Tetsuya Kawata

High- and low-LET induced chromosome damage in human lymphocytes : a time-course of aberrations in metaphase and interphase

Purpose : To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. Materials and methods : Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u -1 iron particles, 400MeV u -1 neon particles or γ-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or γ-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/Hoechst 33258 harlequin staining. Results : There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. Conclusion : The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Diagnostic value of FDG PET and salivary gland scintigraphy for parotid tumors

Purpose: The purpose of this study was to examine the diagnostic value of the combination of F-18 fluorodeoxyglucose (FDG) PET and Tc-99m pertechnetate salivary gland scintigraphy in parotid tumors. Materials and Methods: Seventy-two patients with benign parotid gland tumors (n = 52), malignant parotid tumors (n = 12), and inflammation (n = 8) underwent both FDG PET and salivary gland scintigraphy within 1 week, and 66 of the patients also underwent gallium scintigraphy. All patients were negative on their first fine-needle aspiration (FNA). Results: Malignant parotid tumors showed significantly higher FDG uptake (standard uptake values [SUVs]) than both benign tumors and inflammation, except in Warthins tumor (5.82 ± 3.95 vs. 2.07 ± 1.33; P <0.01). Although the SUV values of Warthins tumor and malignant parotid tumors overlapped somewhat, Warthins tumor did demonstrate increased radiotracer uptake, and it was reliably distinguished from other parotid gland tumors by the use of salivary gland scintigraphy. Considering a SUV value >3 as being positive for malignancy and excluding Warthins tumor on the basis of salivary gland scintigraphy, sensitivity and specificity of FDG PET were 75% and 80%, respectively. These results were superior to those of gallium scintigraphy (58% and 72%, respectively). Conclusions: Although the diagnostic value of FDG PET in the differentiation of malignant from benign parotid gland tumors was limited because of the high FDG uptake in some benign tumors, and particularly pleomorphic adenomas, combining salivary gland scintigraphy with FDG PET may help to negate this drawback, and this combination may be a more promising approach for differentiation of various parotid gland tumors in patients compared with nondiagnostic needle aspiration.

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

PURPOSEnTo 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.nnnMATERIALS AND METHODSnHuman 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.nnnRESULTSnCalyculin 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.nnnCONCLUSIONSnChemically 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.PURPOSEnTo 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.nnnMATERIAL AND METHODSnExponentially 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnCompared 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.

X-rays vs. carbon-ion tumor therapy : Cytogenetic damage in lymphocytes

PURPOSEnTo measure chromosomal aberrations in peripheral blood lymphocytes from cancer patients treated with X-rays or carbon ions (C-ions).nnnMETHODS AND MATERIALSnBlood samples from patients diagnosed for esophageal or uterine cervical cancer were obtained before, during, and at the end of the radiation treatment. The novel technique of interphase chromosome painting was used to detect aberrations in prematurely condensed chromosomes 2 and 4. The fraction of aberrant lymphocytes was measured as a function of the dose to the tumor volume. For comparison, blood samples were also exposed in vitro to X-rays or to carbon ions accelerated at the HIMAC.nnnRESULTSnC-ions were more efficient than X-rays in the induction of chromosomal aberrations in vitro. In patients with similar pathologies, tumor positions, and radiation field sizes, however, C-ions induced a lower fraction of aberrant lymphocytes than X-rays during the treatment. The initial slope of the dose-response curve for the induction of chromosomal aberrations during the treatment was correlated to the relative decrease in the number of white blood cells and lymphocytes during the treatment.nnnCONCLUSIONnC-ions induce a lower level of cytogenetic damage in lymphocytes than X-rays, reducing the risk of bone marrow morbidity.

Dose–response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions

Purpose : To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. Material and methods : Exponentially growing human fibroblast cells AG1522 were irradiated with γ-rays, energetic carbon (13 keV/ μ m, 80keV/ μ m), silicon (55 keV/ μ m) and iron (140 keV/ μ m, 185keV/ μ m, 440keV/ μ m) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. Results : The dose-response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80 keV/ μ m and decreasing at higher LET. The dose-response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for γ-rays and 13 keV/ μ m carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13 keV/ μ m (about 7) and 80 keV/ μ m carbon (about 71), and decreased gradually until 440keV/ μ m iron ions (about 66). Conclusions : High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.

Comparison of F ratios generated from interphase and metaphase chromosome damage induced by high doses of low- and high-LET radiation

Abstract Wu, H., George, K., Kawata, T., Willingham, V. and Cucinotta, F. A. Comparison of F Ratios Generated from Interphase and Metaphase Chromosome Damage Induced by High Doses of Low- and High-LET Radiation. Although biophysical models predict a difference in the ratio of interchromosomal to intrachromosomal interarm exchanges (F ratio) for low- and high-LET radiations, few experimental data support this prediction. However, the F ratios in experiments to date have been generated using data on chromosome aberrations in samples collected at the first postirradiation mitosis, which may not be indicative of the aberrations formed in interphase after exposure to high-LET radiations. In the present study, we exposed human lymphocytes in vitro to 2 and 5 Gy of γ rays and 3 Gy of 1 GeV/nucleon iron ions (LET = 140 keV/μm), stimulated the cells to grow with phytohemagglutinin (PHA), and collected the condensed chromosomes after 48 h of incubation using both chemically induced premature chromosome condensation (PCC) and the conventional metaphase techniques. The PCC technique used here condenses chromosomes mostly in the G2 phase of the cell cycle. The F ratio was calculated using data on asymmetrical chromosome aberrations in both the PCC and metaphase samples. It was found that the F ratios were similar for the samples irradiated with low- and high-LET radiation and collected at metaphase. However, for irradiated samples assayed by PCC, the F ratio was found to be 8.2 ± 2.0 for 5 Gy γ rays and 5.2 ± 0.9 for 3 Gy iron ions. The distribution of the aberrations indicated that, in the PCC samples irradiated with iron ions, most of the centric rings occurred in spreads containing five or more asymmetrical aberrations. These heavily damaged cells, which were either less likely to reach mitosis or may reach mitosis at a later time, were responsible for the difference in the F ratios generated from interphase and metaphase analysis after exposure to iron ions.

Measurements of the equivalent whole-body dose during radiation therapy by cytogenetic methods

Estimates of equivalent whole-body dose following partial body exposure can be performed using different biophysical models. Calculations should be compared with biodosimetry data, but measurements are complicated by mitotic selection induced in target cells after localized irradiation. In this paper we measured chromosomal aberrations in peripheral blood lymphocytes during radiotherapy, and estimated the equivalent whole-body dose absorbed, by using the novel technique of interphase chromosome painting. Premature chromosome condensation was induced in stimulated lymphocytes by incubation in calyculin A, and slides were hybridized in situ with whole-chromosome DNA probes specific for human chromosomes 2 and 4. Reciprocal exchanges were used to estimate the equivalent whole-body dose, based on individual pre-treatment in vitro calibration curves. Equivalent whole-body dose increased as a function of the number of fractions, and reached a plateau at high fraction numbers. Chromosomal aberration yields were dependent on field size, tumour position and concurrent chemotherapy. Results suggest that interphase chromosome painting is a simple technique able to give a reliable estimate of the equivalent whole-body dose absorbed during therapeutic partial-body irradiation.

Complex-type chromosomal exchanges in blood lymphocytes during radiation therapy correlate with acute toxicity

The new method of chemical-induced premature chromosome condensation combined with fluorescence in situ hybridization was used to analyze chromosomal damage in peripheral blood mononuclear lymphocytes of patients undergoing radiation treatment for esophageal cancer with high-energy X-rays or accelerated carbon ions at the National Institute of Radiological Sciences (Chiba, Japan). Total number of aberrant cells correlated with radiation field size, but no correlation was found with acute toxicity. A high frequency of complex-type exchanges were also recorded. This aberration type presented a high individual variability, and correlated well with the acute morbidity. Cytogenetic analysis by interphase chromosome painting is proposed as a useful tool for monitoring normal tissue effects during radiotherapy.

A Comparison of Chromosome Repair Kinetics in G0 and G1 Reveals that Enhanced Repair Fidelity under Noncycling Conditions Accounts for Increased Potentially Lethal Damage Repair

Abstract Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G0 phase or allowed to enter G1 phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G0. By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G1. The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G1) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G1) than for noncycling cells (G0). Since most repair in G0/G1 occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.