Frequencies of Chromosome Aberrations are Lower in Splenic Lymphocytes from Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays Compared with Non-Irradiated Control Mice.

Chromosome aberrations have been one of the most sensitive and reliable biomarkers of exposure to ionizing radiation. Using the multiplex fluorescence in situ hybridization (M-FISH) technique, we compared the changes, over time, in the frequencies of translocations and of dicentric chromosomes in the splenic lymphocytes from specific pathogen-free (SPF) C3H/HeN female mice continuously exposed to 0.05 mGy/day (18.25 mGy/year) gamma rays for 125 to 700 days (total accumulated doses: 6.25-35 mGy) with age-matched non-irradiated controls. Results show that the frequencies of translocations and of dicentric chromosomes increased significantly over time in both irradiated and non-irradiated control mice, and that the frequencies were significantly lower, not higher, in the irradiated mice, which differs from our previous reports of increased chromosome aberration frequencies at higher radiation dose rates of 1 mGy/day and 20 mGy/day. These results will be useful when considering the radiation risk at very low-dose rates comparable to regulatory dose limits.

Frequencies of Chromosome Aberrations are Lower in Splenic Lymphocytes from Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays Compared with Non-Irradiated Control Mice.

Chromosome aberrations have been one of the most sensitive and reliable biomarkers of exposure to ionizing radiation. Using the multiplex fluorescence in situ hybridization (M-FISH) technique, we compared the changes, over time, in the frequencies of translocations and of dicentric chromosomes in the splenic lymphocytes from specific pathogen-free (SPF) C3H/HeN female mice continuously exposed to 0.05 mGy/day (18.25 mGy/year) gamma rays for 125 to 700 days (total accumulated doses: 6.25-35 mGy) compare with age-matched non-irradiated controls. Results show that the frequencies of translocations and of dicentric chromosomes increased significantly over time in both irradiated and non-irradiated control mice, and that the frequencies were significantly lower, not higher, in the irradiated mice, which differs from our previous reports of increased chromosome aberration frequencies at higher radiation dose rates of 1 mGy/day and 20 mGy/day. These results will be useful when considering the radiation risk at very low-dose rates comparable to regulatory dose limits.

Life Span, Cause of Death and Neoplasia in B6C3F1 Mice Exposed In Utero to Low- and Medium-Dose-Rate Gamma Rays.

Previously, we reported that while low-dose-rate (LDR) gamma-ray exposure to 20 mGy/day for the entire gestation period (gestation days 0-18) did not result in any significant effect in B6C3F1 pups up to 10 weeks of age when compared to the non-irradiated controls, exposure to medium-dose-rates (MDR, 200 and 400 mGy/day) resulted in growth retardation and gonadal hypoplasia, in addition to delayed ossification (only at 400 mGy/day). In the present work, we investigated the late effects of continuous in utero exposure to gamma rays at LDRs (0.05, 1.0 and 20 mGy/day) and at an MDR of 400 mGy/day, on life span, causes of death, neoplastic and non-neoplastic disease incidences in B6C3F1 mice. Reproductive parameters such as litter size and weaning rates was not significantly different among the LDR groups, but was significantly decreased in the MDR group, when compared to the non-irradiated controls. Mean life spans were not significantly different among the LDR exposed groups compared to the non-irradiated controls, whereas the life spans of those exposed to the MDR were significantly shorter than the non-irradiated controls. There was no significant difference in tumor spectra between the non-irradiated and LDR nor MDR irradiated groups. In mice exposed to MDR in utero, the over-all incidence rates shifted with increased incidences in the number of neoplasms of liver (both sexes) and endocrine (adrenals, pituitary and ovaries in females) origin with corresponding decreases in the incidence of malignant lymphomas (both sexes) and lung neoplasms (males). Multiple primary neoplasms were significantly increased only in females exposed to MDR. Results show that B6C3F1 mice exposed to gamma-rays in utero at LDRs of 0.05, 1 and 20 mGy/day for the entire gestation period (18 days) does not significantly alter lifespan, cause of death, neoplasm incidence rates and tumor spectra.

LOW DOSE-RATE RADIATION-SPECIFIC ALTERATIONS FOUND IN A GENOME-WIDE GENE EXPRESSION ANALYSIS OF THE MOUSE LIVER.

Life span shortening and increased incidences of cancer and non-cancer diseases were observed in B6C3F1 mice irradiated with gamma-rays at a low dose-rate (LDR) of 20 mGy/d for 400 d. A genome-wide gene expression profiling of livers from mice irradiated at a LDR (20 mGy/d, 100-400 d) was performed. LDR radiation affected specific pathways such as those related to lipid metabolism, e.g. 'Cholesterol biosynthesis' and 'Adipogenesis' in females irradiated for 200 and 300 d at 20 mGy/d, with increased expression of genes encoding cholesterol biosynthesis enzymes (Cyp51, Sqle, Fdps) as age and radiation dose increased. No significant alterations in the expression of these genes were observed in male mice exposed similarly. However, the genes encoding adipogenesis regulators, Srebf1 and Pparg, increased with age and radiation dose in both sexes. Comparison between LDR-irradiated and medium dose-rate (400 mGy/d) male mice revealed quite different gene expression profiles. These results seem to be consistent with the increased incidence of fatty liver and obesity in female mice exposed to LDR radiation and suggest that metabolism is an important target of LDR radiation.

ADAPTIVE RESPONSE IN MICE CONTINUOUSLY IRRADIATED WITH LOW DOSE-RATE RADIATION.

Previous reports showed a reduction in hematopoietic death in mice exposed to a high (challenge) radiation dose if exposed two weeks prior with a relatively small (priming) radiation dose (0.3-0.5 Gy). This in vivo acquisition of radioresistance, known as "adaptive response" or the "Yonezawa effect," was shown in the experiments performed using high dose-rates (HDR) for priming. In the present study, we used low (LDR) and medium dose-rates (MDR) of radiation for priming in male C57BL mice. A total dose of 0.45-0.46 Gy (LDR, 20 mGy/day × 23 days or MDR, 18 mGy/hour × 25 hours) was used for priming, and was followed by challenge exposure 12 days later at an HDR (0.8 Gy/min) to a total dose of 6.75 Gy. Increased survival rates were observed in mice exposed to priming radiation delivered at LDR or MDR, suggesting that the adaptive responses induced are comparable with those induced at HDR.

Increased Frequency of Copy Number Variations Revealed by Array Comparative Genomic Hybridization in the Offspring of Male Mice Exposed to Low Dose-Rate Ionizing Radiation.

There is very little information on the transgenerational or genetic effects of low dose-rate ionizing radiation. We report the detection of the transgenerational effects of chronic low dose-rate irradiation in mice, at the molecular level in the whole genome, using array comparative genomic hybridization technology. We observed that the number of the mice with de novo copy number variations (specifically, deletions) was significantly increased in the offspring of C57BL/6J male mice exposed to 20 mGy/day gamma-rays for 400 days (total dose: 8000 mGy), as compared to non-irradiated controls. We did not detect any difference in the size of the de novo deletions between the irradiated and the non-irradiated groups. An analysis of the life span of the offspring suggested a possibility that de novo copy-number variations may be associated with shorter life spans.

Effects of Continuous In Utero Low- and Medium-Dose-Rate Gamma-Ray Exposure on Fetal Germ Cells.

The effects of radiation exposure on germ cells and the gonads have been well studied at acute high-dose exposures, but the effects of chronic low-dose-rate (LDR) irradiation, particularly relevant for radiation protection, on germ cells and the gonads are largely unknown. Our previous study revealed that chronic exposure of mice to medium-dose-rate (MDR, 200 or 400 mGy/day) gamma-rays in utero for the entire gestation period (18 days) induced only a mild degree of general growth retardation, but with very drastic effects on the gonads and germ cells. In the current study, we further investigated the histomorphological changes in the gonads and the number of germ cells from gestation day (GD) 18 fetuses irradiated with MDR throughout the entire gestation period. The germ cells in the testes and ovaries of the MDR-irradiated fetuses were almost obliterated. Gestation day 18 fetuses exposed to LDR (20 mGy/day) radiation for the entire gestation period showed decreases in the number of the germ cells, which were not statistically significant or only marginally significant at most. Further investigations on the effects of LDR irradiation in utero using more sensitive methods are necessary.

Life-Shortening Effect of Chronic Low-Dose-Rate Irradiation in Calorie-Restricted Mice.

Calorie restriction is known to influence several physiological processes and to alleviate the late effects of radiation exposure such as neoplasm induction and life shortening. However, earlier related studies were limited to acute radiation exposure. Therefore, in this study we examined the influence of chronic low-dose-rate irradiation on lifespan. Young male B6C3F1/Jcl mice were divided randomly into two groups, which were fed either a low-calorie (65 kcal/ week) or high-calorie (95 kcal/week) diet. The latter is comparable to ad libitum feeding. The animals in the irradiated group were continuously exposed to gamma rays for 400 days at 20 mGy/day, resulting in a total dose of 8 Gy. Exposure and calorie restriction were initiated at 8 weeks of age and the diets were maintained for life. The life-shortening effects from chronic whole-body irradiation were compared between the groups. Body weights were reduced in calorie-restricted mice irrespective of radiation treatment. Radiation induced a shortened median lifespan in both groups, but to a greater extent in the calorie-restricted mice. These results suggest that calorie restriction may sensitize mice to chronic low-dose-rate radiation exposure to produce a life-shortening effect rather than alleviating the effects of radiation.

Minimum environmental enrichment is effective in activating antitumor immunity to transplanted tumor cells in mice.

Studies of environmental enrichment are progressing in the fields of nervous system, stress and exercise. Recently, housing in enriched environment have shown to influence to carcinogenesis and life span. However, the study for antitumor effect of environmental enrichment are difficult to reproduce due to the complexity of the experimental technique. Thus, a simpler experiment system is needed for antitumor study using environmental enrichment. In this research, we propose a minimum environmental enrichment, which is an experimental system by placing one mouse igloo which is normally used as a mouse shelter in the rearing environment. The experimental system of minimum environmental enrichment is not only easy to reproduce but also have enhanced activity to suppress the growth of transplanted tumor significantly. It was found that the activation of NK cells is involved also in the immune system related to tumor immunity of minimum environmental enrichment. Because minimum environmental enrichment is effective in activating antitumor immunity to transplanted tumor cells in mice, we believe this will be useful for promoting antitumor studies using environmental enrichment.

Chronic exposure to gamma irradiation at low-dose rates accelerates blood pressure decline associated with aging in female B6C3F1 mice.

PURPOSE: Many studies are focusing on the biological effects of gamma irradiation at low-dose rates. Studies have shown that chronic exposure to gamma irradiation at low-dose rates shortened the lifespan of mice due to neoplasm formation. The aim of this study was to clarify the physiological effects of long-term exposure to gamma irradiation at low-dose rates in mice, measured with noninvasive parameters such as blood pressure. MATERIALS AND METHODS: Specific-pathogen-free female B6C3F1 mice were irradiated with gamma rays at a low dose of 20 mGy/day - a dose rate shown to shorten the life span in previous studies. The blood pressure parameters (systolic, diastolic, and mean blood pressure), heart rate, tail blood volume, and blood flow of the mice were measured every 7 weeks. Age-matched, non-irradiated mice were used as controls. RESULTS AND CONCLUSION: The blood pressure levels of the irradiated mice decreased at an earlier age compared to the non-irradiated control mice. The expression levels of the marker genes of aging that are also associated with regulation of blood pressure showed significant differences between non-irradiated and irradiated mice. These results indicated that long-term exposure to gamma irradiation at low-dose rates induce the expression levels of Rap1a and reduces Panx1 and Sirt3, which may have contributed to the accelerated blood pressure decline in female mice.

Experimental studies on the biological effects of chronic low dose-rate radiation exposure in mice: overview of the studies at the Institute for Environmental Sciences.

This review summarizes the results of experiments conducted in the Institute for Environmental Sciences for the past 21 years, focusing on the biological effects of long-term low dose-rate radiation exposure on mice. Mice were chronically exposed to gamma rays at dose-rates of 0.05, 1 or 20 mGy/day for 400 days to total doses of 20, 400 or 8000 mGy, respectively. The dose rate 0.05 mGy/day is comparable to the dose limit for radiation workers. The parameters examined were lifespan, neoplasm incidence, antineoplasm immunity, body weight, chromosome aberration(s), gene mutation(s), alterations in mRNA and protein levels and trans-generational effects. At 20 mGy/day, all biological endpoints were significantly altered except neoplasm incidence in the offspring of exposed males. Slight but statistically significant changes in lifespan, neoplasm incidences, chromosome abnormalities and gene expressions were observed at 1 mGy/day. Except for transient alterations in the mRNA levels of some genes and increased liver neoplasm incidence attributed to radiation exposure, the remaining biological endpoints were not influenced after exposure to 0.05 mGy/day. Results suggest that chronic low dose-rate exposure may induce small biological effects.

Screening of biomarkers for liver adenoma in low-dose-rate γ-ray-irradiated mice.

PURPOSE: Chronic low-dose-rate (20 mGy/day) γ-irradiation increases the incidence of hepatocellular adenomas (HCA) in female B6C3F1 mice. The purpose of this study is to identify potential serum biomarkers for these HCAs by a new approach. MATERIAL AND METHODS: Microarray analysis were performed to compare the gene expression profiles of HCAs from mice exposed to low-dose-rate γ-rays with those of normal livers from non-irradiated mice. From the differentially expressed genes, those for possibly secretory proteins were selected. Then, the levels of the proteins in sera were analysed by ELISA. RESULTS: Microarray analysis identified 4181 genes differentially expressed in HCAs (>2.0-fold). From these genes, those for α-fetoprotein (Afp), α-1B-glycoprotein (A1bg) and serine peptidase inhibitor Kazal type-3 (Spink3) were selected as the genes for candidate proteins. ELISA revealed that the levels of Afp and A1bg proteins in sera significantly increased and decreased, respectively, in low-dose-rate irradiated mice with HCAs and also same tendency was observed in human patients with hepatocellular carcinomas. CONCLUSION: These results indicate that A1bg could be a new serum biomarker for liver tumor. This new approach of using microarray to select genes for secretory proteins is useful for prediction of novel tumor markers in sera.

Preservation of chromosomal integrity in murine spermatozoa derived from gonocytes and spermatogonial stem cells surviving prenatal and postnatal exposure to γ-rays in mice.

Pre- and postnatal male mice were acutely (659-690 mGy/min) and continuously (0.303 mGy/min) exposed to 2 Gy γ-rays to evaluate spermatogenic potential and chromosome damage in their germ cells as adults. Acute irradiation on Days 15.5, 16.5, and 17.5 post-coitus affected testicular development, as a result of massive quiescent gonocyte loss; the majority of the seminiferous tubules in these testes were devoid of germ cells. Acute irradiation on Days 18.5 and 19.5 post-coitus had less effect on testicular development and spermatogenesis, even though germ cells were quiescent gonocytes on these days. Adverse effects on testicular development and spermatogenesis were observed following continuous irradiation between Days 14.5 and 19.5 post-coitus. Exposure to acute and continuous postnatal irradiation after the differentiation of spermatogonial stem cells and spermatogonia resulted in nearly all of the seminiferous tubules exhibiting spermatogenesis. Neither acute nor continuous irradiation was responsible for the increased number of multivalent chromosomes in primary-spermatocyte descendents of the exposed gonocytes. In contrast, a significant increase in cells with multivalent chromosomes was observed following acute irradiation on Days 4 and 11 post-partum. No significant increases in unstable structural chromosomal aberrations or aneuploidy in spermatozoa were observed, regardless of cell stage at irradiation or the radiation dose-rate. Thus, murine germ cells that survive prenatal and postnatal irradiation can restore spermatogenesis and produce viable spermatozoa without chromosome damage. These findings may provide a better understanding of reproductive potential following accidental, environmental, or therapeutic irradiation during the prenatal and postnatal periods in humans.

Role of the Msh2 gene in genome maintenance and development in mouse fetuses.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5 dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5 dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin.

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.

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.

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.

Induction of mitosis delay, apoptosis and aneuploidy in human cells by phenyl hydroquinone, an Ames test-negative carcinogen.

Ortho-phenyl phenol and its hepatic derivative, phenyl hydroquinone, do not generate base-substitution-type mutations, but cause bladder cancer in rats and mice. The mechanism of their carcinogenic effect is unknown. We have previously shown that o-phenyl phenol and phenyl hydroquinone induce mitotic arrest and aneuploidy in Saccharomyces cerevisiae. To further delineate the mechanism of action of phenyl hydroquinone, we examined its effect on human cells. Treatment of the colon cancer cell line HCT116 with 0 to 150 microM phenyl hydroquinone caused a concentration-dependent inhibition of growth, accumulation of cells having G2/M DNA content, and an increase in the mitotic index. Moreover, a dose-dependent increase in apoptotic cells was observed. Finally, a high frequency of aneuploid cells was found. On the other hand, no increase in gamma-H2AX foci was observed. The results show that phenyl hydroquinone does induce mitotic arrest, apoptosis and aneuploidy in the absence of DNA damage. Our results may be useful to understand the mechanisms of action of chemical substances that are Ames test-negative carcinogens.

Absence of Ku70 gene obliterates X-ray-induced lacZ mutagenesis of small deletions in mouse tissues.

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.

UVA1 genotoxicity is mediated not by oxidative damage but by cyclobutane pyrimidine dimers in normal mouse skin.

UVA1 induces the formation of 8-hydroxy-2'-deoxyguanosines (8-OH-dGs) and cyclobutane pyrimidine dimers (CPDs) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA1 has not been clarified. We irradiated living mouse skin with 364-nm UVA1 laser light and analyzed the DNA damage formation and mutation induction in the epidermis and dermis. Although dose-dependent increases were observed for both 8-OH-dG and CPD, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C --> T transition at a dipyrimidine site. Moreover, these UV-specific mutations occurred preferentially at the 5'-TCG-3' sequence, suggesting that CpG methylation and photosensitization-mediated triplet energy transfer to thymine contribute to the CPD-mediated UVA1 genotoxicity. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA1 exposure. We also found differences in the responses to the UVA1 genotoxicity between the epidermis and the dermis: the mutation induction after UVA1 irradiation was suppressed in the dermis at all levels of irradiance examined, whereas it leveled off from a certain high irradiance in the epidermis.