Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Genomic profile of radiation-induced early-onset mouse B-cell lymphoma recapitulates features of Philadelphia chromosome-like acute lymphoblastic leukemia in humans.

Epidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome-like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL.

Brca1L63X /+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer.

Women who are heterozygous for deleterious BRCA1 germline mutations harbor a high risk of hereditary breast cancer. Previous Brca1-heterozygous animal models do not recapitulate the breast cancer phenotype, and thus all currently used knockout models adopt conditional, mammary-specific homozygous Brca1 loss or addition of Trp53 deficiency. Herein, we report the creation and characterization of a novel Brca1 mutant rat model harboring the germline L63X mutation, which mimics a founder mutation in Japan, through CRISPR-Cas9-based genome editing. Homozygotes (Brca1L63X/L63X ) were embryonic lethal, whereas heterozygotes (Brca1L63X/+ ) showed apparently normal development. Without carcinogen exposure, heterozygotes developed mammary carcinoma at a comparable incidence rate with their wild-type (WT) littermates during their lifetime. Intraperitoneal injection of 1-methyl-1-nitrosourea (25 or 50 mg/kg) at 7 weeks of age induced mammary carcinogenesis at comparable levels among the heterozygotes and their littermates. After exposure to ionizing radiation (0.1-2 Gy) at 7 weeks of age, the heterozygotes, but not WT littermates, displayed dose-dependent mammary carcinogenesis with 0.8 Gy-1 excess in hazard ratio during their middle age; the relative susceptibility of the heterozygotes was more prominent when rats were irradiated at 3 weeks of age. The heterozygotes had tumors with a lower estrogen receptor α immunopositivity and no evidence of somatic mutations of the WT allele. The Brca1L63X/+ rats thus offer the first single-mutation, heterozygous model of BRCA1-associated breast cancer, especially with exposure to a DNA break-inducing carcinogen. This implies that such carcinogens are causative and a key to breast cancer prevention in individuals who carry high-risk BRCA1 mutations.

Interstitial chromosomal deletion of the tuberous sclerosis complex 2 locus is a signature for radiation-associated renal tumors in Eker rats.

Ionizing radiation can damage DNA and, therefore, is a risk factor for cancer. Eker rats, which carry a heterozygous germline mutation in the tumor-suppressor gene tuberous sclerosis complex 2 (Tsc2), are susceptible to radiation-induced renal carcinogenesis. However, the molecular mechanisms involved in Tsc2 inactivation are unclear. We subjected Fischer 344 × Eker (Long Evans Tsc2+/- ) F1 hybrid rats to gamma-irradiation (2 Gy) at gestational day 19 (GD19) or postnatal day 5 (PND5) and investigated the patterns of genomic alterations in the Tsc2 allele of renal tumors that developed at 1 year after irradiation (N = 24 tumors for GD19, N = 10 for PND5), in comparison with spontaneously developed tumors (N = 8 tumors). Gamma-irradiation significantly increased the multiplicity of renal tumors. The frequency of LOH at the chromosome 10q12 region, including the Tsc2 locus, was 38%, 29% and 60% in renal carcinomas developed from the nonirradiated, GD19 and PND5 groups, respectively. Array comparative genomic hybridization analysis revealed that the LOH patterns on chromosome 10 in renal carcinomas were classified into chromosomal missegregation, mitotic recombination and chromosomal deletion types. LOH of the interstitial chromosomal deletion type was observed only in radiation-associated carcinomas. Sequence analysis for the wild-type Tsc2 allele in the LOH-negative carcinomas identified deletions (nonirradiated: 26%; GD19: 21%) and base-substitution mutations (GD19: 4%). Reduced expression of Tsc2 was also observed in the majority of the LOH-negative carcinomas. Our results suggest that interstitial chromosomal deletion is a characteristic mutagenic event caused by ionizing radiation, and it may contribute to the assessment of radiation-induced cancer risk.

The effect of radiation on the ability of rat mammary cells to form mammospheres.

As classical transplantation repopulation assays for studying the radiobiology of rat mammary stem/progenitor cells are extremely time-consuming, this study aimed to characterize the radiobiological properties of mammospheres, spherical clumps of mammary cells formed under non-adherent culture conditions, which are a simple and widely used technique for assessing progenitor cell activity. Rat mammary cells were dissociated and used in transplantation repopulation assays and for the formation of mammospheres. Immunofluorescence for cytokeratin 14 and 18 was used to identify basal and luminal mammary epithelial cells, respectively. Incorporation of 5-bromo-2'-deoxyuridine was used to evaluate cell proliferation. The repopulating activity of the transplanted primary rat mammary cells demonstrated their radiosensitivity, reproducing previous data, with a significant reduction in repopulating activity at ≥ 2 Gy. Cells constituting rat mammospheres were positive for either cytokeratin 14 or 18, with occasional double-positive cells. Both proliferation and aggregation contributed to sphere formation. Cells obtained from the spheres showed lower repopulating activity after transplantation than primary cells. When primary cells were irradiated and then used for sphere formation, the efficiency of sphere formation was significantly decreased at 8 Gy but not at ≤ 6 Gy, indicating radioresistance of the formation process. Irradiation at 8 Gy reduced the proliferation of cells during sphere formation, whereas the cellular composition of the resulting spheres was unaffectes. Thus, mammosphere formation assays may measure a property of putative mammary progenitors that is different from what is measured in the classic transplantation repopulation assay in radiobiology.

[Questionnaire Survey of Fukushima Daiichi Nuclear Power Plant Workers in 2016 on Knowledge and Anxiety About Radiation].

The results of a survey of radiation workers suggest that they are worried about the effects of radiation exposure on health, and approximately 30% of Fukushima Daiichi Nuclear Power Plant (FDNPP) workers have anxiety. This questionnaire survey reveals that the higher the frequency of radiation education, the higher the knowledge of radiation the workers will have, and that the higher the level of knowledge, the lower the anxiety. To reduce anxiety, it is important to increase knowledge about radiation through radiation education. However, even those workers who had radiation education several times still had anxiety. According to the Ordinance on the Prevention of Ionizing Radiation Hazards, the time spent on education about the effects of radiation on the human body is only about 30 minutes. This education is not enough to reduce anxiety. FDNPP workers needed more effective education to increase their knowledge and to reduce their anxiety.

Mutational landscape of T-cell lymphoma in mice lacking the DNA mismatch repair gene Mlh1: no synergism with ionizing radiation.

Biallelic germline mutations in the DNA mismatch repair gene MLH1 lead to constitutional mismatch repair-deficiency syndrome and an increased risk for childhood hematopoietic malignancies, including lymphoma and leukemia. To examine how Mlh1 dysfunction promotes lymphoma as well as the influence of ionizing radiation (IR) exposure, we used an Mlh1-/- mouse model and whole-exome sequencing to assess genomic alterations in 23 T-cell lymphomas, including 8 spontaneous and 15 IR-associated lymphomas. Exposure to IR accelerated T-cell lymphoma induction in the Mlh1-/- mice, and whole-exome sequencing revealed that IR exposure neither increased the number of mutations nor altered the mutation spectrum of the lymphomas. Frequent mutations were evident in genes encoding transcription factors (e.g. Ikzf1, Trp53, Bcl11b), epigenetic regulators (e.g. Suv420h1, Ep300, Kmt2d), transporters (e.g. Rangap1, Kcnj16), extracellular matrix (e.g. Megf6, Lrig1), cell motility (e.g. Argef19, Dnah17), protein kinase cascade (e.g. Ptpro, Marcks) and in genes involved in NOTCH (e.g. Notch1), and PI3K/AKT (e.g. Pten, Akt2) signaling pathways in both spontaneous and IR-associated lymphomas. Frameshift mutations in mononucleotide repeat sequences within the genes Trp53, Ep300, Kmt2d, Notch1, Pten and Marcks were newly identified in the lymphomas. The lymphomas also exhibited a few chromosomal abnormalities. The results establish a landscape of genomic alterations in spontaneous and IR-associated lymphomas that occur in the context of mismatch repair dysfunction and suggest potential targets for cancer treatment.

Kinetics of cytokine mRNA and protein expression by plastic adherent cells in the thymus after split-dose irradiation.

Whole body irradiation causes significant apoptosis in various tissues such as the thymus. If apoptotic cells outnumber the phagocytic capacity of macrophages, apoptosis becomes secondary necrosis, inducing inflammatory cytokine expression in macrophages. Radiation also induces thymic lymphomas in C57BL/6 mice after four consecutive irradiations with 1.6 Gy X-rays with nearly 100% incidence. Since cancer development is modulated by a microenvironment involving macrophages, we examined the kinetics of thymocyte number and plastic adherent cell number in the thymus as well as cytokine mRNA expression by plastic adherent cells in the thymus after split-dose irradiation. Upon split-dose irradiation, thymocyte number changed dramatically, whereas plastic adherent cell number did not. Among cytokine mRNAs tested, IL-1ß, IL-11 and IL-12p40 mRNAs were up regulated 2 days after the 1st and 2nd, 3rd and 4th, and 2nd and 3rd irradiations, respectively. On the other hand, TNF-α mRNA was up regulated 2 days after the 3rd irradiation and 2 weeks after the 4th irradiation. The level of IL-11 protein was also increased 2 days after 3rd and 4th irradiations. These results suggest that, upon split-dose irradiation, macrophages in the thymus produce various cytokines in a time-dependent manner, thereby contributing to induction of thymic lymphomas.

Phosphorus Localization and Its Involvement in the Formation of Concentrated Uranium in the Renal Proximal Tubules of Rats Exposed to Uranyl Acetate.

Although the kidneys comprise a critical target of uranium exposure, the dynamics of renal uranium distribution have remained obscure. Uranium is considered to function physiologically in the form of uranyl ions that have high affinity for phosphate groups. The present study applied microbeam-based elemental analysis to precisely determine the distribution of phosphorus and uranium in the kidneys of male Wistar rats exposed to uranium. One day after a single subcutaneous injection of uranyl acetate (2 mg/kg), areas of concentrated phosphorus were scattered in the S3 segments of the proximal tubule of the kidneys, whereas the S3 segments in control rats and in rats given a lower dose of uranium (0.5 mg/kg) contained phosphorus without concentrated phosphorus. Areas with concentrated phosphorus contained uranium 4- to 14-fold more than the mean uranium concentration (126-472 vs. 33.1 ± 4.6 µg/g). The chemical form of uranium in the concentrated phosphorus examined by XAFS was uranium (VI), suggesting that the interaction of uranyl ions with the phosphate groups of biomolecules could be involved in the formation of uranium concentration in the proximal tubules of kidneys in rats exposed to uranium.

Epigenetic dysregulation of key developmental genes in radiation-induced rat mammary carcinomas.

With the increase in the number of long-term cancer survivors worldwide, there is a growing concern about the risk of secondary cancers induced by radiotherapy. Epigenetic modifications of genes associated with carcinogenesis are attractive targets for the prevention of cancer owing to their reversible nature. To identify genes with possible changes in functionally relevant DNA methylation patterns in mammary carcinomas induced by radiation exposure, we performed microarray-based global DNA methylation and expression profiling in γ-ray-induced rat mammary carcinomas and normal mammary glands. The gene expression profiling identified dysregulation of developmentally related genes, including the downstream targets of polycomb repressive complex 2 (PRC2) and overexpression of enhancer of zeste homolog 2, a component of PRC2, in the carcinomas. By integrating expression and DNA methylation profiles, we identified ten hypermethylated and three hypomethylated genes that possibly act as tumor-suppressor genes and oncogenes dysregulated by aberrant DNA methylation; half of these genes encode developmental transcription factors. Bisulfite sequencing and quantitative PCR confirmed the dysregulation of the polycomb-regulated developmentally related transcription-factor genes Dmrt2, Hoxa7, Foxb1, Sox17, Lhx8, Gata3 and Runx1. Silencing of Hoxa7 was further verified by immunohistochemistry. These results suggest that, in radiation-induced mammary gland carcinomas, PRC2-mediated aberrant DNA methylation leads to dysregulation of developmentally related transcription-factor genes. Our findings provide clues to molecular mechanisms linking epigenetic regulation and radiation-induced breast carcinogenesis and underscore the potential of such epigenetic mechanisms as targets for cancer prevention.

Uranium XAFS analysis of kidney from rats exposed to uranium.

The kidney is the critical target of uranium exposure because uranium accumulates in the proximal tubules and causes tubular damage, but the chemical nature of uranium in kidney, such as its chemical status in the toxic target site, is poorly understood. Micro-X-ray absorption fine-structure (µXAFS) analysis was used to examine renal thin sections of rats exposed to uranyl acetate. The U LIII-edge X-ray absorption near-edge structure spectra of bulk renal specimens obtained at various toxicological phases were similar to that of uranyl acetate: their edge position did not shift compared with that of uranyl acetate (17.175 keV) although the peak widths for some kidney specimens were slightly narrowed. µXAFS measurements of spots of concentrated uranium in the micro-regions of the proximal tubules showed that the edge jump slightly shifted to lower energy. The results suggest that most uranium accumulated in kidney was uranium (VI) but a portion might have been biotransformed in rats exposed to uranyl acetate.

Nationwide survey of radiation exposure during pediatric computed tomography examinations and proposal of age-based diagnostic reference levels for Japan.

BACKGROUND: Diagnostic reference levels (DRLs) have not been established in Japan. OBJECTIVE: To propose DRLs for CT of the head, chest and abdomen for three pediatric age groups. MATERIALS AND METHODS: We sent a nationwide questionnaire by post to 339 facilities. Questions focused on pediatric CT technology, exposure parameters, CT protocols, and radiation doses for age groups <1 year, 1-5 years, and 6-10 years. RESULTS: For the three age groups in the 196 facilities that responded, the 75th percentile values of volume CT dose index based on a 16-cm phantom (CTDIvol 16 [mGy]) for head, chest and abdominal CT were for infants 39.1, 11.1 and 12.0, respectively; for 1-to 5-year-olds 46.9, 14.3 and 16.7, respectively; and for 6-to 10-year-olds 67.7, 15.0 and 17.0, respectively. The corresponding dose­length products (DLP 16 [mGy・cm]) for head, chest and abdominal CT were for infants 526.1, 209.1 and 261.5, respectively; for 1-to 5-year-olds 665.5, 296.0 and 430.8, respectively; and for 6-to 10-year-olds 847.9, 413.0 and 532.2, respectively. CONCLUSION: The majority of CTDIvol 16 and DLP 16 values for the head were higher than DRLs reported from other countries. For risk reduction, it is necessary to establish DRLs for pediatric CT in Japan.

Ionizing radiation, inflammation, and their interactions in colon carcinogenesis in Mlh1-deficient mice.

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and ß-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency.

Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity.

Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney.

Molecular characterization of cancer reveals interactions between ionizing radiation and chemicals on rat mammary carcinogenesis.

Although various mechanisms have been inferred for combinatorial actions of multiple carcinogens, these mechanisms have not been well demonstrated in experimental carcinogenesis models. We evaluated mammary carcinogenesis initiated by combined exposure to various doses of radiation and chemical carcinogens. Female rats at 7 weeks of age were γ-irradiated (0.2-2 Gy) and/or exposed to 1-methyl-1-nitrosourea (MNU) (20 or 40 mg/kg, single intraperitoneal injection) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (40 mg/kg/day by gavage for 10 days) and were observed until 50 weeks of age. The incidence of mammary carcinoma increased steadily as a function of radiation dose in the absence of chemicals; mathematical analysis supported an additive increase when radiation was combined with a chemical carcinogen, irrespective of the chemical species and its dose. Hras mutations were characteristic of carcinomas that developed after chemical carcinogen treatments and were overrepresented in carcinomas induced by the combination of radiation and MNU (but not PhIP), indicating an interaction of radiation and MNU at the level of initiation. The expression profiles of seven classifier genes, previously shown to distinguish two classes of rat mammary carcinomas, categorized almost all examined carcinomas that developed after individual or combined treatments with radiation (1 Gy) and chemicals as belonging to a single class; more comprehensive screening using microarrays and a separate test sample set failed to identify differences in gene expression profiles among these carcinomas. These results suggest that a complex, multilevel interaction underlies the combinatorial action of radiation and chemical carcinogens in the experimental model.

Cancer prevention by adult-onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice.

Children are especially sensitive to ionizing radiation and chemical carcinogens, and limiting their cancer risk is of great public concern. Calorie restriction (CR) is a potent intervention for suppressing cancer. However, CR is generally not appropriate for children. This study, therefore, examined to see if adult-onset CR influences the lifetime cancer risk in mice after early-life exposure to ionizing radiation. Infant male mice (1-week-old) were exposed to 3.8 Gy X-rays, fed a control 95 kcal/week or CR 65 kcal/week diet from 7 weeks of age (adult stage), and their lifespan and tumor development were assessed. Irrespective of CR, X-rays shortened lifespan by 38%, and irrespective of irradiation CR extended lifespan by 20%. Thymic lymphoma (TL) and early-occurring non-TL were induced by radiation. The liver and Harderian gland were more susceptible to radiation-induced tumors than the lungs and non-thymic lymphoid tissues (late occurring). CR reduced the risk of hepatocellular carcinoma, late-occurring non-TL, lung tumor, Harderian tumor, and hemangioma but had less impact on TL and early-occurring non-TL. Most notably, the effects of X-rays on induction of lung tumors, late-occurring non-TL and hemangioma were essentially canceled by CR. The ability of CR to prevent late-occurring tumors was the same for non-irradiated and irradiated mice, indicating that the mechanism by which CR influences cancer is independent of irradiation. Our results indicate that adult-onset CR significantly inhibits late-occurring tumors in a tissue-dependent manner regardless of infant radiation exposure.

[Summary of a survey on radiation exposure during pediatric computed tomography examinations in Japan, focusing on the computed tomography examination environment].

We carried out a nationwide questionnaire survey of pediatric computed tomography (CT) in 339 facilities. Most facilities operated multi detector-row CT (MDCT), and over half operated 64, 128, 256 and 320-slice MDCT. In 32% of facilities, pediatric CT protocols were set taking image quality and dose into consideration. However, in the other facilities, pediatric CT protocols may not be optimized because there is no clear standard for image quality or dosage for pediatric CT examinations in Japan. To promote the optimization of pediatric CT protocols, we regard it as an urgent task to establish diagnostic reference levels for pediatric CT examinations.

Relative Biological Effectiveness of Carbon Ion Beams for Induction of Medulloblastoma with Radiation-specific Chromosome 13 Deletion in Ptch1+/- Mice.

Carbon ion radiotherapy (CIRT) for pediatric cancer is currently limited because of the unknown risk of induction of secondary cancers. Medulloblastoma of Ptch1+/- mice offers a unique experimental system for radiation-induced carcinogenesis, in which tumors are classified into spontaneous and radiation-induced subtypes based on their features of loss of heterozygosity (LOH) that affect the wild-type Ptch1 allele. The present study aims to investigate in young Ptch1+/- mice the carcinogenic effect, and its age dependence, of the low-linear energy transfer (LET, ∼13 keV/µm) carbon ions, to which normal tissues in front of the tumor are exposed during therapy. We irradiated Ptch1+/- mice at postnatal day (P) 1, 4, or 10 with 290 MeV/u carbon ions (0.05-0.5 Gy; LET, 13 keV/µm) and monitored them for medulloblastoma development. Loss of heterozygosity of seven genetic markers on chromosome 13 (where Ptch1 resides) was studied to classify the tumors. Carbon ion exposure induced medulloblastoma most effectively at P1. The LOH patterns of tumors were either telomeric or interstitial, the latter occurring almost exclusively in the irradiated groups, allowing the use of interstitial LOH as a biomarker of radiation-induced tumors. Radiation-induced tumors developed during a narrow age window (most strongly at P1 and only moderately at P4, with suppressed tumorigenesis at P10). Calculated using previous results using 137Cs gamma rays, the values for relative biological effectiveness (RBE) regarding radiation-induced tumors were 4.1 (3.4, 4.8) and 4.3 (3.3, 5.2) (mean and 95% confidence interval) for exposure at P1 and 4, respectively. Thus, the RBE of carbon ions for medulloblastoma induction in Ptch1+/- mice was higher than the generally recognized RBE of 1-2 for cell killing, chromosome aberrations, and skin reactions.

Ikaros is a critical target during simultaneous exposure to X-rays and N-ethyl-N-nitrosourea in mouse T-cell lymphomagenesis.

Cancer risk associated with radiation exposure is considered the result of concurrent exposure to other natural and manmade carcinogens. Available data on the molecular characteristics of cancer after simultaneous exposure to radiation and chemicals are insufficient. In our study, we used a mouse thymic lymphoma (TL) model that was synergistically induced by simultaneous exposure to X-rays and N-ethyl-N-nitrosourea (ENU) at subcarcinogenic doses and analyzed the mutation frequency and spectrum of the TL-associated genes Ikaros, Notch1, p53 and Kras. We found that the point mutation frequency in Ikaros was significantly increased to 47% for simultaneous exposure compared to 13 and 0% for X-ray and ENU exposure alone, respectively. These mutations were mostly G:C > A:T at non-CpG sites and T:A > C:G, both of which are characteristic of ENU mutagenesis. About half of the point mutations were accompanied by loss of heterozygosity (LOH), typical of X-irradiation. The remaining half did not include LOH, which suggests that they were dominant-negative mutations. In Notch1, the frequency of abnormalities was high (>58%) regardless of the treatment, suggesting that Notch1 aberration may be important for T-cell lymphomagenesis. The p53 and Kras mutation frequencies were low for all treatments (<23%). Importantly, the frequency of TLs containing mutations in multiple genes, especially both Ikaros and Notch1, increased after simultaneous exposure. Thus, after simultaneous exposure, Ikaros is a critical target and is inactivated by ENU-induced point mutations and/or X-ray-induced LOH in T-cell lymphomagenesis. Furthermore, concomitant alterations of multiple tumor-associated genes may contribute to enhanced lymphomagenesis after simultaneous exposure.

Co-operative effects of thoracic X-ray irradiation and N-nitrosobis(2-hydroxypropyl) amine administration on lung tumorigenesis in neonatal, juvenile and adult Wistar rats.

Assessment of risks associated with childhood exposure to ionizing radiation when combined with chemical carcinogens is of great importance. We studied the age-dependence of the effect of combined exposure to ionizing radiation (IR) and a chemical carcinogen on lung carcinogenesis. Female 1-, 5-, and 22-week-old Wistar rats were locally irradiated on the thorax with X-rays (3.18 Gy) and/or were injected intraperitoneally with N-nitrosobis(2-hydroxypropyl)amine (BHP) (1g/kg body weight) 1 week after X-ray exposure or at 23 weeks of age. Rats were terminated at 90 weeks of age. We found that: (i) the incidence of lung tumors (adenoma and adenocarcinoma) increased slightly as a function of age at X-ray exposure, although this was not statistically significant, while the incidence induced by BHP decreased with increasing age at administration; (ii) combined exposure to X-rays at 5 or 22 weeks with BHP 1 week later enhanced the tumor incidence, and the effect at early-life stage (5 weeks irradiation) was more effective than that at late-life stage (22 weeks irradiation); (iii) combined exposure preferentially enhanced malignant transformation; (iv) although a longer interval between the X-ray and BHP treatments reduced the combined effect, risks of early-life irradiation at 1 or 5 weeks of age lasted into adulthood; (v) adenomas and adenocarcinomas induced by X-ray and/or BHP originated from surfactant apoprotein A-positive alveolar type II cells; and (vi), extracellular signal-regulated kinase pathway activation was observed in half the adenocarcinomas, regardless of the exposure schedule. In conclusion, combined exposure may enhance lung tumorigenesis more synergistically at early-life stage (5 weeks of age) than later-life stage.