Chizuru Tsuruoka

LET and Ion Species Dependence for Cell Killing in Normal Human Skin Fibroblasts

Abstract Tsuruoka, C., Suzuki, M., Kanai, T. and Fujitaka, K. LET and Ion Species Dependence for Cell Killing in Normal Human Skin Fibroblasts. Radiat. Res. 163, 494–500 (2005). We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/μm. The RBE of carbon ions at 98 keV/μm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/μm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/β2 (where Z* is the effective charge and β is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/β2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/β2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.

The difference in LET and ion species dependence for induction of initially measured and non-rejoined chromatin breaks in normal human fibroblasts.

Abstract Tsuruoka, C., Suzuki, M., Hande, M. P., Furusawa, Y., Anzai, K. and Okayasu, R. The Difference in LET and Ion Species Dependence for Induction of Initially Measured and Non-rejoined Chromatin Breaks in Normal Human Fibroblasts. Radiat. Res. 170, 163–171 (2008). We studied the LET and ion species dependence of the induction of chromatin breaks measured immediately after irradiation as initially measured breaks and after 24 h postirradiation incubation (37°C) as non-rejoined breaks in normal human fibroblasts with different heavy ions, such as carbon, neon, silicon and iron, generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS). Chromatin breaks were measured as an excess number of fragments of prematurely condensed chromosomes using premature chromosome condensation (PCC). The results showed that the number of excess fragments per cell per Gy for initially measured chromatin breaks was dependent on LET in the range from 13.3 to 113.1 keV/μm but was not dependent on ion species. On the other hand, the number of non-rejoined chromatin breaks detected after 24 h postirradiation incubation was clearly dependent on both LET and ion species. No significant difference was observed in the cross section for initially measured breaks, but a statistically significant difference was observed in the cross section for non-rejoined breaks among carbon, neon, silicon and iron ions. This suggests that the LET-dependent structure in the biological effects is reflected in biological consequences of repair processes.

Yield of Single- and Double-Strand Breaks and Nucleobase Lesions in Fully Hydrated Plasmid DNA Films Irradiated with High-LET Charged Particles

We measured the yield and spectrum of strand breaks and nucleobase lesions produced in fully hydrated plasmid DNA films to determine the linear energy transfer (LET) dependence of DNA damage induced by ion-beam irradiation in relation to the change in the atomic number of ions. The yield of isolated damage was revealed as a decrease in prompt SSBs with increasing LET of He2+, C5+,6+ and Ne8+,10+ ions. On the other hand, the yields of prompt DSBs increased with increasing ion LET. SSBs were additionally induced in ion-irradiated DNA film by treatment with two kinds of base excision repair proteins (glycosylases), Nth and Fpg, indicating that base lesions are produced in the hydrated DNA film. This result shows that nucleobase lesions are produced via both chemical reactions with diffusible water radicals, such as OH radicals, and direct energy deposition onto DNA and the hydrated water layer. Nth-sensitive sites deduced to be pyrimidine lesions, such as 5,6-dihydrothymine (DHT), showed a relatively larger yield than Fpg-sensitive sites deduced to be purine lesions, such as 7,8-dihydro-8-oxo-2′deoxyguanine (8-oxoGua), for all ion exposures tested. The yield of SSBs or DSBs observed by enzyme treatment decreased noticeably with increasing LET for all tested ions. These results indicated that higher-LET ions preferentially produce a complex type of damage that might compromise the activities of the glycosylases used in this study. These findings are biologically important since, under cell mimicking conditions, persistent DNA damage occurs in part due to direct energy deposition on the DNA or hydrated water shell that is specifically induced by dense ionization in the track.

Genetic Analysis of T Cell Lymphomas in Carbon Ion-Irradiated Mice Reveals Frequent Interstitial Chromosome Deletions: Implications for Second Cancer Induction in Normal Tissues during Carbon Ion Radiotherapy

Monitoring mice exposed to carbon ion radiotherapy provides an indirect method to evaluate the potential for second cancer induction in normal tissues outside the radiotherapy target volume, since such estimates are not yet possible from historical patient data. Here, male and female B6C3F1 mice were given single or fractionated whole-body exposure(s) to a monoenergetic carbon ion radiotherapy beam at the Heavy Ion Medical Accelerator in Chiba, Japan, matching the radiation quality delivered to the normal tissue ahead of the tumour volume (average linear energy transfer = 13 keV.μm-1) during patient radiotherapy protocols. The mice were monitored for the remainder of their lifespan, and a large number of T cell lymphomas that arose in these mice were analysed alongside those arising following an equivalent dose of 137Cs gamma ray-irradiation. Using genome-wide DNA copy number analysis to identify genomic loci involved in radiation-induced lymphomagenesis and subsequent detailed analysis of Notch1, Ikzf1, Pten, Trp53 and Bcl11b genes, we compared the genetic profile of the carbon ion- and gamma ray-induced tumours. The canonical set of genes previously associated with radiation-induced T cell lymphoma was identified in both radiation groups. While the pattern of disruption of the various pathways was somewhat different between the radiation types, most notably Pten mutation frequency and loss of heterozygosity flanking Bcl11b, the most striking finding was the observation of large interstitial deletions at various sites across the genome in carbon ion-induced tumours, which were only seen infrequently in the gamma ray-induced tumours analysed. If such large interstitial chromosomal deletions are a characteristic lesion of carbon ion irradiation, even when using the low linear energy transfer radiation to which normal tissues are exposed in radiotherapy patients, understanding the dose-response and tissue specificity of such DNA damage could prove key to assessing second cancer risk in carbon ion radiotherapy patients.

Rejoining kinetics of G1-PCC breaks induced by different heavy-ion beams with a similar LET value

In previous studies we have shown that the linear energy transfer (LET)-relative biological effectiveness (RBE) curves were affected by LET and ion species [1,2]. In this paper we have examined the difference in the repair kinetics of G1-prematurely condensed chromosome breaks in normal human fibroblasts following irradiation with different heavy-ion beams of similar LET values. Normal human fibroblasts were irradiated with about 110 keV/microm of carbon (135 MeV/n), neon (400 MeV/n) and silicon ions (490 MeV/n), and the doses of carbon (3.25 Gy), neon (2.94+/-0.01 Gy) and silicon (2.31 Gy) were chosen to produce approximately the same number of initially measured G1-premature chromosome condensation (PCC) breaks (about 37 excess fragments per cell). The number of G1-PCC breaks was counted as excess fragments of prematurely condensed chromosomes using the PCC technique in the G1/G0 phase. The fractions of residual G1-PCC breaks after 24 h post-irradiation and half time, which is the time point where 50% of initially measured G1-PCC breaks are rejoined (t1/2), of the slow components of rejoining in carbon- and neon-ion irradiated cells were different from those of silicon-ion irradiated cells. However, no difference was observed in the half time of the fast components of rejoining in each ion beam. The results suggest that the difference in the fractions of residual G1-PCC breaks after 24 h post-irradiation reflect the result of the slow repair process for induced G1-PCC breaks, and that the repair process is dependent on the ion species, not the LET values.

Reduction in Life Span of Normal Human Fibroblasts Exposed to Very Low-Dose-Rate Charged Particles

Abstract Suzuki, M., Tsuruoka, C., Uchihori, Y., Ebisawa, S., Yasuda, H. and Fujitaka, K. Reduction in Life Span of Normal Human Fibroblasts Exposed to Very Low-Dose-Rate Charged Particles. Radiat. Res. 164, 505–508 (2005). We studied the effect of chronic low-dose irradiation with heavy ions on the life span of normal human fibroblasts in vitro. Cells were cultured in a CO2 incubator that was placed in the irradiation room for biological studies of heavy ions in the Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS) and were exposed to scattered radiations produced by heavy-ion beams for the life span of the cell population. The absorbed dose, which was measured using a thermoluminescence dosimeter (TLD) and a silicon semiconductor detector, was 1.4 mGy per day when the HIMAC was operated for biological experiments. The total number of population doublings of the exposed cells as reduced to 79–93% of that of nonexposed control cells. However, the life span of cells exposed to low-dose 137Cs γ rays (∼1 mGy/day) in the CO2 incubator in the γ-irradiation room in NIRS was prolonged to 104–106% of that of nonexposed control cells. Thus there is evidence that exposure to chronic low-dose heavy-ion radiation reduces the life span of cells.

Sensitive Detection of Radiation-Induced Medulloblastomas after Acute or Protracted Gamma-Ray Exposures in Ptch1 Heterozygous Mice Using a Radiation-Specific Molecular Signature

Recently reported studies have led to a heightened awareness of the risks of cancer induced by diagnostic radiological imaging, and in particular, the risk of brain cancer after childhood CT scans. One feature of Ptch1+/– mice is their sensitivity to radiation-induced medulloblastomas (an embryonic cerebellar tumor) during a narrow window of time centered on the days around birth. Little is known about the dynamics of how dose protraction interacts with such narrow windows of sensitivity in individual tissues. Using medulloblastomas from irradiated Ptch1+/– mice with a hybrid C3H × C57BL/6 F1 genetic background, we previously showed that the alleles retained on chromosome 13 (which harbors the Ptch1 gene) reveal two major mechanisms of loss of the wild-type allele. The loss of parental alleles from the telomere extending up to or past the Ptch1 locus by recombination (spontaneous type) accounts for almost all medulloblastomas in nonirradiated mice, while tumors in irradiated mice often exhibited interstitial deletions, which start downstream of the wild-type Ptch1 and extend up varying lengths towards the centromere (radiation type). In this study, Ptch1+/– mice were exposed to an acute dose of either 100 or 500 mGy gamma rays in utero or postnatally, or the same radiation doses protracted over a four-day period, and were monitored for medulloblastoma development. The results showed dose- and age-dependent radiation-induced type tumors. Furthermore, the size of the radiation-induced deletion differed with the dose rate. The results of this work suggest that tumor latency may be related to the size of the deletion. In this study, 500 mGy exposure produced radiation-induced type tumors at all ages and dose rates, while 100 mGy exposure did not significantly produce radiation-induced type tumors. The radiation signature allows for unique mechanistic insight into the action of radiation to induce DNA lesions with known causal relationship to a specific tumor type, particularly for doses and dose rates that are relevant to both diagnostic and accidental radiological exposures.

Relative biological effectiveness of therapeutic proton beams for HSG cells at Japanese proton therapy facilities

We investigated the relative biological effectiveness (RBE) of therapeutic proton beams at six proton facilities in Japan with respect to cell lethality of HSG cells. The RBE of treatments could be determined from experimental data. For this purpose, we used a cell survival assay to compare the cell-killing efficiency of proton beams. Among the five linear accelerator (LINAC) X-ray machines at 4 or 6 MeV that were used as reference beams, there was only a small variation (coefficient of variation CV = 3.1% at D10) in biological effectiveness. The averaged value of D10 for the proton beams at the middle position of the spread-out Bragg peak (SOBP) was 4.98. These values showed good agreement, with a CV of 4.3% among the facilities. Thus, the average RBE10 (RBE at the D10 level) at the middle position of the SOBP beam for six facilities in Japan was 1.05 with a CV of 2.8%.

PO-117 Increased risk of in utero x-ray exposure to mice treated with n-ethyl-n-nitrosourea postnatally

Introduction A-bomb survivor study reports that in utero exposure to radiation increases risks of not only childhood cancers but also adult-onset cancers. However, little is known about whether the risk of in utero exposure is influenced by postnatal exposure to other carcinogens. In this study, we examined the lifespan shortening and cancer risk of mice after irradiation in utero and treatment with N-ethyl-N-nitrosourea postnatally. Material and methods Female B6C3F1 mice were either irradiated with 2 Gy X-rays at embryonic day 17 (X-ray alone) or administrated with 125 ppm N-ethyl-N-nitrosourea (ENU) for 4 weeks from 5, 9, or 13 weeks old (ENU alone). Another groups of mice were both exposed to X-rays in utero and administrated with ENU postnatally, i.e., 5, 9, or 13 weeks old (X-rays+ENU). Control group were treated with sham-irradiation and vehicle-only. All mice were analysed for the life-span shortening and tumour spectrum histopathologically at moribund or just after death. Results and discussions The mean lifespan of control mice was 797+/-143 days. In utero X-ray exposure shortened lifespan by 8.5%. The mean lifespan of mice treated with ENU alone at 5, 9 and 13 weeks of age were 366+/-117, 461+/-104, and 475+/-123 days, respectively. In utero exposure shortened lifespan of mice postnatally treated with ENU at 5, 9 and 13 weeks of age by 16.8, 9.0 and 7.5%, respectively, indicating that estimated risk of in utero exposure was enhanced by twofold in mice treated with ENU at juvenile (5 weeks). This enhancement is in part ascribed to acceleration of tumour development such as thymic lymphoma. In contrast, the risk of lifespan shortening after in utero exposure was not influenced by ENU treatment when ENU was treated after adults (9 and 13 weeks). Histopathological examination is now undertaken in order to clarify the tumours whose risk is increased by in utero exposure in control and ENU treated mice. Conclusion The risk of in utreo exposure to X-rays was influenced by postnatal treatment with ENU, which depends on the age of ENU treatment. Increase in risk of in utero exposure by juvenile ENU treatment was ascribed to acceleration of tumours such as thymic lymphoma.

Radiation Exposure Enhances Hepatocyte Proliferation in Neonatal Mice but not in Adult Mice

There is a natural tendency to expect that irradiation of an infant organ prior to development-related expansion will result in a higher risk of developing cancer than that of fully-developed adult tissue, and this has generally been observed. However, if tissues also vary in their initial responses to radiation depending on age, the interplay between tissue- and age-dependent risk would potentially be quite complex. We have previously shown opposing age-dependent induction of apoptosis for the intestinal epithelium and hematopoietic cells in mice, but such data are not yet available for the liver. Here, we have examined markers of DNA damage, initiation of DNA damage responses, cell cycle arrest, apoptosis and proliferation, as well as gene expression, in the B6C3F1 mouse liver over the hours and days after irradiation of mice at 1 or 7 weeks of age. We found that induction and resolution of radiation-induced DNA damage is not accompanied by significant changes in these cellular end points in the adult liver, while in infant hepatocytes modest induction of p53 accumulation and p21-mediated cell cycle arrest in a small fraction of damaged cells was overshadowed by a further stimulation of proliferation over the relatively high levels already found in the neonatal liver. We observed distinct expression of genes that regulate cell division between the ages, which may contribute to the differential responses. These data suggest that the growth factor signaling environment of the infant liver may mediate radiation-induced proliferation and increased liver cancer risk after irradiation during early life.