Kazumi Yamauchi

Differential effects of low- and high-dose X-rays on N-ethyl-N-nitrosourea-induced mutagenesis in thymocytes of B6C3F1 gpt-delta mice.

Carcinogenesis in humans is thought to result from exposure to numerous environmental factors. Little is known, however, about how these different factors work in combination to cause cancer. Because thymic lymphoma is a good model of research for combined exposure, we examined the occurrence of mutations in thymic DNA following exposure of B6C3F1 gpt-delta mice to both ionizing radiation and N-ethyl-N-nitrosourea (ENU). Mice were exposed weekly to whole body X-irradiation (0.2 or 1.0 Gy), ENU (200 ppm) in the drinking water, or X-irradiation followed by ENU treatment. Thereafter, genomic DNA was prepared from the thymus and the number and types of mutations in the reporter transgene gpt was determined. ENU exposure alone increased mutant frequency by 10-fold compared to untreated controls and over 80% of mutants had expanded clonally. X-irradiation alone, at either low or high dose, unexpectedly, reduced mutant frequency. Combined exposure to 0.2 Gy X-rays with ENU dramatically decreased mutant frequency, specifically G:C to A:T and A:T to T:A mutations, compared to ENU treatment alone. In contrast, 1.0 Gy X-rays enhanced mutant frequency by about 30-fold and appeared to accelerate clonal expansion of mutated cells. In conclusion, repeated irradiation with 0.2 Gy X-rays not only reduced background mutation levels, but also suppressed ENU-induced mutations and clonal expansion. In contrast, 1.0 Gy irradiation in combination with ENU accelerated clonal expansion of mutated cells. These results indicate that the mode of the combined mutagenic effect is dose dependent.

Mild inflammation accelerates colon carcinogenesis in Mlh1-deficient mice

Objective: Inflammatory bowel disease, which frequently accompanies silencing of Mlh1, plays a key role in the pathogenesis of colorectal cancer. The interaction between inflammation and mismatch repair deficiency, however, remains unclear. The aim of this study was to determine the effect of inflammation on colorectal carcinogenesis in Mlh1-deficient mice. Method: Inflammatory colitis was induced by treatment with 1% dextran sodium sulfate (DSS) in drinking water for 1 week in Mlh1 knockout (Mlh1–/–), Mlh1 heterozygous (Mlh1+/–) and wild-type (Mlh1+/+) mice at 10 weeks of age. The development of colon tumors was followed for a subsequent 15 weeks and the tumors were analyzed immunohistochemically for the expression and localization of iNOS, β-catenin and p53. Results: Male and female Mlh1–/– mice with DSS showed a 63 and 44% incidence of tumors, respectively, whereas no tumors were observed in Mlh1+/– and Mlh1+/+ mice. The mice without DSS treatment did not develop any tumors regardless of the genotype. While aberrant expression of β-catenin was not detected in colonic neoplasms, p53 and iNOS expression was increased in 100 and 77%, respectively. These immunohistochemical changes were consistent with those of human colon cancers associated with ulcerative colitis. Conclusion: Our results indicate that Mlh1 deficiency strongly accelerates colon carcinogenesis when combined with inflammation. Thus the cells with Mlh1 deficiency, either inherently or colitis associated, may be at an increased risk of cancer under inflammatory conditions.

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.

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.

Combined exposure to X-irradiation followed by N-ethyl-N-nitrosourea treatment alters the frequency and spectrum of Ikaros point mutations in murine T-cell lymphoma.

Ionizing radiation is a well-known carcinogen, but its potency may be influenced by other environmental carcinogens, which is of practical importance in the assessment of risk. Data are scarce, however, on the combined effect of radiation with other environmental carcinogens and the underlying mechanisms involved. We studied the mode and mechanism of the carcinogenic effect of radiation in combination with N-ethyl-N-nitrosourea (ENU) using doses approximately equal to the corresponding thresholds. B6C3F1 mice exposed to fractionated X-irradiation (Kaplans method) followed by ENU developed T-cell lymphomas in a dose-dependent manner. Radiation doses above an apparent threshold acted synergistically with ENU to promote lymphoma development, whereas radiation doses below that threshold antagonized lymphoma development. Ikaros, which regulates the commitment and differentiation of lymphoid lineage cells, is a critical tumor suppressor gene frequently altered in both human and mouse lymphomas and shows distinct mutation spectra between X-ray- and ENU-induced lymphomas. In the synergistically induced lymphomas, we observed a low frequency of LOH and an inordinate increase of Ikaros base substitutions characteristic of ENU-induced point mutations, G:C to A:T at non-CpG, A:T to G:C, G:C to T:A and A:T to T:A. This suggests that radiation doses above an apparent threshold activate the ENU mutagenic pathway. This is the first report on the carcinogenic mechanism elicited by combined exposure to carcinogens below and above threshold doses based on the mutation spectrum of the causative gene. These findings constitute a basis for assessing human cancer risk following exposure to multiple carcinogens.

Complicated biallelic inactivation of Pten in radiation-induced mouse thymic lymphomas

Inactivation of the phosphatase and tensin homolog gene (Pten) occurs via multiple tissue-dependent mechanisms including epigenetic silencing, point mutations, insertions, and deletions. Although frequent loss of heterozygosity around the Pten locus and plausible involvement of epigenetic silencing have been reported in radiation-induced thymic lymphomas, the proportion of lymphomas with inactivated Pten and the spectrum of causal aberrations have not been extensively characterized. Here, we assessed the mode of Pten inactivation by comprehensive analysis of the expression and alteration of Pten in 23 radiation-induced thymic lymphomas developed in B6C3F1 mice. We found no evidence for methylation-associated silencing of Pten; rather, complex structural abnormalities comprised of missense and nonsense mutations, 1- and 3-bp insertions, and focal deletions were identified in 8 of 23 lymphomas (35%). Sequencing of deletion breakpoints suggested that aberrant V(D)J recombination and microhomology-mediated rearrangement were responsible for the focal deletions. Seven of the 8 lymphomas had biallelic alterations, and 4 of them did not express Pten protein. These Pten aberrations coincided with downstream Akt phosphorylation. In conclusion, we demonstrate that Pten inactivation is frequently biallelic and is caused by a variety of structural abnormalities (rather than by epigenetic silencing) and is involved in radiation-induced lymphomagenesis.

Tissue‐specific and time‐dependent clonal expansion of ENU‐induced mutant cells in gpt delta mice

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N‐ethyl‐N‐nitrosourea (ENU) with the persistence of ENU‐induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU‐induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592–606, 2017.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.

Combined Effect of Ionizing Radiation and N-Ethyl-N-Nitrosourea on Mutation Induction and Lymphoma Development

Carcinogenesis in humans is thought to result from exposure to numerous environmental factors. Little is known, however, about how these different factors work in combination to cause cancer. Mouse thymic lymphoma is a good model for research on radiation and chemical carcinogenesis. We examined here the occurrence of thymic lymphoma and mutation induction following exposure to both X-rays and N-ethyl-N-nitrosourea (ENU) in B6C3F1 mice. Mice were exposed weekly to whole-body X-irradiation (0.2 or 1.0 Gy per each exposure) for 4 consecutive weeks, ENU (200 ppm) in the drinking water for 4 weeks, or X-irradiation followed by ENU treatment. The incidence of lymphoma after 0.2 and 1.0 Gy were 0% and 10%, respectively. ENU treatment induced lymphoma in 20% of exposed mice. When ENU was combined with 1.0 Gy, lymphoma incidence increased up to 94%, showing a synergistic effect. In contrast, combination of ENU with 0.2 Gy resulted in a decrease in lymphoma incidence, that is, an antagonistic effect. Mutant frequency of the reporter transgene gpt after ENU exposure alone increased by tenfold compared to untreated controls. Combined exposure of ENU with 0.2 Gy X-rays dramatically decreased mutant frequency. In contrast, 1.0 Gy X-rays combined with ENU further enhanced mutant frequency and accelerated clonal expansion of mutated cells. In conclusion, the mutagenic and carcinogenic effect of combined exposure of X-rays with ENU is dose dependent.