Effects of 2.45 GHz microwaves on meiotic chromosomes of male CBA/CAY mice

Male CBA/CAY mice were exposed daily (6 days a week) for 30 minutes in an environmentally controlled waveguide to continuous 2.45 GHz microwave radiation for 2 weeks at average whole body absorbed dose rates of 0.05, 0.5, 10, and 20 mW/g. Shan exposed animals served as controls. Chain translocations were observed at diakinesis at metaphase I in microwave exposed animals. The yield of translocations increased with exposure, and varied nonlinearly with dose rate. An increase in incidence of univalents was seen after exposure at 10 and 20 mW/g. The findings are interpreted to indicate interference with normal spermatogenesis during the exposure period.     

Lifetime persistence and clonality of chromosome aberrations in the peripheral blood of mice acutely exposed to ionizing radiation

As the measurement of chromosomal translocations increases in popularity for quantifying prior radiation exposure, information on the possible decline of these "stable" aberrations over time is urgently needed. We report here information about the persistence of radiation-induced chromosome aberrations in vivo over the life span of a rodent. Female C57BL/6 mice were given a single whole-body acute exposure of 0, 1, 2, 3 or 4 Gy (137)Cs gamma rays at 8 weeks of age. Chromosome aberrations were analyzed from peripheral blood samples at various intervals between 1 day and 21 months after exposure. Aberrations were detected by painting chromosomes 2 and 8. Translocations decreased dramatically during the first 3 months after irradiation, beyond which time the frequencies remained relatively constant out to 1 year, when the effects of aging and clonal expansion became significant. Both reciprocal and nonreciprocal translocations increased with age in the unexposed control animals and were involved in clones. As expected of unstable aberrations, dicentrics decreased rapidly after exposure and reached baseline levels within 3 months. These results indicate that the persistence of translocations induced by ionizing radiation is complicated by aging and clonal expansion and that these factors must be considered when quantifying translocations at long times after exposure. These results have implications for biological dosimetry in human populations.     

Genetic injury in hybrid male mice exposed to low doses of 60Co gamma-rays or fission neutrons. III. Frequencies of abnormal sperm and reciprocal translocations measured during and following long-term weekly exposures

Male B6CF1 mice were exposed to either fission neutrons or 60Co gamma-rays at once-weekly doses approaching occupational levels for periods up to 60 weeks. Both during and after the irradiation sequence, the mice were screened to determine the incidence of abnormal epididymal sperm and of reciprocal chromosome translocations in metaphase spermatocytes. Abnormal sperm frequencies equilibrated with dose/week by 10 weeks and also showed an additive nonlinear seasonal increment. The relative biological effectiveness (RBE) for these damages is 12 +/- 1. After exposures ended, sperm frequencies in gamma-irradiated mice quickly returned to near-normal levels. Neutron-irradiated males showed a significantly elevated level of abnormalities for approximately 30 weeks--a paradoxical finding--as no clear evidence of cumulative injury was seen during exposure. When assayed at 10 and 25 weeks of exposure but not later, translocation frequencies demonstrated an increment, significant in the neutron series, attributed to irradiated spermatocytes. Dose-response analysis with cumulative dose up to the 60-week maximum gave an RBE of 45 +/- 10. Post exposure, the incidence of translocations subsided slightly, but the RBE remained above 30.     

The mutagenic effect of different types of irradiation on the sex cells of male mice. VIII. Frequency of development of reciprocal translocations in the spermatogonia of mice subjected to chronic gamma-irradiation]

The frequency of reciprocal translocations in mice spermatogonia after the exposure to chronic gamma-irradiation at doses of 100, 200, 300, 600, 920 r, at the dose rate of 4,2 r/day was investigated. It was shown that the mutation frequency increased insignificantly with the increase of the radiation dose (y =0,8+0.0011x). The comparison of the data obtained with earlier results revealed no changes in the yield of translocations at the reduction of the dose rate from 10 r/day to 4,2 r/day. The investigation of the genetic radiosensitivity of mice spermatogonia after a chronic gamma-irradiation showed a tendency to increase in their radioresistance.     

Generation and Characterization of Heritable Reciprocal Translocations in Mice

Reciprocal translocations have provided crucial tools for the localization of genes associated with a variety of human cancers and hereditary diseases. Although heritable translocations are relatively rare in humans, they can be easily induced in mice through exposure of male germ cells at specific spermatogenic stages to different types of radiation and chemicals. Mutagenesis schemes that produce translocations at high frequencies in the progeny of treated males are summarized, and the use of these valuable mutations for analyzing developmental consequences of partial aneuploidy, for identification of mutant genes, and for other purposes is reviewed. Preliminary studies of a large collection of translocation mutants, including several stocks that display dominantly or recessively inherited phenotypes caused by the disruption of critical genes are described. These combined studies demonstrate that several mutagenesis protocols can be used to generate easily mapped, novel mouse mutations with high efficiency and highlight the unique value of reciprocal translocations as tools for gaining access to the biological functions of mammalian genes.     

AID is required for c-myc/IgH chromosome translocations in vivo

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.     

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.     

Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53^−/− mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53^−/− mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53^−/− mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.     

Chromosome rearrangements from spermatogonial chronic neutron irradiation in mice.

Adult male mice were given a range of neutron doses at 80 +/- 20 mrad/h from a plutonium-beryllium source. Cytogenetic analysis indicated that chronic spermatogonial exposure to a mean total dose of 10, 30, 52, 98 or 150 rad produced translocations, sampled in spermatocytes four months later, amounting to 0.32, 0.99. 1.69, 1.91 and 1.65%, respectively. The dose response for the 0-52 rad range was linear. For higher doses, a better fit to the data was an expression with dose exponent above unity.     

Chromosomal polymorphism in the house mouse (Mus domesticus) of Greece and Yugoslavia

A total of 88 wild mice from the Dalmatian coast of Yugoslavia (35 animals), and Peloponnesus (30 animals) and Thebes (23 animals) on mainland Greece were karyotyped. In all but five animals Robertsonian translocations were found. Mice from the Dalmatian region were homozygous for translocations Rb(5.15), Rb(6.12), Rb(8.17), Rb(9.13), and Rb(10.14); they were homo-or heterozygous for the translocation Rb(1.11). Some of them lacked the Rb(1.11) translocation altogether so that the diploid numbers in the Yugoslavian mice were 2n=28, 29, 30, or 40. The mice from the vicinity of Olympia in northwestern Peloponnesus were homozygous for eight Robertsonian translocations: Rb(1.3), Rb(2.5), Rb(4.6), Rb(8.12), Rb(9.16), Rb(10.14), Rb(11.17), and Rb(13.15). Their diploid chromosome number was therefore 2n=24. Mice from the vicinity of Patras in northwest Peloponnesus carried all except the first three of these eight translocations; their chromosome number was 2n=30. Finally, the mice from Thebes were homozygous for translocations Rb(2.15), Rb(4.14), Rb(5.12), and Rb(10.13). They were homo- or heterozygous for Rb(6.9), Rb(8.17), and Rb(1.11); some mice lacked the Tb(1.11) translocation altogether. The translocations Rb(6.9)40Tu and Rb(10.13)42Tu represent new arm combinations not found previously in any wild mouse population. the remaining translocations have previously been found in different Mediterranean countries, in Scotland and in southern Germany. The findings suggest that each translocation arose only once and that different translocations have come together in different populations to generate a unique karyotype characterizing this population.     

Low-dose ionizing radiation and chromosome translocations: a review of the major considerations for human biological dosimetry

Chromosome translocations are a molecular signature of ionizing radiation exposure. Translocations persist significantly longer after exposure than other types of chromosome exchanges such as dicentrics. This persistence makes translocations the preferred aberration type for performing radiation dosimetry under conditions of protracted exposure or when exposure assessments are temporally delayed. Low doses of radiation are inherently difficult to quantify because the frequency of induced events is low and the background level of translocations among unexposed subjects can show considerable variability. Analyses of translocation frequencies can be confounded by several factors, including age of the subject, lifestyle choices such as cigarette smoking, the presence of clones of abnormal cells, and possibly genotypic variability among subjects. No significant effects of gender or race have been observed, but racial differences have not been completely ruled out. Translocation analyses may be complicated by the presence of different types of exchanges, i.e., reciprocal or non-reciprocal, and because translocations sometimes occur as a component of complex exchanges that include other forms of chromosome rearrangements. Rates of radiation exposure, ranging from acute to chronic, are known to influence the accumulation of translocations and may also affect their persistence. The influences on translocation frequencies of low-dose radiation hypersensitivity as well as the bystander effect and the adaptive response remain poorly characterized. Thus, quantifying the relationship between radiation dose and the frequency of translocations in any given subject requires attention to multiple issues. Part of the solution to understanding the in vivo dose-response relationship is to have accurate estimates of the baseline levels of translocations in healthy unexposed subjects, and some work in this area has been accomplished. Long-term cytogenetic follow-up of exposed subjects is needed to characterize translocation persistence, which is especially relevant for risk analyses. More work also needs to be done in the area of quantifying the role of known confounders. Characterizing the role of genotype will be especially important. Improvements in the ability to use translocation frequencies for low-dose biological dosimetry will require scoring very large numbers of cells per subject, which may be accomplished by developing a rapid automated image analysis system. This work would enhance our comprehension of the effects of low-dose radiation exposure and could lead to significant improvements in understanding the relationship between chromosome damage and human health.     

The cellular lethality of radiation-induced chromosome translocations in human lymphocytes

Recent evidence has shown that translocation frequencies decline over time. This phenomenon might be explained by the co-occurrence of translocations in cells that also contain dicentrics, in which case translocations would be eliminated as a by-product of selection against dicentrics. Alternatively, a fraction of translocations may themselves be lethal. Here we describe our initial approaches to develop mathematical models to test whether the decline in translocation frequencies results from the first, the second, or a combination of these two possibilities. The models assumed that all chromosome exchanges were simple, i.e., were comprised of dicentrics as well as one-way and two-way translocations. Complex aberrations (three or more breaks in two or more chromosomes) were not modeled, nor were fragments or intrachromosomal exchanges (rings, inversions). We tested the models using Monte Carlo simulations, and then we fitted the models to data describing chromosome aberration frequencies induced by a single acute in vitro exposure to (137)Cs gamma rays in human peripheral blood lymphocytes from two donors. Chromosome painting was used to enumerate translocations and dicentrics from 2 to 7 days after exposure. Our results indicate that in donor no. 2, the decline in translocation frequencies occurs as a by-product of selection against dicentrics. However, in donor no. 1, whose cells appeared more radiosensitive than cells from donor no. 2, up to 40% of the one-way translocations may themselves be lethal at high doses, although calculations indicate that two-way translocations do not cause lymphocyte mortality. Individual variation in the probability that translocations are lethal to cells appears to be important, and one-way translocations appear to be lethal more often than two-way translocations. Within the limits of these models, these findings indicate that both postulated mechanisms, i.e. inherent lethality and selection against dicentrics in the same cells, contribute to the loss of both one-way and two-way translocations.     

Enhanced recovery of radiation-induced translocations from spermatogonial stem cells of p53 null mice

X-ray-induced (4Gy) chromosomal translocations were studied in mouse spermatogonial stem cells with different p53 status by meiotic analysis at the spermatocyte stage, many cell generations after the moment irradiation. The results show enhanced recovery of translocations from p53 -/- mice relative to +/- and +/+ littermates. The enhanced recovery is probably due to an altered cell cycle distribution of the stem cells in the -/- mice leading to less radioresistant G(0)-G(1) transition cells, rather than differences in apoptotic response. Experiments with the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide (3-AB) indicate that, in contrast to the situation in +/+ mice, no sensitization in the p53-deficient mice occurred for both testis weight loss and the recovery of induced translocations. This result also points to the presence of less radioresistant stem cells in the testis of p53 null mice.     

Mutagenic effect of different types of irradiation on the sex cells of male mice. IX. Duration of preservation of radiation-induced reciprocal translocations during the course of the reproductive period]

The rate of induction of reciprocal translocations by gamma-irradiation of mouse spermatogonia was studied by cytological examination of descendent spermatocytes. The CBA mice were given a total single acute dose of 300 r or 3 times per 300 r with 7 and 28 days intervals. The irradiated mice were killed within 3,7 and 12 month after the irradiation. The frequency of translocations in 3 and 7 month after the treatment was the same. A 25% decrease in the yield of reciprocal translocations was observed in 12 months after the irradiation. It suggests that the genetic risk from ionizing radiation remains high a year after the irradiation.     

Effects of stock differences in the acceleration of puberty in female mice

Newly born TO strain female mice were exposed daily to the urine from male albino mice of the same and CFLP strains, from feral mice carrying Robertsonian translocation chromosomes and to water as a control condition. At 21 days of age, when exposure was discontinued, there were differences in body weight between treatments which were not present when adult. Exposure to urine from mice with Robertsonian translocations did not accelerate puberty and the interval between vaginal opening and first oestrus was longer (4.2 days) than in mice exposed to the urine from the albino strains (1.8 days). Mice exposed to the urine from the Robertsonian stock were in dioestrus more often than those exposed to the urine from laboratory strains. The Robertsonian mice also differed in their behaviour in an open arena in that they passed fewer faecal pellets than those exposed to the urine from the albino mice. The water control mice defecated the least frequently. The mice exposed to the Robertsonian urine were less active than the laboratory strains but the differences did not reach an acceptable level (P less than 0.06) of significance.     

Causes of oncogenic chromosomal translocation

Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematologic malignancies and childhood sarcomas. In addition to their diagnostic utility, chromosomal translocations are increasingly being used in the clinic to guide therapeutic decisions. However, the mechanisms that cause these translocations remain poorly understood. Illegitimate V(D)J recombination, class switch recombination, homologous recombination, non-homologous end-joining and genome fragile sites all have potential roles in the production of non-random chromosomal translocations. In addition, mutations in DNA-repair pathways have been implicated in the production of chromosomal translocations in humans, mice and yeast. Although initially surprising, the identification of these same oncogenic chromosomal translocations in peripheral blood from healthy individuals strongly suggests that the translocation is not sufficient to induce malignant transformation, and that complementary mutations are required to produce a frank malignancy.     

The effect of chronic exposure to cadmium and gamma radiation at low doses on the genetic structures in mice

The DNA-protein cross-links (DPC) in mouse thymocytes and spleen lymphocytes, the number of abnormal sperm heads (ASH) and the number of micronuclei (MN) in normochromatic erythrocytes (NCE) of peripheral blood were studied in mice exposed to long-term low-intensity gamma radiation (0.072 cGy/days) and/or cadmium with drinking water (0.01 mg Cd2+/l) for 20, 40 and 80 days. The dependence of DPC level on the total dose (exposure time) of gamma radiation and/or cadmium is nonlinear. The maximal level of DPC in cells of lymphoid organs upon exposure to gamma radiation or cadmium was recorded on the 40-th day, and under combined exposure on the 20-th day of the experiment. The long-term exposure to cadmium or gamma radiation causes an increase in the ASH frequency. The increase in frequencies of MN in NCE and reciprocal translocations in spermatocytes was not found.     

Response of mouse spermatogonial stem cells to X-ray induction of heritable reciprocal translocations

Although heritable translocations are an important endpoint for the assessment of genetic risk from radiation, there has been a serious information gap with regard to thier induction in spermatogonical stem cells, the most important cell stage in males for risk considerations. This led to uncertainty in estimating the magnitude of risk per unit exposure. Further, the relationship between the frequency of r eciprocal exchanges scored by cytological analysis of the exposed male's meiocytes and the frequency of those transmitted to first-generation offspring needed to be re-examined. In order to fill in these gaps, two radiation studies, i.e., dose response and dose fractionation, were conducted on spermatogonial stem cells in which heritable and cytologically detected translocations were scored.The present data are by far the most extensive, to date, for heritable translocation induction in spermatogonial stem cells. The linearity of the rising portion of the dose-effect curve and the additivity of effects observed in the fractionation study allow a direct estimation of the number of transmissible translocations expected per unit exposure. Thus,t he expected increase in heritable translocations per rad of acute X-rays in 3.89 × 10^?5 per gamete. The data also show a lack of consistensy between cytologically and genetically scored translocations.     

Increased frequency of chromosome translocations associated with diagnostic x-ray examinations

Informative studies of cancer risks associated with medical radiation are difficult to conduct owing to low radiation doses, poor recall of diagnostic X rays, and long intervals before cancers occur. Chromosome aberrations have been associated with increased cancer risk and translocations are a known radiation biomarker. Seventy-nine U.S. radiologic technologists were selected for blood collection, and translocations were enumerated by whole chromosome painting. We developed a dose score to the red bone marrow for medical radiation exposure from X-ray examinations reported by the technologists that they received as patients. Using Poisson regression, we analyzed translocations in relation to the dose scores. Each dose score unit approximated 1 mGy. The estimated mean cumulative red bone marrow radiation dose score was 42 (range 1-265). After adjustment for age, occupational radiation, and radiotherapy for benign conditions, translocation frequencies significantly increased with increasing red bone marrow dose score with an estimate of 0.007 translocations per 100 CEs per score unit (95% CI, 0.002 to 0.013; P = 0.01). Chromosome damage has been linked with elevated cancer risk, and we found that cumulative radiation exposure from medical X-ray examinations was associated with increased numbers of chromosome translocations.     

Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse

Transgenic mice have been used to explore the role of chromosomal translocations in the genesis of tumors. But none of these efforts has actually involved induction of a translocation in vivo. Here we report the use of Cre recombinase to replicate in vivo the t(8;21) translocation found in human acute myeloid leukemia (AML). As in the human tumors, the murine translocation fuses the genes AML1 and ETO. We used homologous recombination to place loxP sites at loci that were syntenic with the break points for the human translocation. Cre activity was provided in mice by a transgene under the control of the Nestin promoter, or in cultured B cells by infecting with a retroviral vector encoding Cre. In both instances, Cre activity mediated interchromosomal translocations that fused the AML1 and ETO genes. Thus, reciprocal chromosomal translocations that closely resemble rearrangements found in human cancers can be achieved in mice.