Influence of Age on Leukemia Mortality Associated with Exposure to γ rays and 2-MeV Fast Neutrons in Male C3H Mice.

The relative biological effectiveness (RBE) of densely ionizing radiation can depend on the biological context. From a radiological perspective, age is an important factor affecting health risks of radiation exposure, but little is known about the modifying impact of age on the effects of densely ionizing radiation. Herein, we addressed the influence of age on leukemogenesis induced by accelerator-generated fast neutrons (mean energy, ∼2 MeV). Male C3H/HeNrs mice were exposed to 137Cs γ rays (0.2-3.0 Gy) or neutrons (0.0485-0.97 Gy, γ ray contamination 0.0105-0.21 Gy) at 1, 3, 8, or 35 weeks of age and observed over their lifetimes under specific pathogen-free conditions. Leukemia and lymphoma were diagnosed pathologically. Hazard ratio (HR) and RBE for myeloid leukemia mortality as well as the age dependence of these two parameters were modeled and analyzed using Cox regression. Neutron exposure increased HR concordant with a linear dose response. The increase of HR per dose depended on age at exposure, with no significant dose dependence at age 1 or 3 weeks but a significant increase in HR of 5.5 per Gy (γ rays) and 16 per Gy (neutrons) at 8 weeks and 5.8 per Gy (γ rays) and 9 per Gy (neutrons) at 35 weeks. The RBE of neutrons was 2.1 (95% confidence interval, 1.1-3.7), with no dependence on age. The development of lymphoid neoplasms was not related to radiation exposure. The observed increasing trend of radiation-associated mortality of myeloid leukemia with age at exposure supports previous epidemiological and experimental findings. The results also suggest that exposure at the susceptible age of 8 or 35 weeks does not significantly influence the RBE value for neutrons for induction of leukemia, unlike what has been documented for breast and brain tumors.

Interstitial deletion of the Apc locus in ß-catenin-overexpressing cells is a signature of radiation-induced intestinal tumors in C3B6F1 ApcMin/+ mice†.

Recent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanning Apc in a subset of sporadic and radiation-induced intestinal tumors of ApcMin/+ mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1 ApcMin/+ mice that were either left untreated or irradiated with 2 Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable ß-catenin, which is a hallmark of Apc+ allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving the Apc+ allele in ß-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involving Apc+ was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving the Apc+ allele as a causative radiation-related event in intestinal tumors of ApcMin/+ mice, providing an accurate approach for attributing individual tumors to radiation exposure.

Lung-Cancer Risk in Mice after Exposure to Gamma Rays, Carbon Ions or Neutrons: Egfr Pathway Activation and Frequent Nuclear Abnormality.

Lung is one of the high-risk organs for radiation-induced carcinogenesis, but the risk of secondary lung-cancer development after particle-beam therapy and the underlying mechanism(s) remain to be elucidated. To investigate the effects of particle-beam radiation on adjacent normal tissues during cancer therapy, 7-week-old male and female B6C3F1 mice were irradiated with 0.2-4 Gy of gamma rays (for comparison), carbon ions (290 MeV/u, linear energy transfer 13 keV/µm), or fast neutrons (0.05-1 Gy, mean energy, ∼2 MeV), and lung-tumor development was assessed by histopathology. Mice irradiated with ≥2 Gy of carbon ions or ≥0.2 Gy of neutrons developed lung adenocarcinoma (AC) significantly sooner than did non-irradiated mice. The relative biological effectiveness values for carbon ions for lung AC development were 1.07 for male mice and 2.59 for females, and the corresponding values for neutrons were 4.63 and 4.57. Genomic analysis of lung ACs revealed alterations in genes involved in Egfr signaling. Hyperphosphorylation of Erk and a frequent nuclear abnormality (i.e., nuclear groove) were observed in lung ACs of mice irradiated with carbon ions or neutrons compared with ACs from non-irradiated or gamma-ray-irradiated groups. Our data indicate that the induction of lung AC by carbon ions occurred at a rate similar to that for gamma rays in males and approximately 2-to 3-fold greater than that for gamma rays in females. In contrast, the effect of neutrons on lung AC development was approximately 4- to 5-fold greater than that of gamma rays. Our results provide valuable information concerning risk assessment of radiation-induced lung tumors after particle-beam therapy and increase our understanding of the molecular basis of tumor development.

High Relative Biological Effectiveness of 2 MeV Fast Neutrons for Induction of Medulloblastoma in Ptch1+/- Mice with Radiation-specific Deletion on Chromosome 13.

Neutron radiation, a high-linear energy transfer radiation, has a high relative biological effectiveness (RBE) for various end points. The age at exposure is an important modifier of the effects of radiation, including carcinogenesis, with infants being generally more radiosensitive. Ptch1+/- mice offer a unique experimental system for assessing radiation carcinogenesis. Spontaneous development of medulloblastoma tumors occurs in nonirradiated animals that lose their Ptch1+ allele, most frequently by a loss of heterozygosity (LOH) of chromosome 13 via recombination or non-disjunction (referred to as S-type tumors). In contrast, tumors occur in irradiated Ptch1+/- mice as a result of chromosome 13 LOH with an interstitial deletion (R-type), making spontaneous and radiation-induced tumors discernible. To elucidate the influence of age on the effect of fast neutrons, we irradiated Ptch1+/- mice with neutrons (mean energy, ∼2 MeV) or γ rays on embryonic day (E)14 and E17 and on postnatal day (P)1, 4 or 10 and classified the resulting medulloblastomas based on chromosome 13 aberrations. Instead of LOH, some tumors harbored mutations in their Ptch1+ gene via a nonirradiation-associated mechanism such as duplication, insertion, base substitution or deletion with microhomology-mediated end joining; thus, these tumors were classified as S-type. The RBE regarding the induction of R-type tumors was 12.9 (8.6, 17.2), 9.6 (6.9, 12.3), 21.5 (17.2, 25.8), and 7.1 (4.7, 9.5) (mean and 95% confidence interval) for mice irradiated on E14, E17, P1 and P4, respectively, with the highest value seen during the most active development of the tissue and P10 being completely resistant. These results indicate that the developmental stage at exposure of the tissue influences the RBE of neutrons.

Calorie Restriction Suppresses the Progression of Radiation-Induced Intestinal Tumours in C3B6F1 Apc Min/+ Mice.

BACKGROUND/AIM: Progress in cancer treatment and diagnosis has made second cancer after medical radiation exposure a particular concern among childhood cancer survivors. Calorie restriction (CR) is a broadly effective cancer prevention strategy, although its effects on radiation-induced intestinal tumours are unclear. Here we examined the cancer-preventative efficacy of a CR diet at different starting ages on radiation induction of intestinal tumours in mice. MATERIALS AND METHODS: Male C3B6F1 ApcMin/+ mice were irradiated with 0 or 2 Gy of X-rays at 2 weeks of age. After an interval of 2, 8 or 18 weeks, mice were fed with a non-CR (95 kcal/week/mouse) or CR (65 kcal/week/mouse) diet. Intestinal tumours were evaluated for number, size distribution and malignancy. RESULTS: CR suppressed the size and progression of both spontaneous and radiation-induced intestinal tumours depending on age at starting of CR. CR diets were effective even administered to adult mice. CONCLUSION: CR was effective for suppression of tumour progression, which was accelerated by radiation exposure. Use of CR might be a useful cancer-prevention strategy for radiation-induced tumours of the intestinal tract.

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

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

The effect of age at exposure on the inactivating mechanisms and relative contributions of key tumor suppressor genes in radiation-induced mouse T-cell lymphomas.

Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult-irradiation group, with Ikaros frequently (50%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc-finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult-irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2a and Ikaros are a prominent feature of young adult irradiation-induced T-cell lymphoma, tumors arising after irradiation from infancy suffer a second hit in Pten by mis-segregation or recombination. This is the first report showing an influence of age-at-exposure on genomic alterations of tumor suppressor genes and their relative involvement in radiation-induced T-cell lymphoma. These data are important for considering the risks associated with childhood exposure to radiation.

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.

Genomic and gene expression signatures of radiation in medulloblastomas after low-dose irradiation in Ptch1 heterozygous mice.

Accurate cancer risk assessment of low-dose radiation poses many challenges that are partly due to the inability to distinguish radiation-induced tumors from spontaneous ones. To elucidate characteristic features of radiation-induced tumors, we analyzed 163 medulloblastomas that developed either spontaneously or after X-ray irradiation at doses of 0.05-3 Gy in Ptch1 heterozygous mice. All spontaneous tumors showed loss of heterozygosity in broad regions on chromosome 13, with losses at all consecutive markers distal to Ptch1 locus (S-type). In contrast, all tumors that developed after 3 Gy irradiation exhibited interstitial losses around Ptch1 with distal markers retained (R-type). There was a clear dose-dependent increase in the proportion of R-type tumors within the intermediate dose range, indicating that the R-type change is a reliable radiation signature. Importantly, the incidence of R-type tumors increased significantly (P = 0.007) at a dose as low as 50 mGy. Integrated array-comparative genomic hybridization and expression microarray analyses demonstrated that expression levels of many genes around the Ptch1 locus faithfully reflected the signature-associated reduction in genomic copy number. Furthermore, 573 genes on other chromosomes were also expressed differently between S-type and R-type tumors. They include genes whose expression changes during early cerebellar development such as Plagl1 and Tgfb2, suggesting a recapitulation of gene subsets functioning at distinct developmental stages. These findings provide, for the first time, solid experimental evidence for a significant increase in cancer risk by low-dose radiation at diagnostic levels and imply that radiation-induced carcinogenesis accompanies both genomic and gene expression signatures.