Youichi Odagiri

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis‐block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin‐B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5‰ and the interquartile range was between 3 and 12‰. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14–24%). Statistical analyses were performed using random‐effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods. Environ. Mol. Mutagen. 37:31–45, 2001

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.

Accelerated accumulation of somatic mutations in the senescence-accelerated mouse.

116 nature genetics volume 19 june 1998 quantitative fluorescence in situ hybridization technique has been developed and used to analyse telomeres of individual chromosomes of inbred mouse strains9. No discernible genetic determinant appeared to regulate telomere lengths between littermates of the same strain. Rather, telomere length on individual chromosomes was highly variable among siblings despite genotypic identity. This is reminiscent of the effect of the locus reported here, and may be a possible mechanism. At present, however, the role of telomere shortening in organismal ageing, as opposed to cellular replicative senescence10, is unclear11, and deserves further study. The existence of genes affecting variability in expression of other phenotypes in genotypically identical populations has been suggested by others12,13, but to our knowledge this is the first locus to be mapped. An alternate mechanism of action for this type of gene invokes a heightened sensitivity to environmental factors that leads to extended phenotypic variation13. The unique nature of the trait complicates cloning and obscures potential paths to understanding its mechanism of action, primarily because it can only be studied in populations and not in individuals. The simplest cloning strategy is usually the candidate gene approach, which in this case is frustrated by the lack of solid clues concerning a mechanism. If a sequence difference was detected in a candidate gene between the DBA/2 and C57BL/6 progenitor strains, obtaining proof that it was responsible for the phenotype would likely be very time-consuming, but should be achievable. Similarly, a positional-cloning strategy, requiring extensive progeny testing of mice segregating critical recombinant events, would be difficult and require considerable patience. However, the broad implications of this trait for genetic studies in general, and in genetic studies of ageing in particular, may warrant the effort.

Influence of serum micronutrients on the incidence of kinetochore-positive or -negative micronuclei in human peripheral blood lymphocytes

The possible contribution of some selected serum micronutrients (beta-carotene, vitamins B12 and C, folic acid and alpha-tocopherol) to spontaneous chromosomal damage was investigated in human peripheral blood lymphocytes from 33 non-smoking healthy donors by the cytokinesis-block micronucleus assay. Labelling of micronuclei with antikinetochore serum was used to discriminate between kinetochore-positive and -negative micronuclei and thus between micronuclei which arise from whole chromosome loss and those which arise from chromosome breaks. Simple correlation analysis showed that age was significantly associated with the increased frequency of micronucleated cells, and this age-related increase in these cells was due to the increase in cells with both kinetochore-positive and -negative micronuclei. Serum micronutrient levels had no apparent significant effects on incidence of micronucleated cells except for the weak positive correlation between vitamin B12 levels and frequency of kinetochore-positive micronucleated cells. Multiple regression analysis with age and serum micronutrient levels as independent variables showed that (a) age was the most influential variable for the frequency of micronucleated cells, (b) the serum vitamin C level was associated with increased frequency of spontaneous micronucleated cells, and this increase was mainly due to the increase in cells with kinetochore-positive micronuclei, and (c) the serum folic acid level was significantly and negatively related to the frequencies of cells with both kinetochore-positive and -negative micronuclei. To avoid the predominant age-effect, we also performed separate multiple regression analysis with age-adjusted frequency of micronucleated cells as dependent variable. The results from this analysis again showed a significant and positive effect of serum vitamin C level on age-adjusted frequency of kinetochore-positive micronucleated cells, while marginal negative effect of folic acid on age-adjusted frequency of total micronucleated cells (P < 0.06) and kinetochore-positive micronucleated cells (P < 0.051) was detected. These results suggest that age and serum vitamin C are definitely variables for frequencies of spontaneous chromosome loss, and that serum folic acid is perhaps another important micronutrient which influence the frequency of spontaneous chromosomal damage.

DAMAGE TO LYMPHOCYTES BY X-RAY AND BLEOMYCIN MEASURED WITH THE CYTOKINESIS-BLOCK MICRONUCLEUS TECHNIQUE

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.

Gender Differences in Age, Period, and Birth-Cohort Effects on the Suicide Mortality Rate in Japan, 1985-2006

Because suicide is increasingly becoming a public health threat in Japan, it is necessary to identify high-risk groups to develop effective preventive measures. The suicide mortality trends from 1985 to 2006 for Japanese aged between 15 and 79 years were analyzed by a Bayesian age–period–cohort analysis to evaluate the independent effects of age, period, and birth cohort. Age-specific effect showed an overall increase with age in both genders, but a distinct increase was noted only among men aged between 50 and 64 years. The period effect exhibited a sudden rise in 1998; this effect was more apparent in men than in women. The cohort-specific effect increased in male birth cohorts born after 1926 and in female birth cohorts born after 1956. In conclusion, a gender difference was detected in the effects of age, period, and cohort on suicide risk among Japanese.

Interindividual variation in cytogenetic response to X-ray and colchicine measured with the cytokinesis-block micronucleus assay.

Interindividual variation in cytogenetic response to two different types of micronucleus (MN) inducer, X-rays (a clastogen) and colchicine (a spindle poison), was investigated in the peripheral blood lymphocytes of normal healthy donors by the cytokinesis-block MN method. The data for 124 donors between the ages of 19 and 80 years showed that the histogram of individual frequency of X-ray (2 Gy)-induced micronucleated cells followed the normal distribution (Shapiro Wilks W-test) with a significant interindividual variance (ANOVA, p < 0.001). This was, however, not the case for colchicine (0.03 microgram/ml)-induced micronucleated cells. Instead, a skewed distribution illustrating interindividual variation was evident (ANOVA, p < 0.001). Statistical analysis of the effect of age and sex on MN incidence by using the Kruskal-Wallis test indicated that age affected the baseline and colchicine-induced MN incidences strongly but not the X-ray-induced MN incidence. There was no effect of sex on the incidence of micronuclei induced by either agent. In order to avoid any possible effect of age on the MN index, data for young subjects aged less than 30 years old were analyzed separately. The results of this analysis again showed significant interindividual variations in baseline, X-ray-induced, and colchicine-induced micronucleated cell rates. Results of the correlation-coefficient analysis showed that neither X-ray-induced MN incidence nor colchicine-induced MN incidence was related to baseline MN incidence. No correlation between X-ray-induced and colchicine-induced MN incidences was also found by this analysis. These results suggest that interindividual variance in chromosomal response to mutagens in normal populations may be a real phenomenon, as is interindividual variance in baseline MN frequency, and that individual susceptibilities to the two different types of micronucleus inducers (X-ray and colchicine) are unrelated, and the baseline MN level is not of predictive value for the susceptibilities.

Detection of the cytogenetic effect of inhaled aerosols by the micronucleus test

The induction of micronuclei in mice exposed to aerosols of the following 6 genotoxic chemicals by inhalation was examined: cyclophosphamide (CP), methyl methanesulfonate (MMS), mitomycin C (MMC), dimethylnitrosamine (DMN), ethylcarbamate and colchicine. Exposure of mice to CP aerosols at a theoretical concentration of 2426 mg/m3 for 29, 81 and 139 min induced 0.6, 1.0 and 2.3% micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow 24 h after the termination of exposure. The other chemicals except for DMN showed a similar exposure-response relationship following in vivo exposures to their aerosols. The results obtained in this study suggest that the cytogenetic effect of inhaled aerosols can be detected by the micronucleus test, and the method described in the present report is useful as a rapid in vivo test for atmospheric aerosols.

Negative micronucleus tests on caprolactam and benzoin in ICR/JCL male mice

Male ICR/JCL mice were given a single intraperitoneal injection of 125, 250, and 500 mg/kg of caprolactam (CAP), or 250, 500, and 1000 mg/kg of benzoin (ZOIN). Bone marrow preparations were made 24, 30, and 48 h after treatment with the maximum dose, and 30 h after treatment with the other doses. The slides were coded before microscopic examination. No significant increase was found in the incidence of micronucleated polychromatic erythrocytes after treatment with either CAP or ZOIN.

Gender Differences in Projected Life Expectancy in Japan (2023–2047) Determined by Bayesian Age-Period-Cohort Analysis

OBJECTIVES In this study, we aimed to (1) determine the effects of age, period, and cohort on mortality rate trends between 1958 and 2012 in Japan and (2) assess gender differences in projected life expectancy (LE) for the 2023-2047 period. METHODS A time trend study was conducted using age-period-cohort (APC) analysis. A Bayesian APC model was fitted to describe mortality rate trends for the 1958-2012 period and to project mortality rates for 2023-2047. LE was predicted by Chiangs method using projected mortality rates. RESULTS Age, period, and cohort effects showed similar patterns between males and females. As time passes, gender differences in projected LE were larger among individuals over 65 years than among those under 65 years. Time series change rates of the extension of projected LE after excluding specific causes of death showed the following: smaller extension of projected LE in males in terms of mortality risk from malignant neoplasms, heart diseases, pneumonia, and accidents (under 65 years) and in females in terms of mortality risk from heart diseases, cerebrovascular diseases, and suicide (over 65 years). CONCLUSIONS Gender differences in projected LE are expected to be smaller before middle age and to be larger among seniors. These projected gender differences stem in part from the lower mortality risk among men than among women from malignant neoplasms, heart diseases, pneumonia, and accidents (under 65 years), and among women compared to men from heart disease, cerebrovascular disease, and suicide (over 65 years).