Prevalence of adult-onset multifactorial disease among offspring of atomic bomb survivors.

The first study to examine whether parental radiation exposure leads to increased heritable risk of common adult-onset multifactorial diseases (i.e., hypertension, diabetes mellitus, hypercholesterolemia, ischemic heart disease, and stroke) was conducted among 11,951 participants in the clinical examination program out of a potential of 24,673 mail survey subjects who were offspring of survivors born from May 1946 through December 1984. Logistic regression analyses demonstrated no evidence of an association between the prevalence of multifactorial diseases in the offspring and parental radiation exposure, after adjusting for age, city, gender and various risk factors. The odds ratio (OR) for a paternal dose of 1 Gy was 0.91 [95% confidence interval (CI) 0.81-1.01, P = 0.08], and that for a maternal dose of 1 Gy was 0.98 (95% CI 0.86-1.10, P = 0.71). There was no apparent effect of parental age at exposure or of elapsed time between parental exposure and birth, but male offspring had a low odds ratio (OR = 0.76 at 1 Gy) for paternal exposure, but cautious interpretation is needed for this finding. The clinical assessment of nearly 12,000 offspring of A-bomb survivors who have reached a median age of about 50 years provided no evidence for an increased prevalence of adult-onset multifactorial diseases in relation to parental radiation exposure.

Lifespan of human memory T-cells in the absence of T-cell receptor expression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

Nonparametric regression analysis of data from the Ames mutagenicity assay.

The Ames assay has received widespread attention from statisticians because of its popularity and importance to risk assessment. However, investigators have yet to routinely apply modern regression methods that have been available for more than a decade. We study yet another approach, the application of nonparametric regression techniques, not as the ultimate solution but rather as a framework within which to address some of the shortcomings of other methods. But nonparametric regression is itself prone to difficulties when applied to Ames assay data, as we show through the use of two examples and some simulation studies. We argue that there remains a great need for further development of statistical methods suitable to the Ames assay. It is hoped that such work can be stimulated and guided by greater collaboration between statisticians and laboratory investigators.

Monitoring exposure to atomic bomb radiation by somatic mutation.

Atomic bomb survivors are a population suitable for studying the relationship between somatic mutation and cancer risk because their exposure doses are relatively well known and their dose responses in terms of cancer risk have also been thoroughly studied. An analysis has been made of erythrocyte glycophorin A (GPA) gene mutations in 1,226 atomic bomb survivors in Hiroshima and Nagasaki. The GPA mutation frequency (Mf) increased slightly but significantly with age at the time of measurement and with the number of cigarettes smoked. After adjustment for the effect of smoking, the Mf was significantly higher in males than in females and higher in Hiroshima than in Nagasaki. All of these characteristics of the background GPA Mf were in accord with those of solid tumor incidence obtained from an earlier epidemiological study of A-bomb survivors. Analysis of the dose effect on Mf revealed the doubling dose to be about 1.20 Sv and the minimum dose for detection of a significant increase to be about 0.24 Sv. No significant dose effect for difference in sex, city, or age at the time of bombing was observed. Interestingly, the doubling dose for the GPA Mf approximated that for solid cancer incidence (1.59 Sv). And the minimum dose for detection was not inconsistent with the data for solid cancer incidence. The dose effect was significantly higher in those diagnosed with cancer before or after measurement than in those without a history of cancer. These findings are consistent with the hypothesis that somatic mutations are the main cause of excess cancer risk from radiation exposure.

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.

Radiosensitivity of atomic bomb survivors as determined with a micronucleus assay.

If A-bomb survivors include a disproportionately large number of either radioresistant or radiosensitive persons, the surviving population would provide a biased estimate of the true risk of radiogenic cancer. To test this hypothesis, the in vitro X-ray sensitivities of peripheral blood lymphocytes obtained from 937 A-bomb survivors were measured with a cytokinesis-blocking micronucleus assay. Background frequencies (no irradiation in vitro) of micronuclei show a wide distribution. Frequencies in both males and females tend to increase with increasing donor age. Frequencies in females are significantly higher than those in males. Donor age decreases the sensitivity of lymphocytes to in vitro X-ray exposure at a rate of about 0.001 micronuclei per cell per year per gray. There is no effect of donors' sex on in vitro radiation sensitivity. Atomic bomb radiation and cigarette smoking had no significant effect on background and X-ray-induced micronuclei frequencies. Thus there is no difference in radiosensitivity of peripheral blood lymphocytes between proximally and distally exposed survivors.

Study of the titers of anti-Epstein-Barr virus antibodies in the sera of atomic bomb survivors.

Antibody titers to Epstein-Barr virus antigens were determined in the sera of 372 atomic bomb survivors to evaluate the effect of the previous radiation exposure on immune competence against the latent infection of the virus. The proportion of persons with high titers (> or = 1:40) of IgG antibodies to the early antigen was significantly elevated in the exposed survivors. Furthermore, the distribution of IgM titers against the viral capsid antigen was significantly affected by radiation dose with an increased occurrence of titers of 1:5 and 1:10 in the exposed persons, although the dose effect was only marginally suggestive when persons with rheumatoid factor were eliminated from the analysis. These results suggest that reactivation of Epstein-Barr virus in the latent stage occurs more frequently in the survivors, even though this might not be affected by the radiation dose. Otherwise, there was neither an increased trend in the prevalence of high titers (> or = 1:640) of IgG antibodies to the viral capsid antigen among the exposed people nor a correlation between the radiation exposure and distributions of titers of IgA antibodies to the viral capsid antigen or antibodies to the anti-Epstein-Barr virus-associated nuclear antigen.

Effects of radiation on incidence of primary liver cancer among atomic bomb survivors.

We describe the radiation risk for primary liver cancers between 1958 and 1987 in a cohort of atomic bomb survivors in Hiroshima and Nagasaki, Japan. The analysis is based on a comprehensive pathology review of known or suspected liver neoplasms that generated 518 incident, first primary cases, mostly hepatocellular carcinoma. Excess relative risk from atomic bomb radiation was linear: 0.81 per sievert weighted liver dose (95% CI [0.32, 1.43]; P < 0.001). Males and females had similar relative risk so that, given a threefold higher background incidence in males, the radiation-related excess incidence was substantially higher in males. Excess risk peaked for those with age at exposure in the early 20s; there was essentially no excess risk in those exposed before age 10 or after age 45. Whether this was due to a difference in sensitivity or possible confounding by other factors could not be addressed retrospectively in the full cohort. A paucity of cholangiocarcinoma and hemangiosarcoma cases suggested that they are not significantly associated with whole-body radiation exposure, as they are with the internal alpha-particle-emitting radiological contrast medium Thorotrast. Because most of the radiation-related excess cases occurred among males, it is important to ascertain what factors put men at greater risk of radiation-related liver cancer.

Somatic cell mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors: implications for radiation carcinogenesis.

To clarify the relationship between somatic cell mutations and radiation exposure, the frequency of hemizygous mutant erythrocytes at the glycophorin A (GPA) locus was measured by flow cytometry for 1,226 heterozygous atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. For statistical analysis, both GPA mutant frequency and radiation dose were log-transformed to normalize skewed distributions of these variables. The GPA mutant frequency increased slightly but significantly with age at testing and with the number of cigarettes smoked. Also, mutant frequency was significantly higher in males than in females even with adjustment for smoking and was higher in Hiroshima than in Nagasaki. These characteristics of background GPA mutant frequency are qualitatively similar to those of background solid cancer incidence or mortality obtained from previous epidemiological studies of survivors. An analysis of the mutant frequency dose response using a descriptive model showed that the doubling dose is about 1.20 Sv [95% confidence interval (CI): 0.95-1.56], whereas the minimum dose for detecting a significant increase in mutant frequency is about 0.24 Sv (95% CI: 0.041-0.51). No significant effects of sex, city or age at the time of exposure on the dose response were detected. Interestingly, the doubling dose of the GPA mutant frequency was similar to that of solid cancer incidence in A-bomb survivors. This observation is in line with the hypothesis that radiation-induced somatic cell mutations are the major cause of excess cancer risk after radiation exposure. Furthermore, the dose response was significantly higher in persons previously or subsequently diagnosed with cancer than in cancer-free individuals. This may suggest an earlier onset of cancer due to elevated mutant frequency or a higher radiation sensitivity in the cancer group, although the possibility of dosimetry errors should be considered. The findings obtained in the present study suggest that the GPA mutant frequency may reflect the cancer risk among people exposed to radiation.

FISH examination of lymphocytes from Mayak workers for assessment of translocation induction rate under chronic radiation exposures.

PURPOSE: To estimate the translocation-induction rate under chronic exposure conditions by measuring chromosome aberration frequencies in lymphocytes from Mayak nuclear workers using fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: Lymphocytes were examined from 27 nuclear workers at the Mayak Production Association and two control individuals using FISH with probes for chromosomes 1, 2 and 4. Official doses derived from worker film-badge records varied from 0 to 8.50 Gy. RESULTS: The mean (+/-SD) genome-equivalent translocation frequency (F(G)) was 2.30 (+/-0.75)% in the zero-dose group (n = 7), and Poisson regression analysis provided the best-fit equation of F(G)(%) = 2.96(+/-0.39) + 0.69(+/-0.14)D + 0.12(+/-0.05)A, where D is the film-badge-derived dose (Gy), and A is age centred at 67 years. The induction rate would increase to nearly 1% Gy(-1) if the radiation dose to bone marrow, one of the major organs for lymphocytes and where their precursor cells reside, is considered. CONCLUSION: The estimated induction rate in vivo appeared substantially smaller than linear coefficients estimated from various in vitro studies.

Quantitation of skeletal-muscle necrosis in a model compartment syndrome.

UNLABELLED: Skeletal-muscle necrosis was evaluated in previously pressurized canine compartments using technetium-99m stannous pyrophosphate and classic histological criteria. Intracompartmental necrosis was quantitated in the anterolateral muscle compartment of each dog by uptake of 99mTc stannous pyrophosphate using the contralateral anterolateral compartment as an internal control. Representative specimens of muscle were sampled in experimental and control legs of each dog and were analyzed by qualitative histological techniques. Muscle necrosis was assessed in compartments forty-eight hours after pressurization to levels of ten to 120 millimeters of mercury for eight hours in thirty-seven dogs. In another dog, neither anterolateral compartment was pressurized so that both compartments acted as control muscle. The results in these experiments identify a threshold pressure level (thirty millimeters of mercury) and duration (eight hours) at which significant muscle necrosis occurs at normal blood pressure. Our findings imply that a quantitative relationship exists between incorporation of 99mTc stannous pyrophosphate and the level of intracompartmental pressure. This uptake technique, however, is not suitable for diagnosing compartment syndrome in patients with a threatened compartment syndrome. We suggest that intracompartmental pressure measurements by the wick-catheter technique, in conjunction with clinical findings, offer the best means for diagnosing compartment syndrome. CLINICAL RELEVANCE: Significant muscle necrosis associated with an impending compartment syndrome occurs at a threshold intracompartmental pressure of thirty millimeters of mercury after eight hours. Since time variables are often unknown in suspected compartment syndromes, fasciotomy is recommended when intracompartmental pressure exceeds thirty millimeters of mercury in a patient with normal blood pressure. The use of this threshold pressure level as an indication for fasciotomy requires a device for measuring intracompartmental pressure such as the wick catheter.

Effect of radiation and cigarette smoking on expression of FUdR-inducible common fragile sites in human peripheral lymphocytes.

In vitro X-irradiation of human peripheral blood lymphocytes increased the frequencies of fluorodeoxyuridine-induced fragile sites in a dose-related manner. However, the cells from 30 atomic bomb survivors exposed to either high or low radiation doses 47 years earlier showed no demonstrable difference in fragile site expression, indicating that fragile site induction was ephemeral in nature. When fragile sites were analyzed on the basis of tobacco smoking habits, an elevated number was observed in the smokers. The results confirm that fragile sites can be affected by recent exposure to exogenous agents, but the effect is probably of limited duration, based on the atomic bomb survivor experience.

Chromosomal instability in BRCA1- or BRCA2-defective human cancer cells detected by spontaneous micronucleus assay.

The BRCA1 and BRCA2 gene products are believed to play an important part in the onset and/or development of many sporadic mammary cancers. Recently, it has been reported that these two proteins contribute to a centrosome function which is believed to help maintain the integrity of the chromosome segregation process. This may mean a reduced level of the BRCA1 or BRCA2 protein in mammary cells will occasionally lead to nondisjunctional chromosomal loss or gain. We now report that spontaneous micronuclei arising from chromosome(s) which fail to be incorporated into the relevant daughter nuclei during mitosis tend to occur more frequently in BRCA1- or BRCA2-defective human cancer cells than in BRCA-positive cancer cells. Some cases of mammary carcinogenesis may therefore stem from the loss of integrity of chromosome segregation in cells which have a reduced capacity to express either BRCA1 or BRCA2.

Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes.

A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10(-4) and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southern blotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.

Gamma-ray- and fission neutron-induced micronuclei in PHA stimulated and unstimulated human lymphocytes.

Two groups of normal human blood cells, one stimulated with phytohaemagglutinin (PHA) for 24 hr (G1-S phase of the cell cycle) and one unstimulated (G0 phase), were irradiated with 60Co gamma rays or 252Cf radiation. A comparison of radiation-induced micronucleus frequencies showed that the high-dose-rate gamma rays were more effective in inducing micronuclei than low-dose-rate gamma rays. In the cells exposed to low-dose-rate irradiation, there was little difference between the frequency of micronuclei in the G0 phase and the G1-S phase. However, cells in the G1-S phase were more sensitive than G0-phase cells to high-dose-rate gamma rays. The relative biological effectiveness of 252Cf neutron irradiation measured in micronucleus assays was consistent with the value obtained for the lethal effect of 252Cf on cultured cells.

Liver cancer in atomic-bomb survivors: histological characteristics and relationships to radiation and hepatitis B and C viruses.

Histological features of primary liver cancer among atomic-bomb survivors and their relationship to hepatitis B (HBV) and C viral (HCV) infections are of special interest because of the increased risk of liver cancer in persons exposed to ionizing radiation and the high and increasing liver cancer rates in Japan and elsewhere. We conducted a pathology review of liver cancers occurring from 1958 to 1987 among subjects in the 120,321 member cohort of 1945 Hiroshima and Nagasaki residents. A panel of pathologists classified tumor histological types and defined accompanying cirrhotic changes of the liver. Archival tissue samples were assessed for HBV using pathology stains and PCR. Reverse transcriptase (RT) PCR was used to determine HCV status. We used unconditional logistic regression to compare 302 hepatocellular carcinoma (HCC) cases to 53 cholangiocarcinoma (CC) cases, adjusting for age, year of diagnosis, sex and viral status. Cirrhotic changes occurred significantly more often among HCC than CC cases (76% in HCC and 6% in CC). Compared to CC cases, HCC cases were 10.9 times more likely to be HBV-positive (95% confidence interval: 2.1-83.2) and 4.3 times more likely to be HCV-positive (95% confidence interval: 1.1-20.5). No significant differences were found between HCC and CC cases in radiation exposures. The predominance of HCC in the atomic-bomb survivors follows the background liver cancer pattern in Japan. Our findings suggest that HBV and HCV are involved in the pathogenesis of HCC with or without cirrhosis and are significantly less important in that of CC.

Impact of comparison group on cohort dose response regression: an example using risk estimation in atomic-bomb survivors.

Cohort-based dose-response analyses can be biased if based on a comparison group that is not comparable to the exposed persons with respect to uncontrolled factors related to disease incidence or mortality. When data exist over a range of doses including the very low dose region, internal regression standardized analyses based on the regression intercept derived from the exposed subcohort alone can provide risk estimates that are not subject to such comparison-group bias. In the Life Span Study cohort of atomic-bomb survivors, persons with dose estimates of zero comprise a broader geographic distribution than that of persons with non-zero dose estimates. Because there is geographic variation in mortality rates, the zero-dose persons might bias background rate estimates thereby affecting inference about radiation risk. This is illustrated using mortality due to all causes. Restricting the comparison group to certain geographically defined subcohorts resulted in as much as a 6% increase or 8% decrease in the risk estimate. This bias can be corrected using an SMR-type estimate in the regression model, allowing retention of the comparison group in the analysis if it is needed for stability or precision in estimating age, time, and sex effects. Consideration of heterogeneity in comparison groups is particularly important in dose-response studies focused on low doses at which the response may be comparable in magnitude to such heterogeneity.

Statistical issues in biological radiation dosimetry for risk assessment using stable chromosome aberrations.

Biological dosimeters are useful for epidemiologic risk assessment in populations exposed to catastrophic nuclear events and as a means of validating physical dosimetry in radiation workers. Application requires knowledge of the magnitude of uncertainty in the biological dose estimates and an understanding of potential statistical pitfalls arising from their use. This paper describes the statistical aspects of biological dosimetry in general and presents a detailed analysis in the specific case of dosimetry for risk assessment using stable chromosome aberration frequency. Biological dose estimates may be obtained from a dose-response curve, but negative estimates can result and adjustment must be made for regression bias due to imprecise estimation when the estimates are used in regression analyses. Posterior-mean estimates, derived as the mean of the distribution of true doses compatible with a given value of the biological endpoint, have several desirable properties: they are nonnegative, less sensitive to extreme skewness in the true dose distribution, and implicitly adjusted to avoid regression bias. The methods necessitate approximating the true-dose distribution in the population in which biological dosimetry is being applied, which calls for careful consideration of this distribution through other information. An important question addressed here is to what extent the methods are robust to misspecification of this distribution, because in many applications of biological dosimetry it cannot be characterized well. The findings suggest that dosimetry based solely on stable chromosome aberration frequency may be useful for population-based risk assessment.

Longevity of atomic-bomb survivors.

BACKGROUND: Conflicting claims have been made regarding biological and health consequences of exposure to low doses of radiation. Studies have suggested that certain low-dose exposed atomic-bomb survivors live longer than their peers. Earlier studies in other radiation-exposed populations demonstrated life shortening from mortality from cancer but lacked dosimetry and relied on comparison groups which may introduce bias because of lack of comparability. We have re-examined the effect of radiation on life expectancy in one cohort of survivors of the atomic bombings of Hiroshima and Nagasaki, Japan. METHODS: We did a prospective cohort study of 120,321 survivors. The study encompasses 45 years of mortality follow-up with radiation-dose estimates available for most cohort members. We calculated relative mortality rates and survival distribution using internal comparison (cohort-based estimation of background mortality). FINDINGS: Median life expectancy decreased with increasing radiation dose at a rate of about 1.3 years per Gy, but declined more rapidly at high doses. Median loss of life among cohort members with estimated doses below 1 Gy was about 2 months, but among the small number of cohort members with estimated doses of 1 Gy or more it was 2.6 years. Median loss of life among all individuals with greater-than-zero dose estimates was about 4 months. INTERPRETATION: These results are important in light of the recent finding that radiation significantly increases mortality rates for causes other than cancer. The results do not support claims that survivors exposed to certain doses of radiation live longer than comparable unexposed individuals. Because the cohort was intentionally constructed to contain a higher proportion of high-dose atomic-bomb survivors, average loss of life among all exposed atomic-bomb survivors would be less than the 4 months found for the study cohort.

Optimal case-control matching in practice.

We illustrate modern matching techniques and discuss practical issues in defining the closeness of matching for retrospective case-control designs (in which the pool of subjects already exists when the study commences). We empirically compare matching on a balancing score, analogous to the propensity score for treated/control matching, with matching on a weighted distance measure. Although both methods in principle produce balance between cases and controls in the marginal distributions of the matching covariates, the weighted distance measure provides better balance in practice because the balancing score can be poorly estimated. We emphasize the use of optimal matching based on efficient network algorithms. An illustration is based on the design of a case-control study of hepatitis B virus infection as a possible confounder and/or effect modifier of radiation-related primary liver cancer in atomic bomb survivors.