Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Low doses of radiation decrease the level of spontaneous and gamma-induced chromosomal mutagenesis in bone marrow cells of mice in vivo

Low doses of ionizing radiation are known to induce adaptive response (AR), which is characterized in most cases by temporary nature, though the possibility of long-term persistence of AR is not ruled out. In this investigation we studied the effect of low doses of gamma-radiation on both high-dose radiation-induced and spontaneous level of cytogenetic damage throughout the life of mice. SHK male mice 2 months old were used. Priming doses of 0.1 and 0.2 Gy (0.125 Gy/min, gamma-radiation from 60Co) were used. A challenging dose of 1.5 Gy (1 Gy/min) was used in the experiments using a routine AR experimental design. The frequency of micronucleated polychromatic erythrocytes in bone marrow cells of primed, primed and challenged, and control groups was assessed at various times of animal life span. It was shown that: a) single low-dose gamma-irradiation induces a cytogenetic AR which can be revealed at 1, 3, 6, 9, 12 months after priming; b) single low-dose gamma-irradiation decreases the cytogenetic damage to a level below the spontaneous rate at the end of lifetime (20 months) of animals; c) ability to induce adaptive response does not depend on the age of animals at the moment of priming irradiation. In conclusion, the mechanisms underlying AR not only protect from chromosome damage induced by high-dose irradiation but also may play a role in spontaneous mutagenesis during aging of animals.     

Terminal maturation of megakaryocytes and platelet production by hematopoietic stem cells irradiated with heavy-ion beams

Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to high-linear energy transfer (LET) radiations such as heavy-ion beams that have greater biological effects than low-LET radiation. This study examined the terminal maturation of megakaryocytes and platelet production derived from hematopoietic stem cells irradiated with heavy-ion beams. CD34(+) cells derived from human placental/umbilical cord blood were exposed to monoenergetic carbon-ion beams (LET = 50 keV/μm) and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. There was no significant difference in megakaryocyte-specific markers between nonirradiated control and irradiated cells. Expression of Tie-2, a receptor that acts in early hematopoiesis, showed a significant 1.31-fold increase after 2 Gy irradiation compared to control cells on day 7. There was a significant increase in Tie-2 mRNA expression. In addition, the expression of other mRNAs, such as PECAM1, SELP and CD44, was also significantly increased in cells irradiated with heavy-ion beams. However, the adherent function of platelets derived from the irradiated cells showed no difference from that in the controls. These results clarify that the functions of megakaryocytopoiesis and thrombopoiesis derived from hematopoietic stem/progenitor cells irradiated with heavy-ion beams are similar to those in the unirradiated cells, although heavy-ion beams affect the expression of genes associated with cellular adhesion.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.     

Apoptosis in HeLa Hep2 cells is induced by low-dose,low-dose-rate radiation

Radioimmunotherapy with radiolabeled antibodies may cause inhibition of the growth of epithelial tumors, despite low total radiation doses and comparatively low radiosensitivity of epithelial tumor cells. The induction of apoptosis by low-dose radiation, such as delivered in radioimmunotherapy, was investigated in vitro in human HeLa Hep2 carcinoma cells. The cultured cells were exposed to defined radiation doses from a (60)Co radiation therapy source. The radiation source delivered 0.80 +/- 0.032 (mean +/- SD) Gy/min and the cells were given total doses of 1, 2, 5, 10 and 15 Gy. Using fluorescein-labeled Annexin V, followed by flow cytometry and DNA ladder analysis, apoptotic cells were detected and quantified. Radiation doses below 2 Gy did not cause any significant increase in apoptosis. Compared to control cells, apoptosis was pronounced after 5-10 Gy irradiation and was correlated to the radiation dose, with up to 42 +/- 3.5% of the cells examined displaying apoptosis. Clonogenic assays confirmed significantly decreased viability of the cells in the interval 2 to 10 Gy with low-dose-rate radiation, 60 +/- 2% compared to 2 +/- 2%. Lethal effects on the tumor cells were also evaluated by an assay of the cytotoxic effects of the release of (51)Cr. Significant cytotoxicity, with up to 64 +/- 6% dead cells, was observed at 5 Gy. Similar results were obtained when the dose rate was reduced to 0.072 +/- 0.003 Gy/min (mean +/- SD). In the case of the (137)Cs source, the dose rate could be reduced to 0.045 Gy/h, a level comparable to radioimmunotherapy, which induced significant apoptosis, and was most pronounced at 72-168 h postirradiation. It can be concluded that in vitro low-dose and low-dose-rate radiation induces apoptosis in epithelial HeLa Hep2 cells and thus may explain a mechanism by which pronounced inhibition of growth of HeLa Hep2 tumors at doses used in radioimmunotherapy has been obtained previously.     

Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ～ 151 keV/μm), 600 MeV/u silicon ions (LET ～ 51 keV/μm), or 1 GeV protons (LET ～ 0.2 keV/μm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-μm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.     

Variability of the adaptive response to ionizing radiations in humans

Human lymphocytes exposed to low doses of ionizing radiations from incorporated tritiated thymidine ([3H]dThd) or from X-rays become less susceptible to the induction of chromatid aberrations by high doses of X-rays. This indicates that low doses of ionizing radiation can produce an effect similar to the adaptive response observed with alkylating agents in prokaryotes, animal and plant cells. To determine whether there is individual variability in the adaptive response to ionizing radiations we exposed human lymphocytes from 18 different healthy donors to 'adapting' doses of [3H]dThd (0.01 microCi/ml) or X-rays (0.01 Gy) and subsequently to a 'challenge' treatment of 0.75 Gy of X-rays delivered 2 h before fixation. Four of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found. Two of these 4 donors showed no adaptive response in 3 subsequent experiments separated by 4-month intervals. This suggests that the human population exhibits a heterogeneity in the adaptive response to ionizing radiations which might be, at least in part, genetically determined.     

Assessment of genome damage in occupational exposure to ionising radiation and ultrasound

The mutagenic effects of low doses of radiation on occupationally exposed subjects were studied on lymphocyte culture using two methods: analysis of structural chromosome aberrations and micronucleus assay. The results obtained in subjects exposed to ionising radiation alone were compared to those exposed to both ionising radiation and ultrasound. A correlation between the total number of chromosome aberrations and distribution of micronuclei in the genome of somatic cells show higher deviation in the group exposed to X-ray and ultrasound than in the group exposed to X-rays alone. The degree of genome damage in occupational exposure to X-rays and ultrasound were discussed.     

Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/μm), and were then stimulated to obtain dividing cells. PBLs were treated with 100 nM calyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30 Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy⁻¹. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.     

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G₁-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G₁/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.     

Inactivation of hypoxic cells by cisplatin and radiation at clinically relevant doses

We have examined the effects of exposure to cisplatin (cis-diamminedichloroplatinum(II] on the response of exponentially growing V79 cells to low (0-4 Gy) and high (up to 30 Gy) doses of X rays under hypoxic and aerobic conditions. Survival in both dose regions was assessed by clonogenic assays; the low-dose studies were facilitated by a Cell Analyser (B. Palcic and B. Jaggi, Int. J. Radiat. Biol. 50, 345-352 (1986]. The results show that cisplatin, like its isomer trans-DDP, exhibits greater interaction with low than with high radiation doses in hypoxic cells. This increased interaction could be seen even with subtoxic exposures to cisplatin as low as 1 mumol dm-3. In contrast, with cells irradiated in air in the presence of either complex, the interaction seen with high doses of radiation is completely lost or greatly diminished in the low radiation dose region. Further experiments showed that enhanced interaction of hypoxic cells with low doses of radiation could be equally effective with cisplatin pretreatments in air or in hypoxia, even if the cells are exposed to cisplatin only after irradiation. In experiments with nonproliferating plateau-phase cultures, the same enhanced interaction was observed in the low-dose region. These results, for example enhancement ratios of 2.3 and 1.2 at low- and high-dose regions, respectively, for 5 mumol dm-3 cisplatin, are contrasted with those for nitroimidazoles which are better sensitizers in the high-dose region.     

Exposure to low-dose X-rays promotes peculiar autophagic cell death in Drosophila melanogaster, an effect that can be regulated by the inducible expression of Hml dsRNA

We previously reported that to induce an early emergence effect with low-dose X-irradiation in Drosophila, exposure during the prepupae stage is necessary. The present study examined the mechanism by which low-dose radiation rapidly eliminates larval cells and activates the formation of the imaginal discs during metamorphosis. Upon exposure to 0.5 Gy X-rays at 2 h after puparium formation (APF), the larval salivary glands swelled and were surrounded by remarkably thick structures containing an acid phosphatase (Acph) enzyme, implicating a peculiar autophagic cell death. TUNEL staining revealed the presence of DNA fragmentations compared with cells from sham controls which remained unchanged until 12 h APF. Additionally, the salivary glands of exposed flies were completely destroyed by 10 h APF. Furthermore, exposure to 0.5 Gy X-rays also facilitated the activity of the engulfment function of dendritic cells (DCs); they were generated in the larval salivary glands, engulfed the cell corpses and finally moved to the fat body. Data from an experiment demonstrating the inducible expression of Hml double-stranded RNA (dsRNA) indicate that a slow rate of engulfment of larval cells results in a longer time to emergence. Thus, the animals subjected to low-dose X-rays activated autophagic processes, resulting in significantly faster adult eclosion.     

Alpha-particle-induced changes in the stability and size of DNA

The effect of alpha-particle radiation on the thermal stability and size of calf thymus DNA molecules in deoxygenated aqueous solutions was investigated by thermal transition spectrophotometry, pulsed-field gel electrophoresis, and standard agarose gel electrophoresis. The thermal transition of DNA from helix to coil was studied through analysis of the UV A(260) absorbance. The results obtained for alpha particles of mean LET of 128 keV microm(-1) reveal a dual dose response: a tendency for thermal stability of the DNA helix at "low" doses, followed by an increasing instability at higher doses. The same phenomenon was observed for the mean molecular weight of DNA molecules exposed to alpha particles. The results reported here for alpha particles in the low-dose region of 0-16 Gy are consistent with our previous hypothesis of inter- and intramolecular interactions of a covalent character in gamma-irradiated DNA molecules in the dose region of 0-4 Gy.     

Effects of acute low doses of Gamma-radiation on erythrocytes membrane

It is believed that any dose of ionizing radiation may damage cells and that the mutated cells could develop into cancer cells. Additionally, results of research performed over the past century on the effects of low doses of ionizing radiation on biological organisms show beneficial health effects, called hormesis. Much less is known about the cellular response to low doses of ionizing radiation, such as those typical for medical diagnostic procedures, normal occupational exposures or cosmic-ray exposures at flight altitudes. Extrapolating from the effects observed at higher doses to predict changes in cells after low-dose exposure is problematic. We examined the biological effects of low doses (0.01–0.3 Gy) of γ-radiation on the membrane characteristics of erythrocytes of albino rats and carried out osmotic fragility tests and Fourier transform infrared spectroscopy (FTIR). Our results indicate that the lowest three doses in the investigated radiation range, i.e., 0.01, 0.025 and 0.05 Gy, resulted in positive effects on the erythrocyte membranes, while a dose of 0.1 Gy appeared to represent the limiting threshold dose of those positive effects. Doses higher than 0.1 Gy were associated with the denaturation of erythrocyte proteins.     

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantitatively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP-53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implications for estimating the risk associated with low-dose radiation exposure on human health.     

Combined action of X-rays and nonylphenol on mouse sperm

The aim of this study was to assess the effects of 2-weeks’ X-ray and/or nonylphenol (NP) exposure on male mice’s sperm count and quality. Pzh:SFIS mice were exposed to X-rays (0.05 Gy, 0.10 Gy, 0.20 Gy) or to nonylphenol (25 mg/kg bw, 50 mg/kg bw, 100 mg/kg bw) or to both agents (0.05 Gy + 25 mg/kg bw NP, 0.10 Gy + 50 mg/kg bw NP). At 24 h and 5 weeks after the end of exposure the sperm count, morphology and frequency of DNA damage in the male germ cells were estimated. Each agent alone diminished sperm count and morphology. The dose of 0.05 Gy of X-rays decreased the frequency of DNA damage. Combined exposure to lower doses of both agents significantly improved sperm morphology and decreased the level of DNA damage compared to one agent alone. Combined exposure to higher doses reduced the frequency of DNA damage compared to the effect of the appropriate dose of NP. Results of combined exposure to low doses of both agents suggest that 0.05 Gy of X-rays stimulate the DNA damagecontrol system and in consequence repair of DNA caused by X-rays and NP. It may be correlated with increased antioxidant capacity.     

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.