inconsistencies and that further validation of the scoring protocols and experimental techniques may be required before the networks are prepared to deal satisfactorily with a radiological or nuclear emergency. Also, the findings presented here clearly demonstrate the limitations of the dicentric assay for estimating radiation doses after partial-body exposures and bring into question the usefulness of rapid #triage##mode# scoring in such exposure scenarios.

Both occupational exposure to the leukemogen benzene and in vitro exposure to its metabolite hydroquinone (HQ) lead to the induction of numerical and structural chromosome changes. Several studies have shown that HQ can form DNA adducts, disrupt microtubule assembly and inhibit DNA topoisomerase II (topo II) activity. As these are potential mechanisms underlying endoreduplication (END), a phenomenon that involves DNA amplification without corresponding cell division, we hypothesized that HQ could cause END. We measured END in the human lymphoblastoid cell line, TK6, treated with HQ (0-20 microM) and etoposide (0-0.2 microM) for 48 h. Etoposide was used as a positive control as it is a topo II poison and established human leukemogen that has previously been shown to induce END in Chinese hamster ovary cells. Both HQ and etoposide significantly induced END in a dose-dependent manner (P(trend) s.

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10\% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation.

Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood.Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA.Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes.

Karyotyping is currently the #gold##standard# test for the detection of human chromosome abnormalities. Over the past 40 years, changes in techniques have improved the band definition of chromosomes; however, very little has changed with respect to improvements through automation. In this study, we compare chromosome analysis by traditional microscopy with semi-automatic karyotyping using robotic equipment from MetaSystems (Altlussheim, Germany). Analysis using MetaSystems was significantly quicker than using the microscope with an average reduction in analysis time of 26.5 minutes; for the average analyst, this equates to a reduction of 27 percent. Analysis checking times using MetaSystems showed even greater improvement with an average reduction in checking time of 11.4 minutes; for the average checker, this equates to a reduction of 48 percent. The MetaSystems semi-automatic karyotyping equipment offers increased throughput of cases for karyotype analysis while maintaining accuracy.

The present study comprised a biomonitoring study in 95 workers occupationally exposed to styrene and 98 unexposed controls, employing an integrated approach involving biomarkers of exposure, effect, and susceptibility. Airborne styrene was evaluated at workplace, and urinary styrene metabolites, mandelic acid (MA), phenylglyoxylic acid (PGA), vinylphenols (VPTs) and phenylhydroxyethylmercapturic acids (PHEMAs), were measured as biomarkers of internal dose. Cytogenetic alterations were evaluated by analysing the frequency of chromosomal aberrations (CAs) and micronucleated binucleated cells (MNBN) in peripheral blood lymphocytes. The micronucleus assay was coupled with centromeric fluorescence in situ hybridization to distinguish micronuclei (MN) arising from chromosomal breakage (C- MN) from those harboring whole chromosomes (C+ MN). The possible influence of genetic polymorphisms of xenobiotic-metabolizing enzymes involved in styrene biotransformation (EPHX1, GSTT1, GSTM1, GSTP1) and NAT2 on the cytogenetic endpoints was investigated. The exposed workers showed a significantly higher frequency of MNBN (13.8+/-0.5% versus 9.2+/-0.4%; P<0.001) compared to control subjects. The effect appeared to concern both C- and C+ MN. A positive correlation was seen between the frequency of C+ MN and urinary level of MA+PGA (P<0.05) and VPTs (P<0.001). Chromosome-type CAs positively correlated with airborne styrene level and VPTs (P<0.05), whereas chromatid-type CAs correlated with PHEMAs (P<0.05). Workers bearing GSTM1 null genotype showed lowered levels of PHEMAs (P<0.001). The GSTT1 null genotype was associated with increased MNBN frequencies in the exposed workers (P<0.05) and the fast activity EPHX genotype with a moderate decrease in both MNBN and CAs in the controls. Our results suggest that occupational exposure to styrene has genotoxic effects that are potentiated by the GSTT1 gene deletion. These observations may have relevance considering the risk of lymphatic and haematopoietic malignancies tentatively associated with styrene exposure.

In myelodysplastic syndromes (MDS), the karyotype is one of the most significant prognostic markers with profound impact on differential diagnosis and therapeutic decisions. In a retrospective study, we examined karyotypes of bone marrow specimens of an oligocentric cohort comprising 529 patients with MDS to address the question how many metaphases need to be analyzed to detect even small cell clones with an appropriate expenditure. We found a statistically significant difference of the frequency of normal karyotypes in the patient group with 19 or less analyzed metaphases compared to the group with 20 or more metaphases analyzed (56% versus 47%, p=0.041). Furthermore, we demonstrate that the analysis of 25 or more metaphases can further improve the sensitivity of karyotype analysis and leads to the identification of additional clinically relevant abnormal clones or subclones in a substantial proportion of patients. In summary, our data suggest the examination of at least 20 metaphases in MDS.

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.

La technique de référence en dosimétrie biologique est basée sur le dénombrement des aberrations chromosomiques de type dicentrique induit par les rayonnements ionisants. Cet article présente divers systèmesd'analyse d'images utilisés en dosimétrie biologique pour aider la détection de ces aberrations. Les systèmes présentés sont le CYTOGEN de la société IMSTAR, le CYTOSCAN (APPLIED IMAGING) et le METAFER (METASYSTEM). Tous ne présentent pas les mêmes fonctionnalités et chacun peut être utilisé de façon plus ou moins automatique. Certaines fonctionnalités communes de ces systèmes sont comparées. L'aide apportée par les systèmes porte sur 3 points : (1) localisation automatique des métaphases sur les lames, dans ce cas on a un gain de temps d'un facteur 2 à 4 par rapport au comptage manuel ; (2) un outil d'aide au comptage qui apporte un confort de lecture et une meilleure fiabilité des résultats ; (3) la détection automatique des dicentriques est particulièrement utile en cas de tri de population. En effet, dans ce cas il faut estimer très rapidement la dose reçue par un nombre important de personnes. Par contre, l'estimation de dose n'a pas besoin d'être aussi précise que dans le cas de l'expertise individuelle. Des erreurs dans la détection des dicentriques est alors tolérée et une détection automatique des dicentriques est envisageable. Le gain de temps est très appréciable puisqu'il est possible de compter 300 cellules en une demie-heure (METAFER) contre 25 avec la seule aide du chercheur de métaphases. Cependant la qualité de la détection doit encore être améliorée puisque 50 % des dicentriques ne sont pas détectées. Le marquage des centromères par technique FISH devrait permettre d'améliorer la sensibilité de la technique. Les premiers résultats sont encourageant puisque 90 % des centromères sont correctements détectés mais d'autres expériences doivent êtres réalisées pour évaluer le gain de temps.

The efficiency of the automated metaphase finding system METAFER2 is assessed in a routine mutagenicity assay using an aneuploid rat liver cell line treated with various promutagens. Data sets generated by automated and manual selection of metaphases are compared. It is demonstrated that METAFER2 routinely allows an efficient automatic identification of metaphases not only in lymphocyte preparations, but also in preparations from mammalian cell lines with varying chromosome numbers. Although larger slide areas are required for automated compared to manual metaphase scanning, the automatic system is faster by a factor of about 5. The interactive visual elimination of metaphases of insufficient quality is an easy and fast procedure. METAFER2 allows an unbiased selection of metaphases irrespective of their appearance as homogeneously stained first or harlequin-stained second division cells. Random selection of metaphases is neither influenced by various structural chromosome changes nor by increased frequencies of sister-chromatid exchanges.

The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).