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Ionizing radiation can induce mutations in germ cells in various organisms, including fruit flies and mice. However, currently, there is no clear evidence for the transgenerational effects of radiation in humans. This review is an effort to identify possible reasons for the lack of such observations. Literature search and narrative review. 1) In both mice and humans, resting oocytes locate primarily in the cortical region of the ovary where the number of blood vessels is very low especially when young and extra-cellular material is rich, and this region is consequently hypoxic, which probably leads to immature oocytes being resistant to the cell killing and mutagenic effects of radiation. 2) In studies of spermatogonia, the mouse genes used for specific locus test (SLT) studies, which include coat color genes, were hypermutable when compared to many other genes. Recent studies which examined over 1000 segments of genomic DNA indicate that the induction rate of deletion mutation per segment was on the order of 10−6 per Gy, which is one order of magnitude lower than that obtained from the SLT data. Therefore, it appears possible that detecting any transgenerational effects of radiation following human male exposures will be difficult due to a lack of mutable marker genes. 3) Fetal malformations were examined in studies in humans, but the genetic component in such malformations is low, and abnormal fetuses are prone to undergo miscarriage which does not occur in mice, and which leads to difficulties in detecting transgenerational effects. The lack of clear evidence for radiation effects in humans probably does not result from any problem in the methodologies used but may be due largely to biological properties. Currently, whole genome sequencing studies of exposed parents and offspring are planned, but ethical guidelines need to be followed to avoid discrimination, which had once happened to the atomic bomb survivors.

It has been difficult to understand why the relative risk for cancer decreases with an increase in time since an exposure to radiation. It was recently recognized that this decline can be explained by a parallel shift of the age-related cancer mortality curve toward younger ages. In fact, it has been known for many years that mouse survival curves exhibit a parallel shift toward younger ages following an exposure to radiation, but it was not recognized that the mutation induction theory is incompatible with this parallel shift. This is because a parallel shift in the survival curve implies that all the irradiated individuals are affected, but the mutation induction theory assumes that only a fraction of the irradiated individuals is affected following an exposure to radiation. Thus, it seems likely that the target of radiation action, which leads to carcinogenesis, is not restricted to epithelial cells but includes all of the surrounding cells leading to an altered microenvironment. Since it is repeatedly observed that radiation-exposed normal tissues can stimulate transplanted or spontaneously arising tumor cells to grow faster, worsen the malignant phenotypes and finally kill the host earlier than usual, an exposure to radiation seems most likely to cause tissue inflammation, which creates conditions favorable for the growth of spontaneously arising tumor cells. This new concept suggests that it might be possible to attenuate the extent of radiation carcinogenesis by intervening in tissue inflammatory processes.

[Purpose] Ionizing radiation is a well-known carcinogen, and epidemiologic efforts have been made to evaluate cancer risks following a radiation exposure. The basic approach has been to estimate increased levels of cancer mortality resulting from exposures to radiation, which is consistent with the somatic mutation theory of cancer. However, the possibility that an irradiation might cause an earlier onset of cancer has also been raised since the earliest days of animal studies. Recently, the mutation induction model has been challenged because it is unable to explain the observed dose-related parallel shift of entire mouse survival curves toward younger ages following an irradiation. This is because if it is assumed that only a fraction of the irradiated individuals are affected, the irradiated population would consist of two subpopulations with different mean lifespans, which makes the overall distribution of individual lifespans broader, and hence the slope of the survival curves shallower. To explain this parallel shift, it is necessary to assume that all individuals of a population are affected. As a result of these observations, possible mechanisms which could account for the parallel shift of mouse survival curves were sought by examining the radiation induction of various types of tissue damage which could facilitate an earlier onset of spontaneously arising cancers.[Conclusion] A proposed mechanism postulates that a radiation exposure leads to tissue inflammation which subsequently stimulates spontaneously arising cancers and allows them to appear earlier than usual. This notion is not unprecedented, and because the background incidence of cancer increases exponentially with an increase in age, a slight shift of the onset age toward younger ages may make it appear as if the risk is increased. In this scenario, a radiation exposure induces DNA damage, cell death, chromosome aberrations etc., which leads to the multi-pathway responses including activation of stromal fibroblasts, macrophages and various inflammatory factors. Such an inflamed microenvironment favors the growth of spontaneously arising tumor cells although currently, the sequential order or relative importance of the individual factors remains to be known.Biographical NoteNori Nakamura PhD, is a radiation biologist and geneticist working at the Radiation Effects Research Foundation in Hiroshima, Japan, for more than 35 years. He is also interested in biodosimetry methods which include cytogenetic methods and the electron-spin (paramagnetic)-resonance method used to examine tooth enamel.

The frequency of stable chromosome aberrations (sCA) in lymphocytes is a recognized radiation biological dosimeter. Its analysis can provide insights into factors that affect individual susceptibility as well as into the adequacy of radiation dose estimates used in studies of atomic bomb survivors. We analyzed the relationship between atomic bomb radiation exposure using the most recent DS02R1 dose estimates and the frequency of sCA as determined by FISH in 1,868 atomic bomb survivors. We investigated factors that may affect the background sCA rate and the shape and magnitude of the dose response. As in previous analyses of sCA in atomic bomb survivors that were based on Giemsa staining methods and used older DS86 dose estimates, the relationship between radiation dose and sCA rate was significant (P0.0001) with a linear-quadratic relationship at lower doses that did not persist at higher doses. As before, age at the time of the bombing and type of radiation shielding were significant dose-effect modifiers (P0.0001), but in contrast the difference in dose response by city was not so pronounced (P = 0.026) with a city effect not evident at doses below 1.25Gy. Background sCA rate increased with age at the time of examination (P0.0001), but neither sex, city, nor smoking was significantly associated with background rate. Based on FISH methods and recent dosimetry, the relationship between radiation dose and sCA frequency is largely consistent with previous findings, although the lesser importance of city as an effect modifier may reflect better dosimetry as well as more reproducible scoring of sCA. The persisting difference in sCA dose response by shielding category points to remaining problems with the accuracy or precision of radiation dose estimates in some A-bomb survivors.

Genomics of radiation-induced damage The potential adverse effects of exposures to radioactivity from nuclear accidents can include acute consequences such as radiation sickness, as well as long-term sequelae such as increased risk of cancer. There have been a few studies examining transgenerational risks of radiation exposure but the results have been inconclusive. Morton et al. analyzed papillary thyroid tumors, normal thyroid tissue, and blood from hundreds of survivors of the Chernobyl nuclear accident and compared them against those of unexposed patients. The findings offer insight into the process of radiation-induced carcinogenesis and characteristic patterns of DNA damage associated with environmental radiation exposure. In a separate study, Yeager et al. analyzed the genomes of 130 children and parents from families in which one or both parents had experienced gonadal radiation exposure related to the Chernobyl accident and the children were conceived between 1987 and 2002. Reassuringly, the authors did not find an increase in new germline mutations in this population. Science , this issue p. eabg2538 , p. 725

Although mammalian fetuses have been suggested to be sensitive to radiation, an increased frequency of translocations was not observed in blood lymphocytes from atomic bomb (A-bomb) survivors who were exposed to the bomb in utero and examined as adults. Since experiments using hematopoietic cells of mice and rats confirmed this finding, it was hypothesized that either irradiated fetal hematopoietic stem cells (f-HSCs) cannot generate exchange-type chromosomal aberrations or cells bearing induced aberrations are eliminated before the animals reach adulthood. In the present study, pregnant mice (12.5–15.5 days post coitum [dpc]) were irradiated with 2 Gy of X-rays and long-term HSCs (LT-HSCs) were isolated 24 h later. Multicolor fluorescence in situ hybridization (mFISH) analysis of LT-HSC clones proliferated in vitro showed that nine out of 43 (21%) clones from fetuses and 21 out of 41 (51%) clones from mothers bore translocations. These results indicate that cells with translocations can arise in mouse f-HSCs but exist at a lower frequency than in the mothers 24 h after X-ray exposure. Thus, it seems likely that translocation-bearing f-HSCs are generated but subsequently disappear, so that the frequency of lymphocyte translocations may decrease and reach the control level by the time the animals reach adulthood.

This study was performed to investigate the effect of the inclusion rate of corn silage (CS) in ensiled total mixed ration (TMR) on dry matter intake (DMI), nutrient digestibility, and ruminal fermentation in Japanese Wagyu steers. Four ensiled TMRs, in which the crude protein (CP) and total digestible nutrients (TDN) levels were adjusted to be similar, were prepared with 0, 15, 30, and 45% CS, respectively. Four Japanese Wagyu steers during the fattening period were used in a feeding trial with the four dietary treatments of the ensiled TMRs in a 4 × 4 Latin square design. Increasing the inclusion rate of CS resulted in higher pH values and lower lactate production in the ensiled TMR, although all TMR silages showed good fermentative characteristics, such as low pH value and high lactate content. The DMI linearly increase when the inclusion rate of CS in the TMRs increased. There were differences in the apparent digestibilities of CP, non-fibrous carbohydrate, acid detergent fiber, and neutral detergent fiber. The TDN contents were greater in the ensiled TMRs with 15 and 30% CS. The feeding of TMRs with higher inclusion rates of CS gave rise to slower fermentation in the rumen. Corn silage should be utilizable as a TMR material, and its inclusion at approximately 30% in a TMR is a safe upper limit for Japanese Wagyu steers during the fattening period.

It has generally been thought that fetuses are highly sensitive to radiation-induced cancer. Epidemiologic case-control studies indicated that X-ray exposures given to pregnant women (on the order of 1 cGy) could have increased the risk of developing childhood leukemia and solid cancers in the offspring. The authors wished to re-consider this observation. Atomic bomb survivors who were exposed in utero were found to show almost no increase in the frequency of translocations in their blood lymphocytes when the survivors were examined at around 40 years of age. Subsequent animal studies revealed that tissue stem cells in embryos/fetuses may or may not retain radiation-induced chromosome damage depending on the developmental stage at the time of irradiation. Our data are compatible with the model that radiation effects can be recorded only when an exposure occurs after the stem cells have settled in their appropriate niche, and that a small fraction of fetal hematopoietic stem cells began making long-term contributions to the lymphoid cell pool in both mice and humans. It remains to be established whether or not the increased risk of childhood leukemia and other childhood cancers was caused by fetal X-ray exposures of about 1 cGy.

Purpose: Cancer risks for Nagasaki survivors once appeared to be lower than for Hiroshima survivors. The possibility that this was due to overestimation of the doses for the Nagasaki survivors was tested by measuring biological doses of Nagasaki survivors and comparing them with DS02R1 individual doses as previously done for Hiroshima survivors. Materials and methods: The electron spin resonance (ESR) method and cytogenetic method were used to estimate radiation doses for 24 Nagasaki survivors, and the results were compared to calculated DS02R1 doses. Results: Six factory workers and 10 other survivors showed ESR or cytogenetically estimated doses that were in reasonably good agreement with their DS02R1 doses, while one factory worker was found to have an ESR dose estimate of nearly one half of the DS02R1 dose to the eye lens (a proxy organ for teeth). A few outliers were also observed. Conclusions: Although apparently lower cancer risks were observed in the past for Nagasaki survivors when compared to Hiroshima survivors, the present results do not indicate the existence of a trend that DS02R1 doses are overestimated when compared with biologically estimated tooth or cytogenetic doses. This observation is in line with the recent disappearance of the city difference in cancer risks.

The exposure of germ cells to radiation introduces mutations in the genomes of offspring, and a previous whole-genome sequencing study indicated that the irradiation of mouse sperm induces insertions/deletions (indels) and multisite mutations (clustered single nucleotide variants and indels). However, the current knowledge on the mutation spectra is limited, and the effects of radiation exposure on germ cells at stages other than the sperm stage remain unknown. Here, we performed whole-genome sequencing experiments to investigate the exposure of spermatogonia and mature oocytes. We compared de novo mutations in a total of 24 F1 mice conceived before and after the irradiation of their parents. The results indicated that radiation exposure, 4 Gy of gamma rays, induced 9.6 indels and 2.5 multisite mutations in spermatogonia and 4.7 indels and 3.1 multisite mutations in mature oocytes in the autosomal regions of each F1 individual. Notably, we found two types of deletions, namely, small deletions (mainly 1~12 nucleotides) in non-repeat sequences, many of which showed microhomology at the breakpoint junction, and single-nucleotide deletions in mononucleotide repeat sequences. The results suggest that these deletions and multisite mutations could be a typical signature of mutations induced by parental irradiation in mammals.

Genetic effects from radiation have been observed in a number of species to date. However, observations in humans are nearly nonexistent. In this review, possible reasons for the paucity of positive observations in humans are discussed. Briefly, it appears likely that radiation sensitivity for the induction of mutations varies among different genes, and that the specific genes that were used in the past with the specific locus test utilizing millions of mice may have simply been very responsive to radiation. In support of this notion, recent studies targeting the whole genome to detect copy number variations (deletions and duplications) in offspring derived from irradiated spermatogonia indicated that the mutation induction rate per genome is surprisingly lower than what would have been expected from previous results with specific locus tests, even in the mouse. This finding leads us to speculate that the lack of evidence for the induction of germline mutations in humans is not due to any kind of species differences between humans and mice, but rather to the lack of highly responsive genes in humans, which could be used for effective mutation screening purposes. Examples of such responsive genes are the mouse coat color genes, but in human studies many more genes with higher response rates are required because the number of offspring examined and the radiation doses received are smaller than in mouse studies. Unfortunately, such genes have not yet been found in humans. These results suggest that radiation probably induces germline mutations in humans but that the mutation induction rate is likely to be much lower than has been estimated from past specific locus studies in mice. Whole genome sequencing studies will likely shed light on this point in the near future.

Purpose: To review the history of radiation effects in germ cells and to attempt to discern future directions.Materials and methods: Review of published papers.Results: Recent mouse studies using m...

Cancer risks from radiation can be observed as an increase in mortality when compared to a control group. However, it is unknown if this increased risk results from the induction of cancer or from an earlier onset of cancer. In mouse studies, it has been repeatedly shown that after an irradiation, the survival curve is shifted toward lower ages, but remains parallel to the control curve, and the extent of the shift in time to lower ages is dose-dependent. This shift is not satisfactorily explained by the induction model which assumes that cancers in the exposed group consist of spontaneous and induced events. Consequently, it seems that this shift could be interpreted to mean that all animals in the exposed group had suffered from life shortening. Under this scenario, however, it turns out that the radiation effects can no longer be interpreted as the result of oncogenic mutations, because these effects would have to involve all tumors, and the effectiveness of radiation changes with the dose. This leads to the speculation that radiation exposures induce a broad range of tissue injuries, and that these injuries are subsequently subjected to longlasting systemic recovery processes which act as promoters for tumor cells. In other words, potential cancer stem cells which were located in the irradiated field can escape oncogenic damage but undergo stimulation later in life toward the development of malignancy from radiation-induced activated microenvironment. This is an unusual form of the non-targeted or bystander effects of radiation. It is worth noting that this model suggests that there could be a path or paths which could be used to intervene in the process of post-exposure carcinogenesis, and that cancer risks at low doses could be described as days or weeks of life lost.

This study was designed to learn if asymptomatic heterozygotes with mutations in a DNA repair gene are at an increased risk for cancer. To examine this, we focused on carriers of an XPA founder mutation because the frequency of xeroderma pigmentosum (XP) patients is much greater among Japanese than Caucasians, more than half of Japanese XP patients are affected at the XPA gene, and the majority of XP-A patients carry the same founder mutation in the XPA gene. Here we show that the frequency of XPA heterozygote was 14/1698 (0.8%) in cancer-free controls, and the corresponding frequency in patients with nonmelanocytic skin cancer that developed in sun-exposed areas was 11/440 (2.5%, OR = 3.08, p = 0.0097) for basal cell carcinoma, and 3/272 (1.1%, OR = 1.34, p = 0.72) for squamous cell carcinoma. These results suggest a moderately elevated risk for skin cancer among XPA heterozygotes.

The RERF International Low-Dose Symposium was held on 5-6 December 2013 at the RERF campus in Hiroshima, Japan, to discuss the issues facing the Life Span Study (LSS) and other low-dose studies. Topics included the current status of low-dose risk detection, strategies for low-dose epidemiological and statistical research, methods to improve communication between epidemiologists and biologists, and the current status of radiological studies and tools. Key points made by the participants included the necessity of pooling materials over multiple studies to gain greater insight where data from single studies are insufficient; generating models that reflect epidemiological, statistical, and biological principles simultaneously; understanding confounders and effect modifiers in the current data; and taking into consideration less studied factors such as the impact of dose rate. It is the hope of all participants that this symposium be used as a trigger for further studies, especially those using pooled data, in order to reach a greater understanding of the health effects of low-dose radiation.

Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic 60Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the 60Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated 60Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.

Ionizing radiation can induce mutations, and the majority of radiation-induced mutations in mammalian cells are deletions. The most critical types of radiation-induced DNA damage are DNA double-strand breaks, and these breaks are repaired by either the homologous recombination (HR) pathway or the non-homologous end joining (NHEJ) pathway. The HR pathway is not as mutagenic as the NHEJ pathway, and it is expected that radiation-induced deletions would usually have little sequence similarity around the deletion junction points. Here we report sequence data from the regions around the rejoined junctions of 33 de novo copy-number mutations (27 deletions and 6 duplications) obtained from offspring sired by male mice that were irradiated at the spermatogonia stage and from nonirradiated controls. The results indicate that deletions can be classified into three major groups. In group 1, nine deletions were found to share long blocks of similar sequences (200-6,000 bp) at the junctions and the deletion size varied extensively (1 kb to 2 Mb) (e.g., illegitimate recombination). In group 2, five deletions shared short identical sequences (0-7 bp) at the junctions, and the deletion sizes were shorter than 200 kb (e.g., micro-homology-mediated repair). Additional three-deletion candidates of this group were also found but turned out to be inherited from mosaic parents. They are therefore not included in germline mutations. In group 3, twelve deletions shared little sequence similarity (only 0-2 bp) at the junctions (likely due to NHEJ repair) and deletion sizes were longer than 200 kb. Group 1 consisted of deletions found in both spontaneous and irradiated genomes and thus, were probably caused by spontaneous events during meiosis or DNA replication. Group 2 consisted mainly of deletions found in nonexposed genomes. Group 3 consisted primarily of deletions that occurred in the irradiated genomes. Among the duplications, we found no indication of any association with radiation exposures. These results indicate that large size (200 kb) and little sequence similarity around the rejoined sites are likely to be a hallmark of radiation-induced deletions in mice.

Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction.

In experimental organisms such as fruit flies and mice, increased frequencies in germ cell mutations have been detected following exposure to ionizing radiation. In contrast, there has been no clear evidence for radiation-induced germ cell mutations in humans that lead to birth defects, chromosome aberrations, Mendelian disorders, etc. This situation exists partly because no sensitive and practical genetic marker is available for human studies and also because the number of people exposed to large doses of radiation and subsequently having offspring was small until childhood cancer survivors became an important study population. In addition, the genome of apparently normal individuals seems to contain large numbers of alterations, including dozens to hundreds of nonfunctional alleles. With the number of mutational events in protein-coding genes estimated as less than one per genome after 1 gray (Gy) exposure, it is unsurprising that genetic effects from radiation have not yet been detected conclusively in humans.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10(-2)/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10(-5)/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

While it is generally believed that fetuses are at high risk of developing cancers, including leukemia, after low doses of radiation, it has been reported that atomic bomb survivors exposed in utero did not show a dose response for translocations in blood T lymphocytes when they were examined at approximately 40 years of age. Subsequent mouse studies confirmed that animals irradiated during the fetal stage did not show evidence of radiation effects in lymphocytes and bone marrow cells when they were examined after reaching adulthood. However, in a study of rat mammary epithelial cells, radiation effects were clearly observed after fetal irradiation. These results indicate that the fate of chromosome aberrations induced in a fetus could vary among different tissues. Here we report on translocation frequencies in mouse thyroid cells, which were irradiated at different stages of fetal development. Cytogenetic examination was conducted using fluorescence n situ hybridization (FISH) painting of chromosomes 1 and 3. Adult mice, 2 Gy X-ray irradiated at 15.5-day-old fetuses (E15.5), showed a higher translocation frequency (30/1,155 or 25.3 × 10

Purpose: New developments in knowledge of radiation effects on tissue stem cells were discussed in a Workshop held at the Radiation Effects Research Foundation (RERF) in Hiroshima, Japan, 18–19 January 2012.Results: Stem cells and their niche in intestinal mucosa, haemopoietic tissue, hair follicles, and spermatogenesis were discussed variously with regard to radiosensitivity, repair, regeneration, age-dependency of effects, genetic effects, and protection aspects. These tissues all possess a common basic template, but there are structural and hierarchical differences between tissues which continue to be elucidated in terms of a stem-cell age structure and niche regulatory signals which together govern radiation responses.Conclusions: Stem cells and their niche have become much better characterized in recent years, and their radiation response can be elucidated in detail in experimental systems to help underpin both protection and therapeutic recommendations established from human epidemiological evidence...

The electron paramagnetic resonance (EPR, or electron spin resonance) method was used to measure CO₂⁻· radicals recorded in tooth enamel by exposure to atomic-bomb gamma rays. The EPR-estimated doses (i.e. ⁶⁰Co gamma-ray equivalent dose) were generally in good correlation with cytogenetic data of the same survivors, whereas plots of EPR-estimated dose or cytogenetically estimated dose against DS02 doses turned out to scatter more widely. Because those survivors whose EPR doses were higher (or lower) than DS02 doses tended to show also higher (or lower) responses for cytogenetic responses, the apparent variation appears primarily due to problems in individual DS02 doses rather than the measurement errors associated with the EPR or cytogenetic technique. A part of the enamel samples were also used for evaluation of neutron doses by measuring ⁴¹Ca/⁴⁰Ca ratios using the accelerator mass spectrometry technique. The results for the measured ratios were on average ~85 % of the calculated ratios by DS02 (but within the 95 % confidence bounds of the simulated results), which lends support to DS02-derived neutron doses to the survivors.

This paper presents the results of the 4th International Comparison of in vitro electron paramagnetic resonance dosimetry with tooth enamel, where the performance parameters of tooth enamel dosimetry methods were compared among sixteen laboratories from all over the world. The participating laboratories were asked to determine a calibration curve with a set of tooth enamel powder samples provided by the organizers. Nine molar teeth extracted following medical indication from German donors and collected between 1997 and 2007 were prepared and irradiated at the Helmholtz Zentrum Munchen. Five out of six samples were irradiated at 0.1, 0.2, 0.5, 1.0 and 1.5 Gy air kerma; and one unirradiated sample was kept as control. The doses delivered to the individual samples were unknown to the participants, who were asked to measure each sample nine times, and to report the EPR signal response, the mass of aliquots measured, and the parameters of EPR signal acquisition and signal evaluation. Critical dose and detection limit were calculated by the organizers on the basis of the calibration-curve parameters obtained at every laboratory. For calibration curves obtained by measuring every calibration sample three times, the mean value of the detection limit was 205 mGy, ranging from 56 to 649 mGy. The participants were also invited to provide the signal response and the nominal dose of their current dose calibration curve (wherever available), the critical dose and detection limit of which were also calculated by the organizers.

Eleven oribatid species were collected from the Kumaya Cave of Iheya village in Central Ryukyu Arc, South Japan. The cave is a descending horizontal tunnel of a deep-sea abysmal quartziferous deposit formed in the Permian period of the Palaeozoic era and excavated by sea erosion. The floor of the cave was covered with sand, three to four meters deep. Fragments of psephite are found at the end of the cave. Each sample of about 200 ml was collected by hand from the following five marked points of the cave on 17 March 2010: A, Sand and a fragment of psephite; B, Sand and a fragment of psephite; C, Sand, a fragment of psephite, and litter of Ficus microcarpa; D, Litter and humus of F. microcarpa and Cassytha filiformis, and sand; E, Litter and humus of F. microcarpa and C. filiformis, and sand. All species belonged to Brachypylina: Oppiella (O.) nova from A; Mabulatrichus kumayaensis sp. nov., Protoribates kumayaensis sp. nov. and Zygoribatula iheyaensis sp. nov. from B; Tectocepheus kumayaensis sp. nov. from C; Tectocepheus iheyaensis sp. nov., Oribatula kumayaensis sp. nov., Protoribates hirokous sp. nov. and Haplozetes makii sp. nov. from D; Eupelops kumayaensis sp. nov. from E; Neoliodes iheyaensis sp. nov. from C, D and E.

The detection of 41Ca atoms in tooth enamel using accelerator mass spectrometry is suggested as a method capable of reconstructing thermal neutron exposures from atomic bomb survivors in Hiroshima and Nagasaki. In general, 41Ca atoms are produced via thermal neutron capture by stable 40Ca. Thus any 41Ca atoms present in the tooth enamel of the survivors would be due to neutron exposure from both natural sources and radiation from the bomb. Tooth samples from five survivors in a control group with negligible neutron exposure were used to investigate the natural 41Ca content in tooth enamel, and 16 tooth samples from 13 survivors were used to estimate bomb-related neutron exposure. The results showed that the mean 41Ca/Ca isotope ratio was (0.17 ± 0.05) × 10-14 in the control samples and increased to 2 × 10-14 for survivors who were proximally exposed to the bomb. The 41Ca/Ca ratios showed an inverse correlation with distance from the hypocenter at the time of the bombing, similar to values that have been derived from theoretical free-in-air thermal-neutron transport calculations. Given that γ-ray doses were determined earlier for the same tooth samples by means of electron spin resonance (ESR, or electron paramagnetic resonance, EPR), these results can serve to validate neutron exposures that were calculated individually for the survivors but that had to incorporate a number of assumptions (e.g. shielding conditions for the survivors). Fil: Wallner, A.. Ludwig Maximilians Universitat; Alemania. Universitat Technical Zu Munich; Alemania. Universidad de Viena; Austria Fil: Ruhm, W.. Helmholtz Center Munich German Research Center For Environmental Health; Alemania. Ludwig Maximilians Universitat; Alemania Fil: Rugel, G.. Ludwig Maximilians Universitat; Alemania. Universitat Technical Zu Munich; Alemania Fil: Nakamura, N.. Radiation Effects Research Foundation; Japón Fil: Arazi, Andres. Universitat Technical Zu Munich; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Faestermann, T.. Universitat Technical Zu Munich; Alemania Fil: Knie, K.. Universitat Technical Zu Munich; Alemania. Ludwig Maximilians Universitat; Alemania Fil: Maier, H. J.. Ludwig Maximilians Universitat; Alemania Fil: Korschinek, G.. Universitat Technical Zu Munich; Alemania

a Vanderbilt University, Nashville, Tennessee and International Epidemiology Institute, Rockville, Maryland; b Gray Institute for Radiation Oncology and Biology, University of Oxford, United Kingdom; c Radiation Effects Research Foundation, Hiroshima, Japan; d National Institute of Radiological Sciences, Chiba, Japan; e Health Research Foundation, Kyoto, Japan; and f Central Research Institute of Electric Power Industry, Tokyo, Japan

The objective was to evaluate effects of feeding level of milk replacer on body growth, plasma metabolite and insulin concentrations, and allometric growth of visceral organs in suckling calves. Holstein bull calves (n = 8; 3-4 days of age) were fed either a low amount (average 0.63 kgDM/day, LM) or high amount (average 1.15 kgDM/day, HM) of high protein milk replacer until they were slaughtered at 6 weeks of age. Body weight (BW) at 4, 5, and 6 weeks of age, feed intake, average daily gain, and feed efficiency were higher in the HM than LM calves. The HM group had higher plasma glucose at 3 and 4 weeks of age and insulin levels after the age of 4 weeks compared with LM calves whereas no effect was detected on plasma nonesterified fatty acid or urea nitrogen concentrations. The HM calves had greater empty body weight (EBW), viscera-free BW and most of the organs dissected than LM calves. Relative weights (% of EBW) of liver, spleen, kidneys, and internal fat were higher, whereas head and large intestine was lower in HM than LM calves. The results suggest that increased milk feeding levels would accelerate the growth of the body and specific organs.

Genomic instability has been suggested as a mechanism by which exposure to ionizing radiation can lead to cancer in exposed humans. However, the data from human cells needed to support or refute this idea are limited. In our previous study on clonal lymphocyte populations carrying stable-type aberrations derived from A-bomb survivors, we found no increase in the frequency of sporadic additional aberrations among the clonal cell populations compared with the spontaneous frequency in vivo. That work has been extended by using multicolor FISH (mFISH) to quantify the various kinds of chromosome aberrations known to be indicative of genomic instability in cloned T lymphocytes after they were expanded in culture for 25 population doublings. The blood T cells used were obtained from each of two high-dose-exposed survivors (1 Gy) and two control subjects, and a total of 66 clonal populations (36 from exposed and 30 from control individuals) were established. For each clone, 100 metaphases were examined. In the case of exposed lymphocytes, a total of 39 additional de novo stable, exchange-type aberrations [translocation (t) + derivative chromosome (der)] were found among 3600 cells (1.1%); the corresponding value in the control group was 0.6% (17/3000). Although the ratio (39/3600) obtained from the exposed cases was greater than that of the controls (17/3000), the difference was not statistically significant (P = 0.101). A similar lack of statistical difference was found for the total of all structural chromosome alterations including t, der, dicentrics, duplications, deletions and fragments (P = 0.142). Thus there was no clear evidence suggesting the presence of chromosome instabilities among the clonally expanded lymphocytes in vitro from A-bomb survivors.

The histochemical properties, collagen content and architecture of Musculus longissimusthoracis (LT), Musculus pectoralis profundus (PP) and Musculus biceps femoris (BF) were compared in F(1) (half blood) and F(2) (quarter blood) wild boar crossbred pigs and commercial hybrid pigs, and Japanese wild pigs. F(1) pigs showed the lowest growth rate, followed by F(2) pigs. The most rapid growth was shown by the commercial pigs. The percentage weights of LT and PP muscle to body weight were larger in the wild boar crossbred pigs than commercial pigs. The muscles of the crossbred pigs contained type I and IIA myofibers at higher frequency and type IIB at lower frequency than the commercial pigs, except for LT muscle of F(2) pigs. The myofiber diameter in each type of muscle did not differ between pigs except for the smaller type IIA in BF muscle in commercial pigs. The total amount of intramuscular collagen was less in LT muscles than the others. More intramuscular collagen was found in the wild boar crossbred pigs than the commercial pigs in LT and PP muscles. With an increase of collagen content, the perimysial collagen architecture developed but not the endomysial architecture. Traits characteristic of the crossbred pigs seem to be inherited from the wild boar. Our results clarify that cross breeding with wild boar results in pigs with distinctive muscle characteristics in terms of histochemical properties, collagen content and architecture.

Nakamura, Y.-N., Orito, H., Tsuneishi, E., Hirano, K., Kato, N., Shoji, A., Kamiya, M. and Nakanishi, Y. 2008. Changes in plasma composition of Japanese black steers during grazing and fattening periods. J. Appl. Anim. Res., 34: 157–161. The plasma compositions of five Japanese black steers during about 23 months of grazing and fattening period of about 6 months were investigated. Plasma A/G, LDH, Glu, tCho and Ins concentrations increased (P

Nakamura, Y., Shiga, A., Akasaka, S. and Gotoh, T. 2007. Effect of daytime grazing of fresh grass without supplemental feed on serum and milk α-tocopherol concentrations of Holstein dairy cows. J. Appl. Anim. Res., 32: 119–124. The effect of switching 5 Holstein dairy cows from concentrate-fed to roughage-fed conditions without supplemental feed for seven weeks on serum parameters and milk chemical composition in morning and afternoon milk samples was investigated. BW was not affected and blood hematocrit and total protein, glucose and triglyceride concentrations varied within normal values throughout the experiment. During day grazing period, serum non-esterified fatty acid (NEFA), milk somatic cell counts (SCC), total solids, lipids and milk and serum α-tocopherol values increased, whereas, those of milk yield, milk protein, lactose and solids-non-fat decreased. For diurnal changes, NEFA concentrations were lower, whereas, SCC, milk lipid and milk α-tocopherol concentrations were higher in the ...

Using the dorsal ordinary muscle (DOM) of cultured Pacific bluefin tuna (body length [BL]: 47.5–81.8 cm, body weight [BW]: 2.1–13.5 kg, n=15), the changes of proximate compositions and myoglobin (Mb) content with growth were investigated. There was a positive correlation (r=0.9832, P

Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (or =0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated whenor =6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53(-/-) mice and 1.4% in p53(+/-) mice) compared to the frequency in the p53(-/-) mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

We investigated the changes in myoglobin (Mb) content and proximate compositions of the cephalad parts of the dorsal ordinary muscles (Ce-DOM) of full-cycle cultured (FC) Pacific bluefin tuna [body length: 42.6-115.4 cm, body weight (BW): 1.7-38.4 kg, killed from July 2004 to October 2005]. The Mb content of Ce-DOM increased (P

Using the full-cycle cultured (FC) Pacific bluefin tuna, Thunnus orientalis [body weights: 13.1 ± 4.5 (FC1; April in 2004), 20.2 ± 1.8 (FC2; July in 2004), 28.5 ± 6.3 (FC3; November in 2004), 27.0 ± 3.3 (FC4; February in 2005) and 33.5 ± 4.7 kg (FC5; May in 2005), n = 3, respectively] and wild bluefin tuna [33.3 ± 1.5kg (June in 2005), n = 3], proximate and fatty acid compositions of the cephalal (Ce-) and caudal (Ca-) parts of the dorsal (D) and ventral (V) ordinary muscles (OMs) were investigated. Lipid contents of the Ce-DOM and VOMs of FC1-5 increased with growth. In particular, lipid content of the Ce-DOM (23.0%) and VOMs (55.1%) of FC5 was higher ( P P n –3: n –6 (9.4%) of the Ce-VOM of FC5 was lower ( P

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

Using full-cycle cultured (FC) Pacific bluefin tuna (body length [BL], 42.6–66.4 cm; body weight [BW], 1.66–7.40 kg, n=15), the changes in chemical compositions and histological structure of the cephalal parts of the dorsal ordinary muscles (DOM) occurring with growth were investigated. A positive correlation (r=0.9644, P

Using the full-cycle cultured (FC) Pacific bluefin tuna [body weight 16.3±1.9 kg (pre-fasting group, pre-FG), 14.2±0.9 kg (post-fasting group, post-FG)], changes in the physical/chemical properties of the cephalal parts of dorsal (D) and ventral (V) ordinary muscles (OM) by fasting (6 days) during chilled storage (4°C) were investigated. Condition factors were 26.7 (pre-FG) and 20.3 (post-FG, P < 0.05). Fasting changed the liver color to green. Fasting also decreased the amount of protein and lipid contents of the DOM and VOM of FC tuna. The breaking strength and pH of the DOM and VOM of post-FG tuna were higher (P

Using full-cycle cultured Pacific bluefin tuna (BW: 33.5 ± 2.7 kg, cultured for about 33 mo), the physical/ chemical composition and histological properties of the cephalal parts of the dorsal ordinary muscles (DOMs) and ventral ordinary muscles (VOMs) were investigated during chilled storage at 4 °C for 69 h. VOM showed high fat content compared with DOM. The breaking strength of DOM increased up to 33 h but decreased thereafter, whereas VOM decreased gradually after 15 h. The pH and glycogen content of DOMs and VOMs decreased gradually after 9 h. The met-myoglobin (met-Mb)% and K-value of DOMs and VOMs increased gradually during storage. Extension of intercellular spaces of DOM was observed by LM during storage. The abundant quantity of glycogen granules that accumulated around the myofibrils of DOM was confirmed by TEM to decrease during storage (especially, from 15 h).

Collagen content and architecture of the longissimus thoracis (LT) muscle were compared between the concentrate- and forage-fed male goats. During the experimental period of 9 to 26 wks old, the former gained 15.7kg in body weight by feeding on commercial concentrate food and Italian ryegrass hay, while the latter gaincd only 2.4 kg by feeding on hay only. Dry matter, crude protein and crude fat were contained at significantly higher percentages in the LT muscle of the concentrate-fed goats than the forage-fed goats, and conversely, collagen content was significantly higher in the muscle of the latter. However, the collagen pyridinoline content did not differ between them. The myofibers in the concentrate-fed goats (54.0~am) were twlce the size of those in the forage-fed goats (25.5llm). The higher content of crude fat (2.890/0) in the LT muscle of the concentrate-fed g6ats was not enough for adipose tissues to develop wlthin the perimysium as marbling. Immunohistochemical methods revealed types I and HI collagens over both perimysium and endomysium. Thin collagen bundles were loosely distributed in the perimysium of concentrate-fed goats muscles, and conversely, the perimysial architecture in the forage-fed goats muscles appeared as dense stacks of thick collagen fibers. From these results, it was indicated that the development of loose tissues of slender collagen fibers had an intimate relationship with the rapid growih rate of muscle rather than the development of adipose tissue.

The histological properties of the cephalad parts of the dorsal ordinary muscles (CD-OMs) of wild (body weight 33.3 ± 0.9 kg, n = 3) and full-cycle cultured (body weights 13.1 ± 2.6 (FC 1), 20.2 ± 1.0 (FC 2) and 28.5 ± 3.6 (FC 3) kg, n = 3 respectively) Pacific bluefin tuna were investigated. The CD-OMs of FC 1–3 tuna showed low moisture content (P < 0.05) and high fat content (about six times more, P < 0.05) in comparison with wild fish. A large quantity of adipose tissue was observed in the wide spaces between muscle fibres in the CD-OMs of FC 1-3 tuna. The muscle fibre diameter of the CD-OM of FC 3 tuna was significantly lower than that of wild fish (P < 0.05). Myofibrils in the CD-OM of wild tuna were surrounded by a developed sarcoplasmic reticulum and mitochondria; however, there were many glycogen granules among the myofibrils and a large quantity of fat in the CD-OM of full-cycle cultured fish. In particular, the glycogen content of the CD-OM of FC 3 tuna was significantly higher (about 50 times) than that of wild fish. These results indicate that feeding and exercise conditions affect the histological structure and metabolism of the bluefin tuna muscle. Copyright © 2006 Society of Chemical Industry

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.

The effects of conjugated linoleic acid (CLA), γ-linolenic acid (GLA), linoleic acid (LA), and their combinations, on skin composition in mice were investigated. Mice (8 weeks old) were orally administered with either LA, GLA, CLA, LA + GLA, LA + CLA, or CLA + GLA for 4 weeks. Then, the skin was analysed for triacylglycerol content, fatty acid composition and collagen content. Additionally, thicknesses of the dermis layer and subcutaneous tissue layer, and the size and number of adipocytes were measured histologically. The skin fatty acid composition was modified depending upon the fatty acid composition of supplemented oils. In each oil-alone group, skin triacylglycerol content was the highest in LA, followed by GLA and CLA treatments. Combinations with CLA had a similar triacylglycerol content compared with the CLA-alone group. No significant changes in collagen content were observed among any treatments. The effects on subcutaneous thickness were similar to the results obtained in the triacylglycerol contents, where groups supplemented with CLA alone or other fatty acids had significantly thinner subcutaneous tissue compared with the LA-alone group. However, no significant difference was detected in the thickness of the dermis layers. The number of adipocytes was highest in the LA + GLA group and tended to be reduced by CLA with or without the other fatty acids. These results suggest that CLA alone or in combination with other fatty acids strongly modifies skin composition in mice.