Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells.

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Long term depleted uranium exposure in Gulf War I veterans does not cause elevated numbers of micronuclei in peripheral blood lymphocytes.

Depleted uranium (DU) is a high density heavy metal that has been used in military munitions since the 1991 Gulf War. DU is weakly radioactive and chemically toxic. Long term exposure can cause adverse health effects. This study assessed genotoxic effects in DU exposed Gulf War I veterans as a function of uranium (U) body burden. Levels of urine U were used to categorize the cohort into low and high exposure groups. Exposure to DU occurred during friendly fire incidents in 1991 involving DU munitions resulting in inhalation and ingestion exposure to small particles of DU and soft tissue DU fragments from traumatic injuries. All of these Veterans are enrolled in a long term health surveillance program at the Baltimore Veterans Administration Medical Center. Blood was drawn from 35 exposed male veterans aged 36-59 years, then cultured and evaluated for micronuclei (MN) using the cytokinesis block method. The participants were divided into two exposure groups, low and high, based on their mean urine uranium (uU) concentrations. Poisson regression analyses with mean urine U concentrations, current smoking, X-rays in the past year and donor age as dependent variables revealed no significant relationships with MN frequencies. Our results indicate that on-going systemic exposure to DU occurring in Gulf War I Veterans with DU embedded fragments does not induce significant increases in MN in peripheral blood lymphocytes compared to MN frequencies in Veterans with normal U body burdens.

Increased frequency of chromosome translocations in airline pilots with long-term flying experience.

BACKGROUND: Chromosome translocations are an established biomarker of cumulative exposure to external ionising radiation. Airline pilots are exposed to cosmic ionising radiation, but few flight crew studies have examined translocations in relation to flight experience. METHODS: We determined the frequency of translocations in the peripheral blood lymphocytes of 83 airline pilots and 50 comparison subjects (mean age 47 and 46 years, respectively). Translocations were scored in an average of 1039 cell equivalents (CE) per subject using fluorescence in situ hybridisation (FISH) whole chromosome painting and expressed per 100 CE. Negative binomial regression models were used to assess the relationship between translocation frequency and exposure status and flight years, adjusting for age, diagnostic x ray procedures, and military flying. RESULTS: There was no significant difference in the adjusted mean translocation frequency of pilots and comparison subjects (0.37 (SE 0.04) vs 0.38 (SE 0.06) translocations/100 CE, respectively). However, among pilots, the adjusted translocation frequency was significantly associated with flight years (p = 0.01) with rate ratios of 1.06 (95% CI 1.01 to 1.11) and 1.81 (95% CI 1.16 to 2.82) for a 1- and 10-year incremental increase in flight years, respectively. The adjusted rate ratio for pilots in the highest compared to the lowest quartile of flight years was 2.59 (95% CI 1.26 to 5.33). CONCLUSIONS: Our data suggests that pilots with long-term flying experience may be exposed to biologically significant doses of ionising radiation. Epidemiological studies with longer follow-up of larger cohorts of pilots with a wide range of radiation exposure levels are needed to clarify the relationship between cosmic radiation exposure and cancer risk.

Retrospective assessment of radiation exposure using biological dosimetry: chromosome painting, electron paramagnetic resonance and the glycophorin a mutation assay.

Biological monitoring of dose can contribute important, independent estimates of cumulative radiation exposure in epidemiological studies, especially in studies in which the physical dosimetry is lacking. Three biodosimeters that have been used in epidemiological studies to estimate past radiation exposure from external sources will be highlighted: chromosome painting or FISH (fluorescence in situ hybridization), the glycophorin A somatic mutation assay (GPA), and electron paramagnetic resonance (EPR) with teeth. All three biodosimeters have been applied to A-bomb survivors, Chernobyl clean-up workers, and radiation workers. Each biodosimeter has unique advantages and limitations depending upon the level and type of radiation exposure. Chromosome painting has been the most widely applied biodosimeter in epidemiological studies of past radiation exposure, and results of these studies provide evidence that dose-related translocations persist for decades. EPR tooth dosimetry has been used to validate dose models of acute and chronic radiation exposure, although the present requirement of extracted teeth has been a disadvantage. GPA has been correlated with physically based radiation dose after high-dose, acute exposures but not after low-dose, chronic exposures. Interindividual variability appears to be a limitation for both chromosome painting and GPA. Both of these techniques can be used to estimate the level of past radiation exposure to a population, whereas EPR can provide individual dose estimates of past exposure. This paper will review each of these three biodosimeters and compare their application in selected epidemiological studies.

Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

Fish cytogenetics and the future of radiation biodosimetry.

Fluorescence in situ hybridisation (FISH) with whole chromosome paints has greatly facilitated the analysis of structural chromosome aberrations and has led to translocations replacing dicentrics as the aberration of choice for many applications. Major challenges remain if we are to go from translocations to an understanding of the health consequences of radiation exposure. Yet to be surmounted are the roles of individual susceptibility, time since exposure, and the effects of subjects age. Accomplishing these objectives will require automation, reduced costs, improved calibration, and extensive use of baseline samples.

Evaluation of three somatic genetic biomarkers as indicators of low dose radiation effects in clean-up workers of the Chernobyl nuclear reactor accident.

The goals of this study were to assess three biomarkers of genetic effect for their individual and collective ability to detect and estimate radiation exposure in Russian Chernobyl clean-up workers. Work assignments were planned to limit dose to 0.25 Gy. The three biomarkers employed were chromosome translocations detectcd in lynmphocytes by florescence in situ hybridisation (FISH), and mutation at two genes, glycophorin A (GPA) in red blood cells detected by flow cytometry and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes detected by selective cell culture. Samples were Obtained from 1992 to 2000. The time between exposure at Chernobyl and sample acquisition was > or =5 years. The lymphocyte assays detected an elevation over controls in average outcomes it clean-up workers: translocation rates were 46% higher when adjusted for age and smoking and HPRT mutant frequencies were were 16% higher when adjusted for age. The G PA assay did not detect an exposure effect. The results indicate that measuring frequency of translocations by FISH is preferred for low dose radiation, retrospective biochemistry.

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses.

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWRxC57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0cGy 137Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.

The relationship between genotype and chromosome aberration frequencies in a normal adult population.

Cancer susceptibility differences may be attributed in part to genetic variation in genes involved in metabolism of environmental procarcinogens. Increased risks for some cancers have been linked to polymorphisms in certain phase I and II genes, and have been associated with genomic instability and chromosomal aberrations. Aberration frequencies in general, and stable aberration frequencies (translocations and insertions) in particular, are used as biomarkers for disease. Thus, knowledge of the genetic factors that influence the frequency of stable aberrations in a normal population is important for cancer risk determination. In this work, genotypes for a number of xenobiotic enzymes (CYPIA1, CYP2D6, GSTM1, GSTT1, GSTP1, NAT1, NAT2 and epoxide hydrolase) and stable aberration frequencies were determined for 65 normal individuals aged 19-77 years. The population was divided at age 60 years for analysis because there was a significant difference in stable aberration frequencies between these groups. Subjects with low levels (0-66th percentile) of stable aberrations were compared to those with high levels (67th percentile and above). Of all the genotypes studied, only NAT2 showed a notable difference between the high and the low stable aberration groups in the percentage of polymorphisms observed, and this was seen only in the older subjects group. All individuals in the older-high stable aberration group were NAT2 rapid acetylator smokers. NAT2 slow acetylator smokers had significantly lower stable aberration frequencies compared to the NAT2 rapid acetylator smokers. Following previous work showing an increased risk of cancer associated with high levels of aberrations (above the 66th percentile), we hypothesize that smokers with the NAT2 rapid acetylator genotype may be at an increased risk for cancer.

[Effect of age and radiation exposure on the frequency of translocations and dicentrics detected by FISH method in human lymphocytes]. / Vliianie vozrasta i oblucheniia na chastotu translokatsii i ditsentrikov, opredeliaemykh metodom FISH, v limfotsitakh cheloveka.

The frequencies of translocations and dicentrics detected by "chromosome painting" in lymphocytes were estimated in 115 healthy donors and in 273 people exposed to uncontrolled irradiation at low doses 1-4 years ago. Age responses of both types of exchanges at the age range from 3 to 85 years fit to quadratic model. The frequency of translocations grew faster with age than the frequency of dicentrics. The yields of stable exchanges in exposed people was significantly higher than those in control donors of corresponding ages.

Role of maternal exposures and newborn genotypes on newborn chromosome aberration frequencies.

Maternal exposures may induce chromosome damage and birth defects in the fetus. Polymorphic variation in genes coding for enzymes involved in metabolic activation and detoxification of environmental procarcinogens may account for some of the differences in chromosome aberration frequencies in newborns. In this study, 40 mothers completed questionnaires regarding exposures they received during their pregnancy. Umbilical cord blood samples were analyzed for chromosome aberrations. An average of 1020 metaphase cell equivalents (equal to 1020 G-banded cells) were examined from each newborn. In 26 of the newborns, genotyping analysis was performed for genes functioning in metabolic activation and detoxification (cytochrome P450 genes: CYP2D6 and CYP1A1, and phase II genes: NAT1, NAT2, GSTT1, GSTM1, GSTP1, and epoxide hydrolase). A significant association between the CYP1A1 MspI polymorphism and chromosome aberration frequencies was observed in the newborns (p=0.02), with heterozygotes showing higher aberration frequencies than the wild type homozygotes. Some large differences in chromosome aberration frequencies for other genotypes were also noted, but these were not statistically significant. Exposure to tobacco smoke in utero also appeared to increase translocation frequencies. The mean frequency of translocations per 100 cell equivalents from newborns of mothers who smoked during pregnancy was significantly higher than that of newborns whose mothers did not smoke (0.21 vs. 0.11, respectively, p=0.045).

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.

Biological dosimetry of chernobyl cleanup workers: inclusion of data on age and smoking provides improved radiation dose estimates.

We report the results of a study of chromosome translocations in 126 Russian subjects who participated in the cleanup activities at Chernobyl and another 53 subjects, from other places in Russia, who were not exposed at Chernobyl. In agreement with our earlier study, we find increased translocation frequencies among the exposed compared to Russian controls. We describe statistical methods for estimating the dose of ionizing radiation determined by scoring chromosome translocations found in circulating lymphocytes sampled several years after exposure. Two statistical models were fitted to the data. One model assumed that translocation frequencies followed an overdispersed Poisson distribution. The second model assumed that translocation frequencies followed a negative binomial distribution. In addition, the effects of radiation exposure were modeled as additive or as multiplicative to the effects of age and smoking history. We found that the negative binomial model fit the data better than the overdispersed Poisson model. We could not distinguish between the additive and the multiplicative model with our data. Individual dose estimates ranged from 0 (for 43 subjects) to 0.56 Gy (mean 0.14 Gy) under the multiplicative model and from 0 to 0.95 Gy (mean 0.15 Gy) under the additive model. Dose estimates were similar under the two models when the number of translocations was less than 4 per 100 cells. The additive model tended to estimate larger doses when the number of translocations was greater than 4 per 100 cells. We also describe a method for estimating upper 95% tolerance bounds for numbers of translocations in unexposed individuals. We found that inclusion of data on age and smoking history was important for dose estimation. Ignoring these factors could result in gross overestimation of exposures, particularly in older subjects who smoke.

Estimation of the target stem-cell population size in chronic myeloid leukemogenesis.

Estimation of the number of hematopoietic stem cells capable of causing chronic myeloid leukemia (CML) is relevant to the development of biologically based risk models of radiation-induced CML. Through a comparison of the age structure of CML incidence data from the Surveillance, Epidemiology, and End Results (SEER) Program and the age structure of chromosomal translocations found in healthy subjects, the number of CML target stem cells is estimated for individuals above 20 years of age. The estimation involves three steps. First, CML incidence among adults is fit to an exponentially increasing function of age. Next, assuming a relatively short waiting time distribution between BCR-ABL induction and the appearance of CML, an exponential age function with rate constants fixed to the values found for CML is fitted to the translocation data. Finally, assuming that translocations are equally likely to occur between any two points in the genome, the parameter estimates found in the first two steps are used to estimate the number of target stem cells for CML. The population-averaged estimates of this number are found to be 1.86x10(8) for men and 1.21x10(8) for women; the 95% confidence intervals of these estimates are (1.34x10(8), 2. 50x10(8)) and (0.84x10(8), 1.83x10(8)), respectively.

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development.

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.

Radiation-induced breakpoint misrejoining in human chromosomes: random or non-random?

PURPOSE: To investigate whether radiation-induced misrejoining of chromosome breakpoints is randomly or non-randomly distributed throughout the human genome. MATERIALS AND METHODS: Data were combined from as many published cytogenetic studies as possible. The percentage of radiation-induced breaks per megabase (Mb) of DNA between all human chromosomes was calculated, and the observed and expected numbers of breakpoints based on DNA content between and within chromosomes were compared. RESULTS: A DNA-proportional distribution of breakpoints in 14 autosomes and a statistically significant deviation from proportionality in the other eight autosomes and the sex chromosomes was found. Regression analysis showed no significant change in breakpoint frequency per Mb of DNA relative to autosome size. Analysis between chromosome arms showed a non-random distribution of induced breakpoints within certain autosomes, particularly the acrocentrics. In cases of non-random distributions, a prevalence of events was found at heterochromatic regions and/or telomeres, and a clustering of breakpoints was found near the centromeres of many chromosomes. CONCLUSIONS: There is an approximately linear proportionality between autosomal DNA content and observed breakpoint number, suggesting that subsets of autosomes can be used to estimate accurately the overall genomic frequency of misrejoined breakpoints contingent upon a carefully selected subset. However, this conclusion may not apply to the sex chromosomes. The results also support the influence of chromatin organization and/or preferential DNA repair/misrejoining on the distribution of induced breakpoints. However, these effects are not sufficient at a global level to dismiss the value of cytogenetic analysis using a genome subset for biodosimetry.

Detection of structural chromosome damage in rat interphase cells using region-specific fluorescence in situ hybridization probes developed by microdissection.

Cytogenetic analysis using fluorescence in situ hybridization (FISH) was employed to detect structural chromosome aberrations in interphase. We generated DNA probes specific for rat chromosome regions 1q11-12, 1q31-35 and 1q51-53 by microdissection and degenerate oligonucleotide-primed PCR. Targeted regions were labeled in unique colors by FISH. Abnormal cells were identified on the basis of alterations in the physical distance between the hybridization signals. To evaluate the ability of these probes to quantify chromosome aberrations in interphase, rats were acutely exposed whole-body to 0, 1, 2, 3 or 4 Gy of 137Cs gamma rays. Eight days later, peripheral blood, bone marrow, lung and pancreas were removed and hybridized with the probes. Multi-color FISH analysis showed dose-responsive frequencies of abnormal interphase nuclei in peripheral blood and bone marrow cells. In lung and pancreas, on the contrary, no increase in the frequency of the abnormal interphases was observed. However, chromosome damage was observed when primary lung cells, obtained from rats irradiated 8 days previously, were cultured for three days. Detection of rearranged signals after in vitro tissue culture was attributed to the movement of chromosome domains that accompanies mitosis. The use of region-specific painting probes appears useful for detecting structural chromosome damage in interphase cells of rat tissues, although further optimization is still needed to improve the method.

Chronic ingestion of clastogens by mice and the frequency of chromosome aberrations.

Environmental exposure to mutagens is believed to play a significant role in human carcinogenesis. Determination of the in vivo effects of a single mutagen is best done in laboratory animals because humans are exposed to a variety of mutagens both in their diet and in the rest of their environment. In this study, C57BL/6N female mice were used to analyze the effect on chromosomes of chronic ingestion of a mutagen dissolved in drinking water. Cyclophosphamide (CP) or urethane (ethyl carbamate, EC) were dissolved in sterile drinking water at concentrations of 0, 32, 64, and 96 ppm or 0, 5,000, 10,000, and 15,000 ppm, respectively. All exposures began at 8 weeks of age and continued through the 20th week unless terminated earlier due to toxicity. Body weights and water consumption were measured weekly. Blood and bone marrow were taken from approximately five mice per exposure group at 4, 8, and 12 weeks from the start of exposure. All mice remaining after 12 weeks received drinking water without any carcinogen for an additional 6 weeks to determine if induced aberrations persisted. Chromosome translocations, measured by painting, were not induced in blood or bone marrow cells at any time point for either chemical. However, both carcinogens induced significant increases in micronucleated normochromatic erythrocytes, indicating that the carcinogens reached the tissues examined in these experiments. These results indicate that chronic exposure of mice to chemical carcinogens induces chromosome breakage measurable by micronuclei. However, the breakage and reunion necessary to see chromosome exchanges such as translocations were not observed in this study.