Chromosome Aberrations in a Group of People Exposed to Radioactive Releases from the Three Mile Island Nuclear Accident and Inferences for Radiation Effects.

Recently, it has been proposed that the doses received from 133Xe released during the accident in 1979 at the Three Mile Island (TMI) plant in Pennsylvania were much higher than has been conventionally assessed, due to a gross underestimation of the relative biological effectiveness of electrons from beta-particle-emitting radionuclides within the body. The central evidence cited in support of this proposal was the doses derived from cytogenetic analyses of blood sampled in the mid-1990s from people living near TMI at the time of the accident. However, the chromosome aberration data show a marked discrepancy in biodosimetric estimates evaluated from the frequencies of stable translocations and unstable dicentrics (corrected for temporal attenuation), strongly suggesting that exposures to clastogenic agents occurred long after the TMI accident. Few details have been reported on the people providing the blood samples and how they were selected for study. Crucially, this lack of information includes the distributions in the exposed and control groups of age at sampling, which is a critical factor in interpreting translocation data. Contrary to the recent claim, these cytogenetic data offer no support to the suggestion of a serious underestimation of internal doses from beta particles or from 133Xe discharged during the TMI accident.

Chromosomal Abnormalities in Offspring of Young Cancer Survivors: A Population-Based Cohort Study in Denmark.

To examine whether cancer survivors diagnosed before age 35 years are more likely to have offspring with chromosomal abnormalities than their siblings, chromosomal abnormalities were determined in a population-based cohort of 14 611 offspring (14 580 live-born children and 31 fetuses) of 8945 Danish cancer survivors and 40 859 offspring (40 794 live-born children and 65 fetuses) of 19 536 siblings. Chromosomal abnormalities include numeric and structural abnormalities. Odds ratios were estimated by multiple logistic regression models comparing the risk of chromosomal abnormalities among survivors' offspring with that in siblings' offspring. In a subgroup of survivors with gonadal radiation doses (mean = 0.95 Gy for males and 0.91 Gy for females), no indication of a dose response was found. Overall, no increased risk of chromosomal abnormalities among survivors' offspring was observed compared with their siblings' offspring (odds ratio = 0.99, 95% confidence interval = 0.67 to 1.44, two-sided P = .94), with similar risk between male and female survivors. Cancer survivors were not more likely than their siblings to have children with a chromosomal abnormality.

Chromosome analysis in a case of a plutonium contaminated wound.

Chromosome analysis of peripheral blood lymphocytes was undertaken over a 10 year period following an intake of plutonium through a hand wound. Frequencies of cells with unstable complex aberrations remained high throughout this time, probably reflecting direct exposure of lymphocytes as they passed plutonium which had transferred to regional lymph nodes. Analysis at the final sampling time also revealed cells with stable aberrations at a much higher frequency relative to the number of unstable cells than expected from direct exposure, and is therefore most likely to be reflecting exposure to lymphocyte precursor cells from plutonium that has become deposited on bone surfaces.

Chromosome aberrations determined by sFISH and G-banding in lymphocytes from workers with internal deposits of plutonium.

PURPOSE: To examine the influence of α-particle radiation exposure from internally deposited plutonium on chromosome aberration frequencies in peripheral blood lymphocytes of workers from the Sellafield nuclear facility, UK. MATERIALS AND METHODS: Chromosome aberration data from historical single colour fluorescence in situ hybridization (sFISH) and Giemsa banding (G-banding) analyses, together with more recent sFISH results, were assessed using common aberration analysis criteria and revised radiation dosimetry. The combined sFISH group comprised 29 men with a mean internal red bone marrow dose of 21.0 mGy and a mean external γ-ray dose of 541 mGy. The G-banding group comprised 23 men with a mean internal red bone marrow dose of 23.0 mGy and a mean external γ-ray dose of 315 mGy. RESULTS: Observed translocation frequencies corresponded to expectations based on age and external γ-ray dose with no need to postulate a contribution from α-particle irradiation of the red bone marrow by internally deposited plutonium. Frequencies of stable cells with complex aberrations, including insertions, were similar to those in a group of controls and a group of workers with external radiation exposure only, who were studied concurrently. In a similar comparison there is some suggestion of an increase in cells with unstable complex aberrations and this may reflect recent direct exposure to circulating lymphocytes. CONCLUSIONS: Reference to in vitro dose response data for the induction of stable aberrant cells by α-particle irradiation indicates that the low red bone marrow α-particle radiation doses received by the Sellafield workers would not result in a discernible increase in translocations, thus supporting the in vivo findings. Therefore, the greater risk from occupational radiation exposure of the bone marrow resulting in viable chromosomally aberrant cells comes from, in general, much larger γ-ray exposure in comparison to α-particle exposure from plutonium.

Chromosome Aberrations Determined by FISH in Radiation Workers from the Sellafield Nuclear Facility.

Workers from the Sellafield nuclear facility (Cumbria, UK) with occupational exposures to external sources of ionizing radiation were examined for translocation frequencies in peripheral blood lymphocytes using fluorescence in situ hybridization (FISH). This is an extension of an earlier study of retired workers, and includes analyses of additional samples from the earlier collection, bringing the total to 321. Another 164 samples from both current and retired employees, including 26 repeat samples, were obtained from a new collection, thus giving a combined dataset of 459 workers. This all-male population of workers was divided into 6 dose groups comprising 97 with recorded external occupational doses <50 mGy, 118 with 50-249 mGy, 129 with 250-499 mGy, 89 with 500-749 mGy, 17 with 750-999 mGy and 9 with >1,000 mGy. Univariate analysis showed a significant association between external dose and translocation frequency (P < 0.001) with the estimate of slope ± standard error being 1.174 ± 0.164 × 10(-2) translocations per Gy. Multivariate analysis revealed that age increased the rate of translocations by 0.0229 ± 0.0052 × 10(-2) per year (P < 0.001). However, the impact of age adjustment on the radiation dose response for translocation frequencies was minor with the new estimate of slope ± standard error being 1.163 ± 0.162 × 10(-2) translocations per Gy. With the dose response for the induction of translocations by chronic in vivo low-LET radiation now well characterized, cytogenetic analysis can play an integral role in retrospective dose reconstruction of chronic exposure in epidemiological studies of exposed populations.

Chromosome aberrations in workers with exposure to α-particle radiation from internal deposits of plutonium: expectations from in vitro studies and comparisons with workers with predominantly external γ-radiation exposure.

mFISH analysis of chromosome aberration profiles of 47 and 144 h lymphocyte cultures following exposure to 193 mGy α-particle radiation confirmed that the frequency of stable aberrant cells and stable cells carrying translocations remains constant through repeated cell divisions. Age-specific rates and in vitro dose-response curves were used to derive expected translocation yields in nine workers from the Mayak nuclear facility in Russia. Five had external exposure to γ-radiation, two of whom also had exposure to neutrons, and four had external exposure to γ-radiation and internal exposure to α-particle radiation from incorporated plutonium. Doubts over the appropriateness of the dose response used to estimate translocations from the neutron component made interpretation difficult in two of the workers with external exposure, but the other three had translocation yields broadly in line with expectations. Three of the four plutonium workers had translocation yields in line with expectations, thus supporting the application of the recently derived in vitro α-particle dose response for translocations in stable cells. Overall this report demonstrates that with adequate reference in vitro dose-response curves, translocation yield has the potential to be a useful tool in the validation of red bone marrow doses resulting from mixed exposure to external and internal radiation.

Germline minisatellite mutations in workers occupationally exposed to radiation at the Sellafield nuclear facility.

Germline minisatellite mutation rates were investigated in male workers occupationally exposed to radiation at the Sellafield nuclear facility. DNA samples from 160 families with 255 offspring were analysed for mutations at eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern hybridisation. No significant difference was observed between the paternal mutation rate of 5.0% (37 mutations in 736 alleles) for control fathers with a mean preconceptional testicular dose of 9 mSv and that of 5.8% (66 in 1137 alleles) for exposed fathers with a mean preconceptional testicular dose of 194 mSv. Subgrouping the exposed fathers into two dose groups with means of 111 mSv and 274 mSv revealed paternal mutation rates of 6.0% (32 mutations in 536 alleles) and 5.7% (34 mutations in 601 alleles), respectively, neither of which was significantly different in comparisons with the rate for the control fathers. Maternal mutation rates of 1.6% (12 mutations in 742 alleles) for the partners of control fathers and 1.7% (19 mutations in 1133 alleles) for partners of exposed fathers were not significantly different. This study provides evidence that paternal preconceptional occupational radiation exposure does not increase the germline minisatellite mutation rate and therefore refutes suggestions that such exposure could result in a destabilisation of the germline that can be passed on to future generations.

mFISH analysis of chromosome aberrations induced in vitro by α-particle radiation: examination of dose-response relationships.

A multicolored FISH (mFISH) technique was used to characterize the cytogenetic damage associated with exposure to α-particle radiation with particular emphasis on the quality and quantity that is likely to be transmitted through cell division to descendant cells. Peripheral blood lymphocytes were irradiated in vitro with (238)Pu α particles with a range of mean doses up to 936 mGy and were cultured for 47 h. The dose responses for total aberrant cells, stable and unstable cells, and cells with one simple chromosome aberration and multiple chromosome aberrations were predominantly linear for doses that resulted in cell nuclei receiving a single α-particle traversal. However, there was a decrease per unit dose in aberrant cells of all types at higher doses because of cells increasingly receiving multiple traversals. The proportion of radiation-induced aberrant cells containing multiple aberrations ranged from 48 to 74% with little evidence of dose dependency. Ninety-one percent of all cells with multiple aberrations were classified as unstable. Resolving the chromosome rearrangements into simple categories resulted in a linear dose response for dicentrics of 24.9 ± 3.3 × 10(-2) per Gy. The predominant aberration in stable transmissible cells was a single translocation with a dose response for predominantly single hit cell nuclei of 4.1 ± 1.3 × 10(-2) per Gy. Thus, translocations are the most likely aberration to be observed in peripheral blood lymphocytes from individuals with incorporated α-emitting radionuclides resulting in long-term chronic exposure.

The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation.

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.

Germline minisatellite mutations in survivors of childhood and young adult cancer treated with radiation.

PURPOSE: To investigate minisatellite germline mutation rates in survivors of childhood and young adult cancer who received radiotherapy. MATERIALS AND METHODS: DNA samples from 100 families, where one parent was a cancer survivor, were analysed for mutations at eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern hybridisation. RESULTS: No significant difference was observed between the paternal mutation rate of 5.6% in exposed fathers with a mean preconceptional testicular dose of 1.23 Gy (56 mutations in 998 informative alleles) and that of 5.8% in unexposed fathers (17 in 295 informative alleles). Subgrouping the exposed fathers into dose groups of < 0.10 Gy, 0.10-0.99 Gy, 1.00-1.99 Gy, ≥ 2.00 Gy revealed no significant differences in paternal mutation rate in comparison with the unexposed fathers. Maternal mutation rates of 1.6% in cancer survivor mothers with a mean preconceptional ovarian dose of 0.58 Gy (five mutations in 304 informative alleles) and 2.1% in unexposed mothers (21 in 987 informative alleles) were not significantly different. There were no differences in minisatellite mutation rates associated with treatment with chemotherapeutic agents. CONCLUSIONS: This study provides evidence that preconception radiotherapy for childhood or early adulthood cancer does not increase the germline minisatellite mutation rate.

G2 checkpoint control and G2 chromosomal radiosensitivity in cancer survivors and their families.

Significant inter-individual variation in G(2) chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G(2)-M checkpoint control. The hypothesis tested in the current analysis is that increased G(2) chromosomal radiosensitivity is facilitated by a less efficient G(2)-M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G(2) checkpoint delay and G(2) chromosomal radiosensitivity. In Group 1, the extent of G(2) checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G(2) checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G(2) checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G(2)-M checkpoint efficiency and variation in G(2) chromosomal radiosensitivity.

The heritability of G2 chromosomal radiosensitivity and its association with cancer in Danish cancer survivors and their offspring.

PURPOSE: To investigate the relationship between chromosomal radiosensitivity and early-onset cancer under the age of 35 years and to examine the heritability of chromosomal radiosensitivity. MATERIALS AND METHODS: Peripheral blood lymphocytes were cultured for 72 hours prior to being irradiated with 0.5 Gy, 300 kV X-rays. Colcemid was added to cultures 30 min post-irradiation. Cultures were harvested 90 min post-irradiation and analysed for chromatid gaps and breaks. Heritability was estimated using Sequential Oligogenic Linkage Analysis Routines (SOLAR) software and by segregation analysis. RESULTS: Elevated radiosensitivity was seen for seven out of 29 (24.1%) cancer survivors, three out of 29 (10.3%) partners and 10 out of 53 (20.8%) offspring. Although the proportion of individuals displaying enhanced radiosensitivity was twice as high in both the cancer survivor and offspring groups than the partner controls, neither reached statistical significance. Heritability analysis of the radiosensitive phenotype suggested 57.9-78.0% of the variance could be attributed to genetic factors. CONCLUSION: An association between G(2) chromosomal radiosensitivity and childhood and young adult cancer is suggested but was not statistically significant. In contrast, there is strong evidence for heritability of the radiosensitive phenotype. The cancer survivors included a broad range of malignancies and future studies should focus on specific cancers with known or likely faults in deoxyribonucleic acid (DNA) damage recognition and repair mechanisms.

Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification.

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

International study of factors affecting human chromosome translocations.

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation.

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.

Influence of polymorphisms at loci encoding DNA repair proteins on cancer susceptibility and G2 chromosomal radiosensitivity.

Sixteen candidate polymorphisms (13 SNPs and 3 microsatellites) in nine genes from four DNA repair pathways were examined in 83 subjects, comprising 23 survivors of childhood cancer, their 23 partners, and 37 offspring, all of whom had previously been studied for G(2) chromosomal radiosensitivity. Genotype at the Asp148Glu SNP site in the APEX gene of the base excision repair (BER) pathway was associated with childhood cancer in survivors (P = 0.001, significant even after multiple test adjustment), due to the enhanced frequency of the APEX Asp148 allele among survivors in comparison to that of their partners. Analysis of variance (ANOVA) of G(2) radiosensitivity in the pooled sample, as well as family-based association test (FBAT) of the family-wise data, showed sporadic suggestions of associations between G(2) radiosensitivity and polymorphisms at two sites (the Thr241Met SNP site in the XRCC3 gene of the homologous recombinational pathway by ANOVA, and the Ser326Cys site in the hOGG1 gene of the BER pathway by FBAT analysis), but neither of these remained significant after multiple-test adjustment. This pilot study provides an intriguing indication that DNA repair gene polymorphisms may underlie cancer susceptibility and variation in radiosensitivity.

FISH chromosome analysis of plutonium workers from the Sellafield nuclear facility.

Chromosome analysis using a single-color FISH technique to paint three pairs of chromosomes was undertaken on a group of 46 retired plutonium workers with assessed bone marrow doses >60 mSv, 34 of whom were categorized as having robust dosimetry and 12 for whom internal doses were considered less reliable. Comparisons were made with a group of 34 workers with negligible radiation exposure and a group of 34 workers with similar recorded external gamma-ray doses but negligible internal dose. The simple translocation frequency of 17.65 +/- 1.96 x 10(-3) per genome equivalent for the 34 plutonium workers with robust dosimetry was significantly increased in comparison with that of 10.06 +/- 1.16 x 10(-3) per genome equivalent for the unirradiated control group (P = <0.001) and that of 13.55 +/- 1.43 x 10(-3) per genome equivalent for the group with similar external gamma-ray exposure (P = 0.012). Thus, although in vitro studies have indicated that the majority of alpha-particle-irradiated cells suffer complex non-transmissible chromosome damage, in vivo a significant proportion survive with simple exchanges that can be passed on to descendant cells. In contrast, the three groups demonstrated no significant differences in stable complex aberrations. No evidence of an increase in dicentrics or unstable complex aberrations associated with plutonium exposure was observed, and it can therefore be assumed that there is little, if any, ongoing irradiation of mature lymphocytes. The translocation frequency of 12.08 +/- 1.92 x 10(-3) per genome equivalent for the group of 12 plutonium workers with less reliable internal dosimetry could adequately be accounted for by age and external dose and indicates that the internal bone marrow doses are likely to have been overestimated. Cytogenetic analysis can therefore make a valuable contribution to the validation of internal doses from plutonium deposition.

A pilot study examining germline minisatellite mutations in the offspring of Danish childhood and adolescent cancer survivors treated with radiotherapy.

PURPOSE: To investigate germline mutation rate at eight minisatellite loci in 24 Danish families, where one parent is the survivor of childhood or adolescent cancer treated with radiotherapy. MATERIALS AND METHODS: Parents and offspring were profiled for eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern blotting. RESULTS: Seven paternal mutations were observed for 130 informative alleles in 18 offspring from 11 radiation-exposed fathers (mean preconceptional dose for offspring 0.29 Gy, range<0.01-1.2 Gy), compared to six mutations for 146 informative alleles in 21 offspring from 13 unexposed fathers. No statistically significant difference between the total paternal mutation rates was observed (5.4% for exposed fathers and 4.1% for unexposed fathers). Three maternal mutations were observed for 148 informative alleles in 21 offspring from 13 radiation-exposed mothers (mean preconceptional dose for offspring 0.71 Gy, range <0.01-9.2 Gy), compared to one mutation for 130 informative alleles in 18 offspring from 11 unexposed mothers. Again, no statistically significant difference was observed between the total maternal mutation rates (2.0% for exposed mothers and 0.8% for unexposed mothers). CONCLUSIONS: The data from this pilot study demonstrate no statistically significant increase in germline minisatellite mutation rate associated with radiotherapy for childhood and adolescent cancer.