Eisuke Gotoh

A simple method for simultaneous interphase- metaphase chromosome analysis in biodosimetry

PURPOSE To find a simple protocol for measuring chromosome damage both in G1 and in G2/M chromosomes, to overcome problems related to low mitotic index and cell-cycle alterations in biodosimetric tests. MATERIALS AND METHODS The protocol is based on the use of calyculin A to induce premature chromosome condensation in human peripheral blood lymphocytes in different phases of the cell cycle. Chromosome exchanges were measured by fluorescence in situ hybridization (chromosomes 2 and 4) in lymphocytes from four different donors. Cells were exposed to 4Gy X-rays and the results were compared to aberrations in M phase (colcemid block) and G0 (premature chromosome condensation induced by fusion to mitotic hamster cells). RESULTS Treatment with calyculin A produced a high fraction of chromosome condensation in different phases of the cell cycle. Cells in G1 and G2/M could be scored simultaneously for biodosimetry by chromosome painting. The condensation index was 5-20 times higher than the mitotic index (colcemid alone). The calyculin A treatment did not produce a significant increase in the background of chromosomal aberrations or modify the yield of chromosomal aberrations scored after exposure to X-rays. CONCLUSIONS Induction of chromosome condensation by calyculin A is a powerful biodosimetric tool, which provides a high number of spreads for analysis and overcomes problems related to poor in vitro growth or cell-cycle alterations.

Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes

We have studied the induction of chromosomal aberrations in human lymphocytes exposed in G0 to X rays or carbon ions. Aberrations were analyzed in G0, G1, G2 or M phase. Analysis during the interphase was performed by chemically induced premature chromosome condensation, which allows scoring of aberrations in G1, G2 and M phase; fusion-induced premature chromosome condensation was used to analyze the damage in G0 cells after incubation for repair; M-phase cells were obtained by conventional Colcemid block. Aberrations were scored by Giemsa staining or fluorescence in situ hybridization (chromosomes 2 and 4). Similar yields of fragments were observed in G1 and G2 phase, but lower yields were scored in metaphase. The frequency of chromosomal exchanges was similar in G0 (after repair), G2 and M phase for cells exposed to X rays, while a lower frequency of exchanges was observed in M phase when lymphocytes were irradiated with high-LET carbon ions. The results suggest that radiation-induced G2-phase block is associated with unrejoined chromosome fragments induced by radiation exposure during G0.

Chromosome condensation outside of mitosis: mechanisms and new tools

A basic principle of cell physiology is that chromosomes condense during mitosis. However, condensation can be uncoupled from mitotic events under certain circumstances. This phenomenon is known as “premature chromosome condensation (PCC).” PCC provides insights in the mechanisms of chromosome condensation, thus helping clarifying the key molecular events leading to the mitosis. Besides, PCC has proved to be an useful tool for analyzing chromosomes in interphase. For example, using PCC we can visualize genetic damage shortly after the exposure to clastogenic agents. More than 30 years ago, the first report of PCC in interphase cells fused to mitotic cells using Sendai virus was described (virus‐mediated PCC). The method paved the way to a great number of fundamental discoveries in cytogenetics, radiation biology, and related fields, but it has been hampered by technical difficulties. The novel drug‐induced PCC method was introduced about 10 years ago. While fusion‐induced PCC exploits the action of external maturation/mitosis promoting factor (MPF), migrating from the inducer mitotic cell to the interphase recipient, drug‐induced PCC exploits protein phosphatase inhibitors, which can activate endogenous intracellular MPF. This method is much simpler than fusion‐induced PCC, and has already proven useful in different fields. J. Cell. Physiol. 209: 297–304, 2006.

Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation

PURPOSE To analyse the kinetics of chromatid break induction, rejoining, and misrejoining after y-irradiation in G2 phase human cells using premature chromosome condensation induced by calyculin A. MATERIALS AND METHODS Human fibroblast AG1522 cells were irradiated with gamma-rays and chromosomes were then prematurely condensed by calyculin A. The number of chromatid breaks and chromatid exchanges in G2 chromosomes were scored, and fitted curves were calculated. RESULTS Calyculin A induced premature chromosome condensation in cells immediately after irradiation. Kinetics of rejoining of chromatid breaks demonstrated two exponential components with rapid and slow time constants. Within 5 min after irradiation, the number of chromatid breaks fell rapidly to about one-half, then gradually decreased. Chromatid exchanges were formed very quickly, reaching a plateau within 20 min from exposure. CONCLUSIONS Chemically induced premature chromosome condensation technique allows a simple, rapid and precise analysis of chromatid breakage and rejoining. The rapid kinetic component was particularly well characterized.

High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts.

Purpose : To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. Results : The kinetics of chromatid break rejoining following low-or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10 min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. Conclusions : Compared with γ-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.PURPOSE To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.

Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC)

There is a need for quick dose estimation by a simple method in radiation accidents. This study develops a simple and rapid dose estimation protocol for victims of such accidents, in particular those involving high radiation doses. Human peripheral blood lymphocytes (PBL) were γ-irradiated in vitro at several dose points up to 60 Gy, and were stimulated with phytohaemagglutinin-P (PHA-P) for 2 days to obtain dividing cells. PBL were then forced to condense prematurely, using 50 nM calyculin A, and the obtained chromosome spreads were Giemsa stained. The G2-PCC (prematurely condensed chromosomes) index and chromosome number for each radiation dose point were scored. G2-PCC were stably induced using calyculin A within 24 h delays in stimulation of PBL with PHA-P. The chromosome number of G2-PCC increased steeply with radiation doses up to 30 Gy at a rate of 0.31 Gy − 1 and then decreased at 0.30 Gy − 1 up to 40 Gy. More than 10% of G2-PCC index remained up to a 15 Gy dose. Even after 40 Gy irradiation, about 2% PCC index was obtained, and this value was enough to score a sufficient number of chromosome spreads for analysis. Therefore, the combined use of chromosome number and G2-PCC index allows biodosimetry to be done easily and rapidly. If PCC are not induced using calyculin A, it is strongly suggested that the radiation dose is over 50 Gy. A rapid and easy dose estimation for large dose exposure whole-body was realized by combined analysis of Giemsa-stained chromosome number of G2-PCC and PCC index using calyculin A. This simple method will be of use for rapid decision making of therapy for radiation accident victims. This method also has potential for use as a biodosimeter for partial-body exposure accidents.

Simple biodosimetry method for cases of high-dose radiation exposure using the ratio of the longest/shortest length of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC)

The aim was to develop a simple biodosimetry method for as rapid as possible estimation of absorbed radiation doses in victims of radiation accidents, in particular after high-dose exposure. Human peripheral blood lymphocytes (PBL) were gamma-irradiated in vitro with several doses up to 40 Gy stimulated with phytohaemagglutinin-P (PHA-P) for 2 days and their chromosomes condensed prematurely using 50 nm calyculin A. Chromosome lengths of Giemsa-stained G2 prematurely condensed chromosomes (PCC) were measured using image analysing software and the ratio of the longest/shortest chromosome length was calculated. The length ratio (LR) of the longest/shortest Giemsa-stained chromosome s increased with a good correlation to the square root of the radiation dose (D) up to 40 Gy, i.e. LR = (4.90 × D0.5) + 2.14. The LR of the longest/shortest chromosome might be used as an index for estimating the radiation dose. The blood samples should not be cooled until the start of separation/stimulation of the lymphocytes. A rapid and easy estimation of large doses after whole-body exposure was identified by measuring the ratio of the longest/shortest length of Giemsa-stained G2-PCC induced by calyculin A. This simple protocol will be particularly useful for making therapy decisions for victims of ionizing radiation exposure and has potential for use as a biodosimeter for partial-body exposure accidents.

Drug-Induced Premature Chromosome Condensation (PCC) Protocols: Cytogenetic Approaches in Mitotic Chromosome and Interphase Chromatin

Chromosome analysis is a fundamental technique for cytogenetic studies. Chromosomes are conventionally prepared from mitotic cells arrested by colcemid block protocol. However, obtaining the mitotic chromosomes is often hampered under several circumstances. As a result, cytogenetic analysis will be sometimes difficult or even impossible in such cases. Premature chromosome condensation (PCC) is an alternative method that has proved to be a unique and useful way in chromosome analysis. Usually, PCC has been achieved following cell fusion mediated either by fusogenic viruses or by polyethylene glycol (cell-fusion PCC), but the cell-fusion PCC has several drawbacks. The novel drug-induced PCC using protein phosphatase inhibitors was introduced about 10 years ago. This method is much simple and easy even than the conventional mitotic chromosome preparation using colcemid block protocol and obtained PCC index (equivalent to mitotic index for metaphase chromosome) is much higher. Furthermore, this method allows the interphase chromatin to be condensed and visualized like mitotic chromosomes, thus opened the way for chromosome analysis not only in metaphase chromosomes but also in interphase chromatin. The drug-induced PCC has therefore proven the usefulness in cytogenetics and other cell biology fields.

Influence of hilar deposition in the evaluation of the alveolar epithelial permeability on 99mTc-DTPA aerosol inhaled scintigraphy

PurposeWe investigated whether hilar radioaerosol deposition affects the clearance rate of technetium-99m-labeled diethylenetriaminepentaacetic acid (99mTc-DTPA) from peripheral alveolar regions.Materials and methodsA total of 38 patients underwent 99mTc-DTPA inhalation lung scintigraphy. Six region of interest (ROI) patterns were adopted: ROI 1 was outlined around the entire hemithorax, and ROIs 2–6 were outlined around the hemithorax but excluded square ROIs of different size in the hilar region. Half-times (T½) were calculated with time-activity curves using onecompartment and two-compartment analyses. The T½ of ROIs 1–5 were plotted against the T½ of ROI 6, and regression lines were obtained with the least-squares method. The absolute values of the differences between surveyed values and regression line were calculated. The Wilcoxon test for trend and a single linear regression model were used to determine statistical significance.ResultsThere were significant reductions in the absolute values of the differences between surveyed values and regression line from ROIs 1–5 by one-component analysis and the fast component of two-compartment analysis (P < 0.001).ConclusionOur results suggest that the deposition of radioaerosol in the hilar region affects the clearance rate of 99mTc-DTPA from the alveoli in damaged lungs. The hilar region should be excluded from ROIs when alveolar epithelial permeability is evaluated.

Chromosomal Aberrations in Normal Human Cells Induced by the Auger Effect via Ca Atoms

Purpose: To quantify the Auger effect on chromosomal aberrations via Ca atoms in human cells. Material and Methods: Exponentially growing human normal fibroblasts (GM05389) were irradiated with 4.047 (CaK‐P), 4.026 (CaK‐L) and 4.067 (CaK‐H) keV X‐rays (corresponding to the resonance absorption edge of the Ca K‐shell and slightly below and slightly above the edge, respectively) using synchrotron radiation at the photon factory (PF) of the High Energy Accelerator Organization located in Tsukuba. Chromosomal aberrations induced by the irradiation were analyzed by the premature chromosome condensation (PCC) method using calyculin A. The dependency of the chromosomal aberrations on the incubation time post 2 Gy irradiation was observed for each energy. Irradiation using 200 kVp conventional X‐rays was also examined as a reference to CaK irradiation. Results: 1. Soon after irradiation with 2 Gy, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.21, 1.51 and 2.70 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.82, 0.98 and 6.30, for breaks/gaps, isochromatid breaks and exchanges, respectively. 2, After a 6‐hr incubation treatment post 2 Gy irradiation, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.59, 2.03 and 2.14 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.69, 1.66 and 2.00 for breaks/gaps, isochromatid breaks and exchanges, respectively. 3, Soon after irradiation, the ratios of the efficiencies of CaK‐P X‐rays to those of 200 kVp X‐rays were 1.74, 1.29 and 2.51 for breaks/gaps, isochromatid breaks and exchanges, respectively. And after a 6‐hr incubation treatment, the ratios were 5.50, 1.93 and 1.81 for breaks/gaps, isochromatid breaks and exchanges, respectively. Conclusions: An effective enhancement of chromosomal aberrations, such as breaks/gaps, isochromatid breaks and exchanges, was caused by Ca K‐shell ionization or excitation. Auger electrons emitted by Ca atoms in irradiated cells appear to have an important role in causing this enhancement. Comparing these efficiencies of chromosomal aberrations with those produced by 200 kVp conventional X‐rays suggests un‐repaired and complicated damage is induced by the X‐rays around the Ca K‐shell resonance absorption edge.