Honglu Wu

Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes.

Abstract George, K., Durante, M., Willingham, V., Wu, H., Yang, T. C. and Cucinotta, F. A. Biological Effectiveness of Accelerated Particles for the Induction of Chromosome Damage Measured in Metaphase and Interphase Human Lymphocytes. Radiat. Res. 160, 425–435 (2003). Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/μm in the dose range of 0.075–3 Gy. Dose–response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose–response curve for chromosomal damage with respect to low- or high-dose-rate γ rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/μm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for γ rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.

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.

Induction of chromosome aberrations in human cells by charged particles

Chromosome aberrations induced by high-energy charged particles in normal human lymphocytes and human fibroblasts have been investigated. The charged particles included 250 MeV/nucleon protons, 290 MeV/nucleon carbon ions and 1 GeV/nucleon iron ions. The energies of the charged particles were higher than in most of the studies reported in the literature. Lymphocytes were stimulated to grow immediately after irradiation, while fibroblasts were incubated at 37 degrees C for 24 h for repair. Chromosomes were collected at the first mitosis after irradiation and chromosome aberrations were scored using the fluorescence in situ hybridization (FISH) technique with a whole-chromosome 4 probe. Chromosome aberrations were classified as reciprocal exchanges, incomplete exchanges, deletions and complex exchanges. The relative biological effectiveness (RBE) for each type of aberration was calculated by dividing a dose of 4 Gy by the dose of the charged particles producing the same effect as 4 Gy of gamma rays. Results of this study showed that complex aberrations have the highest RBE for radiation of high linear energy transfer (LET) for human lymphocytes, but for fibroblasts, the greatest effect was for incomplete exchanges. For both lymphocytes and fibroblasts, iron ions induced a similar fraction of aberrant cells.

mBAND Analysis of Chromosomal Aberrations in Human Epithelial Cells Exposed to Low- and High-LET Radiation

Abstract Hada, M., Cucinotta, F. A., Gonda, S. R. and Wu, H. mBAND Analysis of Chromosomal Aberrations in Human Epithelial Cells Exposed to Low- and High-LET Radiation. Radiat. Res. 168, 98–105 (2007). Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to 137Cs γ rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and γ rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.

Estimate of the frequency of true incomplete exchanges in human lymphocytes exposed to 1 GeV/u Fe ions in vitro

PURPOSE To study the frequency of true incomplete exchanges induced by high-LET radiation. MATERIALS AND METHODS Human lymphocytes were exposed to 1 GeV/u Fe ions (LET = 140 keV/microm). Chromosome aberrations were analysed by a fluorescence in situ hybridization using a combination of whole-chromosome-specific probes and human telomere probes. Chromosomes 1, 3 and 4 were investigated. RESULTS The percentage of incomplete exchanges was between 23 and 29% if telomere signals were not considered. The percentage decreased to approximately 10% after ruling out false incomplete exchanges containing telomere signals. The final estimation of true incomplete exchanges was <10%. CONCLUSION Within a degree of uncertainty, the percentage of true incomplete exchanges in 1 GeV/u Fe ion-irradiated human lymphocytes was similar to that induced by gamma rays.

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Abstract Wu, H., Durante, M., Furusawa, Y., George, K., Kawata, T. and Cucinotta, F. A. Truly Incomplete and Complex Exchanges in Prematurely Condensed Chromosomes of Human Fibroblasts Exposed In Vitro to Energetic Heavy Ions. Radiat. Res. 160, 418–424 (2003). Confluent human fibroblast cells (AG1522) were irradiated with γ rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37°C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/μm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy γ rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.

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.

mBAND analysis for high- and low-LET radiation-induced chromosome aberrations: A review

During long-term space travel or cancer therapy, humans are exposed to high linear energy transfer (LET) energetic heavy ions. High-LET radiation is much more effective than low-LET radiation in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, and cytogenetic damage can be utilized as a biomarker for radiation insults. Epidemiological data, mainly from survivors of the atomic bomb detonations in Japan, have enabled risk estimation from low-LET radiation exposures. The identification of a cytogenetic signature that distinguishes high- from low-LET exposure remains a long-term goal in radiobiology. Recently developed fluorescence in situ hybridization (FISH)-painting methodologies have revealed unique endpoints related to radiation quality. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. This review will concentrate on recent data obtained with multicolor banding in situ hybridization (mBAND) methods in mammalian cells exposed to low- and high-LET radiations. Chromosome analysis with mBAND technique allows detection of both inter- and intrachromosomal exchanges, and also distribution of the breakpoints of aberrations.

Biological Effects of High-Energy Neutrons Measured In Vivo Using a Vertebrate Model

Abstract Interaction of solar protons and galactic cosmic radiation with the atmosphere and other materials produces high-energy secondary neutrons from below 1 to 1000 MeV and higher. Although secondary neutrons may provide an appreciable component of the radiation dose equivalent received by space and high-altitude air travelers, the biological effects remain poorly defined, particularly in vivo in intact organisms. Here we describe the acute response of Japanese medaka (Oryzias latipes) embryos to a beam of high-energy spallation neutrons that mimics the energy spectrum of secondary neutrons encountered aboard spacecraft and high-altitude aircraft. To determine RBE, embryos were exposed to 0–0.5 Gy of high-energy neutron radiation or 0–15 Gy of reference γ radiation. The radiation response was measured by imaging apoptotic cells in situ in defined volumes of the embryo, an assay that provides a quantifiable, linear dose response. The slope of the dose response in the developing head, relative to reference γ radiation, indicates an RBE of 24.9 (95% CI 13.6–40.7). A higher RBE of 48.1 (95% CI 30.0–66.4) was obtained based on overall survival. A separate analysis of apoptosis in muscle showed an overall nonlinear response, with the greatest effects at doses of less than 0.3 Gy. Results of this experiment indicate that medaka are a useful model for investigating biological damage associated with high-energy neutron exposure.

Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization

Abstract Kawata, T., Ito, H., George, K., Wu, H., Uno, T., Isobe, K. and Cucinotta, F. A. Radiation-Induced Chromosome Aberrations in Ataxia Telangiectasia Cells: High Frequency of Deletions and Misrejoining Detected by Fluorescence In Situ Hybridization. Radiat. Res. 159, 597–603 (2003). The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G2 and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to γ rays and allowed to repair at 37°C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to γ rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.