Caution required for handling genome editing technology.

Genome-editing technology, although a robust tool for genetic engineering, is creating indistinct regulatory boundaries between naturally occurring and modified organisms. However, researchers must act with caution in research and development to avoid misleading society. Furthermore, appropriate regulations should be proactively discussed and established for handling genome-editing technology.

A threshold dose of heavy ion radiation that decreases the oxidative enzyme activity of spinal motoneurons in rats.

The effect of heavy ion radiation exposure of the spinal cord on the properties of the motoneurons innervating the slow soleus and fast plantaris muscles was investigated. A 15-, 20-, 40-, 50-, or 70-Gy dose of carbon ions (5 Gy/min) was applied to the 2nd to the 6th lumbar segments of the spinal cord in rats. After a 1-month recovery period, the number and cell body size of the irradiated motoneurons innervating the soleus and plantaris muscles did not differ from that of the non-irradiated controls, irrespective of the dose received. However, the oxidative enzyme activity of these motoneurons was decreased by heavy ion radiation at doses of 40, 50, and 70 Gy compared to that of the non-irradiated controls. This decrease in oxidative enzyme activity levels in the motoneurons returned to that of the non-irradiated controls after a 6-month recovery period. We conclude that heavy ion radiation at doses of 40-70 Gy reversibly decreases the oxidative enzyme activity of motoneurons in the spinal cord of rats.

Genome-wide expression changes in Saccharomyces cerevisiae in response to high-LET ionizing radiation.

To understand the yeast response to high-linear energy transfer (LET) ionizing radiation (IR), we investigated global gene expression in yeast irradiated by three types of high-LET IR (fast neutrons, heavy ions, and thermal neutrons) and gamma rays using DNA microarray analysis. Stationary cells were irradiated by each IR and recultured in yeast-peptone-dextrose medium to allow repair for 40 min. RNA was then isolated from three independent samples of irradiated yeast. Genes involved in the Mec1p kinase pathway, which functions in DNA damage response, were induced by all forms of high-LET IR and by gamma rays. Some genes related to oxidative stress and the cell wall were induced by all forms of high-LET IRs. Gene expression patterns as a function of each type of high-LET IR were examined statistically by one-way analysis of variance. This analysis demonstrated the existence of irradiation-specific responses. For example, genes involved in ribosomal DNA synthesis were specifically induced by fast neutron irradiation, while the ubiquitin-proteasome system and heat shock response were specifically induced by thermal neutron irradiation. The study characterizes high-LET IR-induced gene expression and provides a molecular understanding of subsequent adaptation in yeast.

Induction of micronuclei in germinating onion seed root tip cells irradiated with high energy heavy ions.

Effects of high LET charged particles on a perfect in-vivo system are an essential theme for the study of the biological effects of radiation. Germinating onion seeds are independent complete organisms and the radiation induced micronuclei in the root chip cells can be examined quantitatively and theoretically. We irradiated with three types of high energy accelerated heavy ions germinating onion seeds using a synchrotron and observed micronuclei in the root tip cells. Micronuclei induction showed characteristic dose responses of an upward convex bell shape and a steep rise near zero doses for all types of the ions. The bell curve dose responses, however, could be explained by a simple mathematical model. A parameter in the model which indicates micronuclei induction frequency and another parameter which indicates induction frequency of lethal damages (or damages delaying cell divisions) per heavy ion track were both proportional to square of the LET. Because we suspected by-stander effect concerning the dose responses rising steeply near zero doses and tapering off for higher doses, we tested acute irradiation to remove time of information transmittance between cells using a single spill (about 0.3 s) of the synchrotron beam. No difference was detected between normal multiple spill irradiations and single spill.

Growth retardation and death of rice plants irradiated with carbon ion beams is preceded by very early dose- and time-dependent gene expression changes.

The carbon-ion beam (CIB) generated by the heavy-ion medical accelerator in Chiba (HIMAC) was targeted to 7-day-old rice. Physiological parameters such as growth, and gene expression profiles were examined immediately after CIB irradiation. Dose-dependent growth suppression was seen three days post-irradiation (PI), and all the irradiated plants died by 15 days PI. Microarray (Agilent rice 22K) analysis of the plants immediately after irradiation (iai) revealed effects on gene expression at 270 Gy; 353 genes were up-regulated and 87 down-regulated. Exactly the same set of genes was affected at 90 Gy. Among the highly induced genes were genes involved in information storage and processing, cellular processes and signaling, and metabolism. RT-PCR analysis confirmed the microarray data.

Extremely low dose ionizing radiation up-regulates CXC chemokines in normal human fibroblasts.

Although the public today could be exposed to X-rays as high as 1 cGy due to diagnostic procedures, the biological effects of this low-dose range have not been well established. We searched through >23,000 transcripts in normal human fibroblasts, HFLIII, using a novel comprehensive expression analysis method. More than 200 genes were up-regulated transiently by 1 cGy of X-rays during the 1-hour period after irradiation. We determined the nucleotide sequence of 10 up-regulated transcripts with the greatest rate of increase in the irradiated HFLIII cells. Three of the 10 transcripts encoded CXC chemokines (CXCL1, CXCL2, and CXCL6). The rest included the transcripts of other secretory products (secretogranin II, thrombospondin type I domain containing 2, amphiregulin, and interleukin-6) and unknown genes. To test the involvement of CXC chemokines in cells irradiated with low doses, we irradiated HFLIII cells with 1 to 20 cGy X-rays and transferred the media from HFLIII culture to two melanoma cell lines characteristic of excessive numbers of the CXC chemokine-specific receptors. The growth of these melanoma lines were significantly stimulated by the medium from HFLIII irradiated at 1 to 5 cGy. Our results indicate that human cells respond to doses of radiation as low as 1 cGy, and mechanisms alternative to those involved in moderate/high-dose studies have to be considered in understanding the biological effects of diagnostic level radiation. In addition, our comprehensive approach using a novel expression profiling method is a powerful strategy to explore biological functions associated with very low levels of toxic agents.

Effects of prenatal irradiation with an accelerated heavy-ion beam on postnatal development in rats: I. Neurophysiological alterations.

Effects on postnatal neurophysiological development in offspring were studied after exposure of pregnant Wistar rats to accelerated carbon-ion beams with an LET of about 13 keV/ mum at doses ranging from 0.1 Gy to 2.5 Gy on the 15th day of gestation. The age at which four physiological markers appeared and five reflexes were acquired was examined prior to weaning. Gain in body weight was monitored until the offspring were 3 months old. Male offspring were evaluated as young adults using two behavioral tests. The effects of X rays estimated for the same biological end points were studied for comparison. For most of the end points at early age, no significant alterations were observed in offspring that received prenatal irradiation with 0.1 Gy of either accelerated carbon ions or X rays compared to the offspring of sham-irradiated dams. However, all offspring whose dams received 2.5 Gy died prior to weaning. Offspring from dams irradiated with accelerated carbon ions generally showed higher incidences of prenatal death and preweaning mortality, markedly delayed accomplishment in their physiological markers and reflexes, and gain in body weight compared to those exposed to X rays at doses of 0.5 to 2 Gy. Significantly reduced ratios of main organ weight to body weight at the postnatal ages of 30, 60 and 90 days were also observed within this dose range. The results indicate that irradiation with 0.5 to 2 Gy on day 15 of gestation caused permanent alterations in offspring that were dependent on dose. The alterations include permanent growth retardation, morphological malformations in main organs, including microcephaly, diminished reflex attainment, delayed appearance of physiological markers, and changes in adult behavior. Exposure to 1 to 2 Gy of radiation resulted in growth retardation and behavioral alterations that persisted throughout life. Accelerated carbon ions generally induced more detrimental effects than X rays.

Germ cell mutagenesis in medaka fish after exposures to high-energy cosmic ray nuclei: A human model.

Astronauts beyond the Earth's orbit are exposed to high-energy cosmic-ray nuclei with high values of linear energy transfer (LET), resulting in much more biological damage than from x-rays or gamma-rays and may result in mutations and cancer induction. The relative biological effectiveness of these nuclei depends on the LET, rising to as high as approximately 50 at LET values of approximately 100-200 keV/microm. An endpoint of concern is germ cell mutations passed on to offspring, arising from exposure to these nuclei. A vertebrate model for germ cell mutation is Medaka fish (Oryzias latipes). We exposed wild type males to doses of 1 GeV per nucleon Fe nuclei or to 290 MeV per nucleon C nuclei. They were mated to females with recessive mutations at five-color loci. The transparent embryos from >100 days of mating (representing exposed sperm, spermatids, or spermatogonia) were observed so as to detect dominant lethal mutations and total color mutations, even though the embryos might not hatch. The relative number of mutant embryos as a function of dose were compared with those induced by gamma-rays. The relative biological effectiveness values for dominant lethal mutations and total color mutations for exposed sperm and spermatids were 1.3-2.1 for exposure to C nuclei and 1.5-3.0 for exposure to Fe nuclei. (The spermatogonial data were uncertain.) These low values, and the negligible number of viable mutations, compared with those for mutations in somatic cells and for neoplastic transformation, indicate that germ cell mutations arising from exposures to cosmic ray nuclei are not a significant hazard to astronauts.

Particle irradiation suppresses metastatic potential of cancer cells.

Particle radiotherapy such as proton and carbon ion has been producing promising clinical results worldwide. The purpose of this study was to compare metastatic capabilities of malignant tumor cells after irradiation with photon, proton, and carbon ion beams to clarify their ion beam-specific biological effects. We examined the biological properties of highly aggressive HT1080 human fibrosarcoma cells to assess their metastatic processes in terms of cell adhesion capability to extracellular matrix, expression of integrins, cell migration, cell invasive capability, and matrix metalloproteinase-2 activity in vitro. We then assessed the metastatic capabilities of LM8 mouse osteosarcoma irradiated with carbon ion or photon beam in the syngeneic mice. Both proton and carbon ion irradiation decreased cell migration and invasion in a dose-dependent manner and strongly inhibited matrix metalloproteinase-2 activity. On the other hand, lower X-ray irradiation promoted cell migration and invasion concomitant with up-regulation of alphaVbeta3 integrin. For cancer cells treated with carbon ion irradiation, the number of pulmonary metastasis was decreased significantly in vivo. These findings suggest that particle irradiation suppresses metastatic potential even at lower dose, whereas photon irradiation promotes cell migration and invasive capabilities at lower dose level, and provide preclinical evidence that ion beam radiotherapy may be superior to conventional photon beam therapy in possible preventive effects on metastases of irradiated malignant tumor cells.

Retrotransposition of limited deletion type of intracisternal A-particle elements in the myeloid leukemia Clls of C3H/He mice.

The murine genome has about 1,000 copies of DNA elements for the intracisternal A-particle (IAP) that resembles a retrovirus. We previously reported that the genomic DNA of the cells from radiation-induced acute myeloid leukemia (AML) lines derived from C3H/He inbred mice was frequently rearranged by the integration of the IAP element. In this study, 8 IAP elements from the characteristic integration sites in 6 cell lines of radiation-induced AML from different mice were characterized and compared in structure with 114 IAP elements isolated from the normal C3H/He genome. One of the 8 elements was a full-length type I IAP, and 7 were of type-I Delta 1 with a common deletion site. Although the type I Delta 1 form is a minor population accounting for about 6% of total genomic IAP elements, it is predominantly retrotransposed in the AML cells from different C3H/He mice. This indicates that limited populations of the IAP elements contribute to the unique retrotransposition in AML cells.

Experimental model for irradiating a restricted region of the rat brain using heavy-ion beams.

Heavy-ion beams have the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like X-rays or gamma-rays. In order to get clarify characteristic effects of heavy-ion beams on the brain, we have developed an experimental system for irradiating a restricted region of the rat brain using heavy-ion beams. The left cerebral hemispheres of the adult rat brain were irradiated at dose of 50 Gy charged carbon particles (290 MeV/nucleon; 5 mm spread-out Bragg peak). After irradiation, the characteristics of the heavy-ion beams and the animal model were studied. Histological examination and measurement showed that extensive necrosis was observed between 2.5 mm and 7.5 mm depth from the surface of the rat head, suggesting a relatively high dose and uniform dose was delivered among designed depths and the spread-out Bragg peak used here successfully and satisfactorily retained its high-dose localization in the defined region. We believe that our experimental model for irradiating a restricted region of the rat brain using heavy-ion beams is a good model for analyzing regional radiation susceptibility of the brain.

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.

HZE radiation effects for hereditary renal carcinomas.

Eker rat known as a model of hereditary renal carcinoma (RC) is an example of Mendelian dominantly inherited predisposition to a specific cancer in experimental animals. We investigate the effects of simulated space radiation on carcinogenesis using HIMAC. We estimated RBE from the Eker rats exposed to the heavy-ions, C (290 MeV/u) and Fe (500 MeV/u) ions, comparing to the effects of X-ray irradiation. Pregnant rats were exposed to C and Fe ions and X-rays with a single dose of 1 Gy, 2 Gy, 3 Gy on day 19 of gestation. The offspring were sacrificed at 8 weeks of age. We evaluated organ weights and tumor genesis. The weights of thymus, lung, liver, spleen were found to be no difference from the control at 1 Gy irradiation but 50% decrease at 3 Gy irradiation. We found in the irradiated animal that kidney, brain and testis were very sensitive organs of which the weight decreased to approximately 80% at 1 Gy and to 40% at 3 Gy irradiations. Based on the dose-response relationship of the radiation-induced carcinoma, averaged RBE ware calculated to be 1.1 for C-ion, 1.6 for Fe-ion.

Loss of heterozygosity in heavy-ion-induced murine T-cell lymphomas.

One of the important concerns for astronauts in space environment is cancer risk associated with cosmic radiation, including heavy particle carbon-ions. But little information on cancer risk is available. In the present study, we investigated the induction of and cellular and molecular characteristics of T-cell lymphomas of B6C3F1 mice induced by carbon-ions and X-rays. The incidence, the latent period and the surface expression of T-cell differentiation antigens were similar between carbon-ion- and X-ray-induced lymphomas. The size of T-cell lymphomas induced by carbon-ions was significantly smaller than that by X-rays. Molecular analysis indicated that high frequency of loss of heterozygosity (LOH) was found on chromosomes 4, 11, 12 and 19 in both lymphomas. Interestingly, the frequency of LOH on chromosome 11 was much higher, but that on chromosome 12 was lower in carbon-ion-induced T-cell lymphomas than in X-ray-induced ones. These results indicate that mechanistic differences may exist between carbon-ion- and X-ray-induced lymphomagenesis.

Effects of low dose particle radiation to mouse neonatal neurons in culture.

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 generated by HIMAC at NIRS and 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 focusing on the dependencies of (1) the animal strains with different radiation sensitivities, and (2) LET with different nuclei. Of the three mouse strains, SCID, B6 and C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to both X-ray and carbon ion ( 290 MeV/n) as evaluated by 10% apoptotic criterion. However, the sensitivity differences among the strains were much smaller in case of carbon ion comparing to that of X-ray. Regarding the LET dependency, the sensitivity was compared with using C3H and B6 cells between the carbon (13 keV/micrometers) and neon (70 keV/micrometers) ions. Carbon (290 MeV/n) did not give a detectable LET dependency from the criterion whereas the neon (400 MeV/n) showed 1.4 fold difference for both C3H and B6 cells. Although a LET dependency was examined by using the most sensitive SCID cells, no significant difference was detected.

Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose.

Angiogenesis is essential for tumor growth and metastasis. Because endothelial cells are genetically stable, they rarely acquire resistance to anticancer modalities, and could, thus, be a suitable target for radiation therapy. Heavy ion radiation therapy has attracted attention as an effective modality for cancer therapy because of its highly lethal effects, but the effects of heavy ion irradiation on in vitro cell function associated with angiogenesis have not been reported. Our study found that in vitro angiogenesis was inhibited by high linear energy transfer carbon ion irradiation even at sublethal dose (0.1 Gy). ECV304 and HUVEC human umbilical vascular endothelial cells were irradiated with 290 MeV carbon ion beams of approximately 110 keV/ micro m or 4 MV X-ray of approximately 1 keV/ micro m. Their adhesiveness and migration to vitronectin or osteopontin were inhibited, and capillary-like tube structures in three-dimensional culture were destroyed after carbon ion irradiation concomitant with the inhibition of matrix metalloproteinase-2 activity, down-regulation of alphaVbeta3 integrin, which is one of the adhesion molecules, slight up-regulation of membrane type1- matrix metalloproteinase, and significant up-regulation of tissue inhibitor of metalloproteinase-2. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary-like tube structure in three-dimensional culture progressed even after 16 Gy irradiation. These results provide an implication that heavy ion beam therapy could be superior to conventional photon beam therapy in preventive effects on in vitro angiogenesis even at sublethal dose, and might inhibit angiogenesis in vivo.

Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center.

PURPOSE: To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS: Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS: RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell-stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION: The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values.

[Radiation-induced apoptosis in vivo: therapeutic significance of apoptosis in radiation therapy].

Apoptotic cell death is frequently found in certain tumor cells after irradiation; however, the incidence is not always high in vivo. Seven tumors were transplanted to nude mice, and their organs were histologically examined after irradiation to study the therapeutic significance of apoptosis in radiation therapy. A high incidence of apoptosis was found only in radiosensitive tumors or normal cells with wild-type p53, but the peak incidence in most cells was only a few percent or less. However, the calculated total incidence of apoptotic cell death was much higher than the actual peak incidence, because the half-life of apoptosis is very short. Even in radioresistant tumors, total radiation-induced apoptosis was estimated to be about 10 percent. These results suggest that apoptotic cell death in radiotherapy may be more important in vivo than previously estimated.

Histological and elemental changes in the rat brain after local irradiation with carbon ion beams.

The left cerebral hemispheres of adult Sprague-Dawley rat brains were irradiated at doses of 30, 50, or 100 Gy with charged carbon particles (290 MeV/nucleon; 5 mm spread-out Bragg peak). The spread-out Bragg peak used here successfully and satisfactorily retained its high-dose localization in the defined region. A histological examination showed that necrotic tissue damage, hemorrhage in the thalamus, and vasodilatations around the necrotic region were induced at 8 weeks after 100 Gy irradiation. The regions with tissue damage correlated well with those expected from the radiation-dose distribution, indicating an advantage of charged carbon particles for irradiating restricted brain regions. An X-ray fluorescent analysis demonstrated a decrease in the concentrations of K and P, and an increase in the concentrations of Cl, Fe, Zn in the damaged region at 8 weeks post-irradiation, though no significant changes were observed before 4 weeks of post-irradiation. This may indicate that even the very high radiation doses used here did not induce acute and immediate neuronal cell death, in contrast with ischemic brain injury where acute neuronal cell death occurred and the elemental concentrations changed within a day after the induction of ischemia.