Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60) Co-γ rays; ～0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G(2) /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G(2) /M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G(2) /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.     

Comparative study with the alkaline Comet assay and the chromosome aberration test

The alkaline Comet assay is becoming a useful tool for early genotoxicity testing of new pharmaceutical drug candidates. The aim of this study was to elucidate the predictive value of Comet assay results for the outcome of the chromosome aberration (CA) test. For this purpose, a validation exercise with 13 drug candidates was carried out utilizing V79 Chinese hamster cells and human lymphocytes. The study demonstrates that results of the Comet assay and the chromosome aberration test show a high degree of agreement, irrespective of the cell type used. In the Comet assay, seven compounds were positive and six were negative, while in the CA test, six were positive and seven were negative. The only discrepancy was found with one compound that was positive in the Comet assay with V79 cells, negative in the Comet assay with human lymphocytes and clearly negative in the CA test with human lymphocytes. For the selection of concentrations for testing in the Comet assay, cytotoxicity by means of cell count after incubation or viability by means of Trypan-blue dye exclusion (TBDE) were used. The results show that either parameter led to analysis of a concentration range in the Comet assay similar to that chosen in the CA test, in which cell count (when using V79 cells) or mitotic index (in case of lymphocytes) were used. However, since cell count after incubation of cells is much more labour-intensive, viability was preferred as the parameter to assess cytotoxicity and for selecting concentrations for analysis in the Comet assay. The data presented in this study may contribute the regulatory acceptance of the Comet assay, e.g. for mechanistic studies.     

Interpreting chromosome aberration spectra.

Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH (multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.     

The statistical analysis of the in vitro chromosome aberration assay using Chinese hamster ovary cells

The purpose of the in vitro chromosome aberration assay (ABS) is to determine whether the test compound is a clastogen, i.e. induces structural changes in chromosomes. Details of this assay can be found in Galloway et al. [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261]. The standard design consists of a negative control and at least three positive dose groups. At each dose, a sample, say 200, of metaphase cells is examined microscopically and cells exhibiting at least one type of chromosome aberration are identified. Using Chinese hamster ovary cells, Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] and Richardson et al. [C. Richardson, D.A. Williams, J.A. Allen, G. Amphlett, D.O. Chanter, B. Phillips, Analysis of data from in vitro cytogenetic assays, in: D.J. Kirkland (Ed.), Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, 1989, pp. 141-154] demonstrated that a binomial sampling model could be used to describe the proportion of cells with chromosome aberrations.Statisticians and toxicologists have also suggested evaluation criteria for the dose response pattern of ABS. Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] suggested one use the Cochran-Armitage trend test. Sofuni et al. [T. Sofuni, A. Matsuoka, M. Sawada, M. Ishidate Jr, E. Zeiger, M.D. Shelby, Mutation Res. 241 (1990) 175-213] considered the dose response to be (strong) positive if it had two significant doses out of three dose groups and decided it was weakly positive if it had only one significant dose and there was a significant trend. The criterion of Galloway et al. for a positive response was a clear dose-related increase in cells with structural aberrations in one experiment or a reproducible single positive dose [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261].We formulate the above three procedures in terms of a Cochran-Armitage trend test and a Dunnett type test. We then compare the performance of these three procedures in terms of a Monte Carlo simulation study. We then develop a software program from the chosen procedure for its ease of use by statisticians and toxicologists.     

Proteus syndrome: a case with clonal chromosome aberration

Proteus syndrome is a disorder characterized by overgrowth of multiple tissues, connective tissue nevi, epidermal nevi and hyperostoses with asymmetric involvement. The clinical expression of the disorder is extremely variable. Molecular pathogenesis of the syndrome is unknown but it is hypothesized that it resulted from a somatic alteration of a gene leading to mosaic effects that would be lethal if the mutation was carried in nonmosaic fashion, and this may explain the variability among patients. We report a new case who presented at birth with asymmetric hypertrophy of the bones and soft tissues of fingers and a tumor of the chest. Cytogenetic analysis of the excised tumor revealed clonal chromosome aberration: mos46, XY, add(9)(p13) [5]/46,XY[30]. During follow up tumors of the rectum and urinary bladder were diagnosed.     

Structural aberration in chromosome No. 2

Summary Two males with gaps and breaks in a chromosome No. 2, at approximately 2q12 are presented. It is concluded that this aberration most probably has no deleterious effect on physical or mental development.

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Zusammenfassung Es wurden zwei Männer mit gaps und Brüchen in einem Chromosomen Nr. 2, annähernd bei 2q12, beobachtet. Diese Aberration hat sehr wahrscheinlich keine nachteilige Wirkung auf die körperliche oder geistige Entwicklung.

     

Vicia faba chromosomal aberration assay

A collaborative study involving laboratories in six countries was initiated under the sponsorship of the International Programme on Chemical Safety (IPCS) to determine the sensitivity, efficiency and reliability of the Vicia faba root tip meristem chromosomal aberration assay using a standardized protocol. The six Laboratories that participated in this study were located in the Slovak Republic, India, Japan, Poland, Sweden and the USA. All laboratories adhered to a standardized protocol for the Vicia faba chromosomal aberartion assay. Four coded chemicals, azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were tested with the Vicia faba chromosomal aberration assay. Of the four chemicals, three (MH, AG and MNU) were found to be clastogenic and gave a concentration related response. However, the results of NaN₃ were equivocal which might be explained by the stability of NaN₃. The conclusions from this study suggest that the Vicia faba chromosomal aberration bioassay is an efficient and reliable short-term bioassay for the rapid screening of chemicals for clastogenicity.     

肿瘤染色体畸变分析方法新进展

摘要: 肿瘤的发生多与染色体畸变有关, 确定染色体畸变与肿瘤的关系, 必然离不开染色体畸变的检测分析。文章简要综述几种常用染色体畸变的检测方法及其新进展, 包括G显带、荧光原位杂交(FISH )、光谱核型分析(SKY)、多色荧光原位杂交(M-FISH)、多色显带分析技术(Rx-FISH)、比较基因组杂交(CGH)和微阵列比较基因组杂交(Array CGH), 以及这些方法在肿瘤诊断和研究方面的应用。     

Effects of pingyanymycin on chromosomes: a possible structural basis for chromosome aberration

Pingyanymycin (PYM), and antitumor-antibiotic complex which belongs to the bleomycin family can induce "G2-free chromatin" and "uncompleted-packing-mitotic figures" (UPM) at increased frequency after treatment of cultured human lymphocytes. PYM can also induce an extraordinarily high frequency of chromosomal breaks but few sister-chromatid exchanges (SCE) in the same experiment, which is similar to the action of bleomycin. To solve this remarkable contradiction we presume that the UPM is related to a basic mechanism for producing chromosomal aberrations. Our results also show that various steps of the chromosomal cycle can be affected by certain chemical agents, and these treatments lead to chromosomal aberrations. Thus, other testing systems should be used in addition to the SCE system.     

Langer-Giedion syndrome,in a child with complex structural aberration of chromosome 8

A patient with typical features of the Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome, type II) is described. In the karyotype an interstitial deletion of the long arm of chromosome 8 (band 8q22) was observed as the result of a complex rearrangement of chromosomes 1 and 8: 46,XY inv(8)(q23 leads to q242), del(8)(q221 leads to q223), ins(8;1) (q221;p321 p341;q242). Previously reported cases of Langer-Giedion syndrome with deletion of 8q are compared with the present one.     

Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.     

Elastin metabolism in rodent lung

Data from the in vivo incorporation of [³H]valine into fractions of elastin obtained from rat or mouse lung suggest that postnatal lung elastin synthesis occurs predominantly in the first 1 to 2 weeks of life. Very little [³H]valine was incorporated into lung elastin obtained from adult animals. When lung elastin from neonatal mice was radiochemically labelled with [¹⁴C]lysine as a single pulse, it was observed that the specific activity of the elastin expressed as the total dpm values as ¹⁴C per mg was not significantly altered over a 6 month period. Elastin appears to turn over very slowly in mouse lung with half-life best estimated in years.     

Dual-energy micro-CT of the rodent lung

The purpose of this work is to investigate the use of dual-energy micro-computed tomography (CT) for the estimation of vascular, tissue, and air fractions in rodent lungs using a postreconstruction three material decomposition method. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact rat lungs were carefully removed from the thorax, injected with an iodine-based contrast agent, and then inflated with different volumes of air (n = 2). Finally, we performed in vivo imaging studies in C57BL/6 mice (n = 5) using fast prospective respiratory gating in end inspiration and end expiration for three different levels of positive end expiratory pressure (PEEP). Before imaging, mice were injected with a liposomal blood pool contrast agent. The three-dimensional air, tissue, and blood fraction maps were computed and analyzed. The results indicate that separation and volume estimation of the three material components of the lungs are possible. The mean accuracy values for air, blood, and tissue were 93, 93, and 90%, respectively. The absolute accuracy in determining all fraction materials was 91.6%. The coefficient of variation was small (2.5%) indicating good repeatability. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end expiration but no significant changes at end inspiration. Our method has applicability in preclinical pulmonary studies where changes in lung structure and gas volume as a result of lung injury, environmental exposures, or drug bioactivity would have important physiological implications.     

Prediction of clonal chromosome aberration frequency in human blood lymphocytes

We recently conducted a large-scale screening for clonal aberrations among atomic bomb survivors and proposed a model for the gross clonal composition of blood lymphocytes. Here we show an application of the model indicating that the number, m,of clones detectable by cytogenetic methods in an individual is predictable by the equation m= (1.8 + 6.4FG) x FP x n/500, where FG represents the estimated translocation frequency in the 46 chromosome set, FP is the observed translocation frequency with FISH or other methods, and nis the number of cells examined. Application of the equation to the results of seven other reports gave close agreement between the observed and calculated numbers of clones. Since the model assumes that clonal expansion is ubiquitous, and any translocation can be the constituent of a clone detectable by cytogenetic means, the vast majority of observed clonal expansions of these somatic cells are likely the result of random-hit events that are not detrimental to human health. Furthermore, since our model can predict the majority of clonal aberrations among Chernobyl workers who were examined 5-6 years after irradiation, clonal expansion seems to occur primarily within a few years after exposure to radiation, most likely being coupled with the process of recovery from radiation-induced injury in the lymphoid and hematopoietic systems.