System of cell irradiation with a defined number of heavy ions (III).

A single cell irradiation system has been developed at JAERI-Takasaki to study radiobiological processes in single-ion-hit mammalian cells and bystander cells, in ways that cannot be achieved using conventional broad field exposures. Individual mammalian cultured cells are irradiated in the atmosphere on the cell dish, the bottom of which is made of ion-track-detector CR-39, with a single or defined numbers of 13.0 MeV/amu 20Ne and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells are performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. Using this system, Chinese hamster ovary (CHO-K1) cells were irradiated with counted number of 20Ne and 40Ar ions. Thereafter, the growth of the cells was observed individually and repeatedly during post-irradiation incubation. The cells hit by a single 40Ar ion on their nucleus showed strong growth inhibition. Meanwhile, the cells in the irradiated dish but not hit by the ion (bystander cells) showed limited cell growth. This might be a bystander effect caused by heavy ion hit cell co-existing in the same dish.     

重离子射线照射对家蚕的生物影响

为解明重离子射线的生物影响,调查了氖、碳及氦(20Ne8+,LET=300keV/μm;12C5+,LET=116keV/μm和4He2+,LET=16.2keV/μm)等重离子射线照射家蚕(Bombyxmori)后的存活率及形态变化。重离子射线照射不同发育时期的幼虫后所引起的生物影响不同,幼虫的发育时期越早,照射后引起的生物影响越大;对同一时期的幼虫,随着剂量的增加,照射的生物影响加大;以化蛹率和羽化率为指标的放射线感受性在供试的3种射线间具有相似的变化倾向,只是射线的射程越长,照射的生物影响越大;对熟蚕卵巢存在部位的局部照射也显示相似的结果。同一射线的不同LET轨迹位置对家蚕的卵巢及真皮细胞的生物影响不同,用Mylar薄膜覆盖调节碳离子射线的射程,卵巢及真皮细胞越是接近射线高LET的Bragg峰,照射个体的鳞毛及卵的形成被强烈抑制。因此,重离子射线对家蚕的生物影响与细胞及植物种子等小个体不同,对于全体照射,重离子射线的射程长短所造成的生物影响比射线的LET大小所引起的生物影响要大;而对于局部照射,目的器官越是接近射线的高LET轨迹,照射的生物影响越大。     

Lethal action of accelerated heavy ions on mammalian cells as affected by inhibitors of DNA synthesis. Results of experimental research

Radiosensitivity of Chinese hamster cells exposed to 137Cs-gamma-radiation and accelerated heavy ions of 4He (L = 22 and 60 keV/micron), 12C (L = 231 keV/micron), and 20Ne (L = 690 keV/micron) was studied in standard conditions and in the presence of arabinosylcytosine and hydroxyurea. These agents were shown to exert a radiosensitizing effect in the case of gamma-radiation. The effect was less pronounced with 4He ion-radiation and was absent upon irradiation with 12C and 20Ne ions. The radiosensitivity was maximum upon irradiation with 4He ions at L = 60 keV/micron. The RBE coefficients of heavy ions under study decreased in the presence of DNA synthesis inhibitors.     

Damage by gamma rays and heavy ions to superhelical structures of nuclear DNA

Chinese hamster cells (V79-4), human lymphocytes and mouse ascites cell were exposed to gamma-rays and heavy ions (4He and 12C). Sedimentation of complexes containing DNA was studied after cell lysis by centrifugation in a neutral sucrose gradient. The distinctions noted after irradiation with gamma-rays and heavy ions are consistent with the idea of the superhelical organization of DNA into discrete and membrane-bound compact units. According to the estimates made the diameter of these complexes was approximately 0.2 micron and DNA content, about 2 X 10(9) dalton.     

Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam

As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy-ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on a thin film of an ion track detector, CR-39, were automatically detected under a fluorescence microscope and irradiated individually with an 40Ar13+ ion (11.5 MeV/nucleon, LET 1260 keV/microm) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch pits by treatment of the CR-39 with an alkaline-ethanol solution at 37 degrees C. The exact positions of ion hits were determined by overlaying images of both cells and etch pits. The cells that were irradiated with argon ions showed a reduced growth in postirradiation observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our verified irradiation method enables us to start a precise study of the effects of high-LET radiation on cells.     

Microbeams of heavy charged particles.

We have established a single cell irradiation system, which allows selected cells to be individually hit with defined number of heavy charged particles, using a collimated heavy-ion microbeam apparatus at JAERI-Takasaki. This system has been developed to study radiobiological processes in hit cells and bystander cells exposed to low dose and low dose-rate high-LET radiations, in ways that cannot be achieved using conventional broad-field exposures. Individual cultured cells grown in special dishes were irradiated in the atmosphere with a single or defined numbers of 18.3 MeV/amu 12C, 13.0 MeV/amu 20Ne, and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells were performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. The actual number of particle tracks that pass through cell nuclei was detected with prompt etching of the bottom of the cell dish made of ion track detector TNF-1 (modified CR-39), with alkaline-ethanol solution at 37 degrees C for 15-30 minutes. Using this system, separately inoculated Chinese hamster ovary cells, confluent normal human fibroblasts, and single plant cells (tobacco protoplasts) have been irradiated. These are the first studies in which single-ion direct hit effect and the bystander effect have been investigated using a high-LET heavy particle microbeam.     

Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions

Ionizing radiation-induced bystander effects have been documented for a multitude of endpoints such as mutations, chromosome aberrations and cell death, which arise in nonirradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. To address this, we employed precise microbeams of carbon and neon ions for targeting only a very small fraction of cells in confluent fibroblast cultures. Conventional broadfield irradiation was conducted in parallel to see the effects in irradiated cells. Exposure of 0.00026% of cells led to nearly 10% reductions in the clonogenic survival and twofold rises in the apoptotic incidence regardless of ion species. Whilst apoptotic frequency increased with time up to 72 h postirradiation in irradiated cells, its frequency escalated up to 24h postirradiation but declined at 48 h postirradiation in bystander cells, indicating that bystander cells exhibit transient commitment to apoptosis. Carbon- and neon-ion microbeam irradiation similarly caused almost twofold increments in the levels of serine 15-phosphorylated p53 proteins, irrespective of whether 0.00026, 0.0013 or 0.0066% of cells were targeted. Whereas the levels of phosphorylated p53 were elevated and remained unchanged at 2h and 6h postirradiation in irradiated cells, its levels rose at 6h postirradiation but not at 2h postirradiation in bystander cells, suggesting that bystander cells manifest delayed p53 phosphorylation. Collectively, our results indicate that heavy ions inactivate clonogenic potential of bystander cells, and that the time course of the response to heavy ions differs between irradiated and bystander cells. These induced bystander responses could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Redox changes induced in hippocampal precursor cells by heavy ion irradiation

Hippocampal precursors retain the capacity to proliferate and differentiate throughout life, and their progeny, immature neurons, can undergo neurogenesis, a process believed to be important in maintaining the cognitive health of an organism. A variety of stresses including irradiation have been shown to deplete neural precursor cells, an effect that inhibits neurogenesis and is associated with the onset of cognitive impairments. Our past work has shown that neural precursor cells exposed to X-rays or protons exhibit a prolonged increase in oxidative stress, a factor we hypothesize to be critical in regulating the function of these cells after irradiation and other stresses. Here we report that irradiation of hippocampal precursor cells with high-linear energy transfer (LET) 1 GeV/nucleon ⁵⁶Fe ions leads to significantly higher levels of oxidative stress when compared to lower LET radiations (X-rays, protons). Irradiation with 1 Gy of ⁵⁶Fe ions elicits twofold to fivefold higher levels of reactive oxygen species (ROS) compared to unirradiated controls, and at lower doses (≤1 Gy) neural precursors exhibit a linear dose response 6 h after heavy ion exposure. The use of the antioxidant lipoic acid (LA) was able to reduce ROS levels below background levels when added before or after ⁵⁶Fe ion irradiation. These results conclusively show that low doses of ⁵⁶Fe ions can elicit significant levels of oxidative stress in neural precursor cells. Given the prevalence of heavy ions in space and the duration of interplanetary travel, these data suggest that astronauts are at risk for developing cognitive decrements. However, our results also indicate that antioxidants delivered before as radioprotective agents or after as mitigating agents hold promise as effective countermeasures for ameliorating certain adverse effects of heavy ion exposure to the CNS.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Tumor cell migration is not influenced by p21 in colon carcinoma cell lines after irradiation with X-ray or 12C heavy ions

Metastasis and recurrences are major problems regarding an effective treatment of solid malignant tumors in clinical oncology. Since the impact of radiation on cell motility is not yet well understood, intrinsic and radiation-induced changes in cell migration have been discussed as possible mechanisms involved in the limitations of radiotherapy. This holds true for conventional radiation treatment and even more for the cellular and molecular effects of therapeutically relevant ¹²C heavy ions. The present study is therefore focused on the investigation of tumor cell migration in vitro after irradiation with X-rays and ¹²C heavy ions and on radiation-induced changes in the expression of proteins that are potentially relevant for motility. Two colon carcinoma cell lines, HCT116 and HCT116 p21−/−, were chosen for this study, which should be isogenic except for their p21-status. We can show here that cells lacking p21 react almost alike to radiation as wild type cells regarding survival and tumor cell migration 24 h after irradiation. Interestingly, differences in protein expression 24 h after irradiation of β₁ integrin and protein kinase B isoforms Akt1 and Akt2 seem to exist. We conclude that tumor cell migration is unaffected by the p21-status in colorectal carcinoma cells and that the expression of the aforementioned proteins alone is not accountable for the differences observed.     

Investigation of lipid peroxidation in liposomes induced by heavy ion irradiation

Lipid peroxidation induced by heavy ion irradiation was investigated in 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) liposomes. Lipid peroxidation was induced using accelerated heavy ions that exhibit linear energy transfer (LET) values between 30 and 15 000 keV/μm and doses up to 100 kGy. With increasing LET, the formation of lipid peroxidation products such as conjugated dienes, lipid hydroperoxides, and thiobarbituric acid-reactive substances decreased. When comparing differential absorption spectra and membrane fluidity following irradiation with heavy ions and x-rays (3 Gy/min), respectively, it is obvious that there are significant differences between the influences of densely and sparsely ionizing radiation on liposomal membranes. Indications for lipid fragmentation could be detected after heavy ion irradiation. Received: 6 March 1997 / Accepted in revised form: 31 March 1998     

Investigation of the bystander effect in CHO-K1 cells

AimInvestigation of the bystander effect in Chinese Hamster Ovary cells (CHO-K1) co-cultured with cells irradiated in the dose range of 0.1–4 Gy of high LET ¹²C ions and X-rays.BackgroundThe radiobiological effects of charged heavy particles on a cellular or molecular level are of fundamental importance in the field of biomedical applications, especially in hadron therapy and space radiation biology.Materials and methodsA heavy ion ¹²C beam from the Heavy Ion Laboratory of the University of Warsaw (HIL) was used to irradiate CHO-K1 cells. Cells were seeded in Petri dishes specially designed for irradiation purposes. Immediately after irradiation, cells were transferred into transwell culture insert dishes to enable co-culture of irradiated and non-irradiated cells. Cells from the membrane and well shared the medium but could not touch each other. To study bystander effects, a clonogenic survival assay was performed.ResultsThe survival fraction of cells co-cultured with cells irradiated with ¹²C ions and X-rays was not reduced.ConclusionsThe bystander effect was not observed in these studies.     

12C6＋离子束辐照对大青叶生理生化特性的影响

利用辐射能量为100 MeV/u的12C6＋ 重离子束辐照大青叶种子,辐照剂量分别为20、40、50和80 Gy研究其对大青叶M1代的生物学效应.研究结果表明,随着辐照剂量的增大,大青叶的根系活力和叶绿素含量显著降低,发现辐照损伤主要抑制了根的生长及营养吸收;酚酸类化合物含量随着辐照剂量的增大而升高的趋势,表明辐照可提高大青叶的抗氧化和抗病虫害能力;可溶性糖、丙二醛、超氧化物歧化酶（SOD）和过氧化物酶（POD）含量变化的总体趋势为随着辐照剂量的增大先升后降.表明重离子辐照能改变大青叶的一些生理生化特征,其中40 Gy的12C6＋离子束辐照有利改善大青叶抗逆性和一些有效化学成分的积累.     

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose (12)C(6+) ions

To investigate the effects of pre-exposure of mouse testis to low-dose (12)C(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation, the testes of outbred Kun-Ming strain mice were irradiated with 0.05Gy of (12)C(6+) ions as the pre-exposure dose, and then irradiated with 2Gy as challenging dose at 4h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2Gy of (12)C(6+) ions. However, pre-exposure of mouse testes to a low dose of (12)C(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose (12)C(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations.     

Cellular and subcellular effects of very heavy ions

The biological effects of irradiation with ions of masses larger than 40 and energies up to 20 MeV per atomic mass unit are reviewed. The objects are viruses, bacterial spores, yeast and mammalian cells. Experimental parameters include loss of colony forming ability, induction of mutants, chromosomal aberrations, cell cycle progression, inhibition of biochemical activities and the formation of strand breaks. Some of the pertinent physical questions--e.g. track structure--are also discussed. It is shown that with very heavy ions the biological effectiveness is no longer unambiguously related to a single parameter like l.e.t. or Z*2/beta 2 but depends strongly on ion energy. This points to the importance of far-reaching delta-electrons. The analysis indicates also that even with very high l.e.t., cells are not killed by the passage of a single particle through their nucleus. Possible implications of the findings for fundamental radiation biology are outlined.     

Effects of heavy ions and energetic protons on normal human fibroblasts

At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirradiated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.     

The effect of accelerated argon ions on the retina

It has been postulated that high energy heavy ions cause a unique form of damage in living tissue, which results from the high linear energy transfer of accelerated single particles. We have searched for these single-particle effects, so-called "microlesions," in composite electron micrographs of retinas of rats which had been irradiated with a dose of 1 Gy of 570 MeV/amu argon ions. The calculated rate of energy deposition of the radiation in the retina was about 100 keV/micron and the influence was four particles per 100 micron 2. Different areas of the irradiated retinas which combined would have been expected to be traversed by approximately 2400 particles were examined. We were unable to detect ultrastructural changes in the irradiated retinas distinct from those of controls. The spatial cellular densities of pigment epithelial and photoreceptor cells remained within the normal range when examined at 24 h and at 6 months after irradiation. These findings suggest that the retina is relatively resistant to heavy-ion irradiation and that under the experimental conditions the passage of high energy argon ions does not cause retinal microlesions that can be detected by ultrastructural analysis.     

Accumulation and removal of heavy metal ions by insolubilized-DNA and its interaction.

DNA was immobilized onto a porous glass bead by a treatment with UV irradiation. The immobilized DNA was insoluble in water and used for accumulation of heavy metal ion. When DNA-immobilized glass bead was added into aqueous solution containing heavy metal ions, such as Hg2+, Cd2+, Pb2+, Zn2+, Cu2+ and Fe3+, the concentration of these metal ions in the solution was decreased. However, the concentration of Mg2+ in the solution was not affected by the addition of the DNA-immobilized glass bead. These results suggested that UV-irradiated DNA selectively accumulated heavy metal ions.     

Effects of heavy ion to the primary [correction of rimary] culture of mouse brain cells.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.