Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

Spontaneous frequency of micronuclei in peripheral blood lymphocytes of Kiev residents]

The level of micronuclei in the peripheral blood lymphocytes of Kyiv residents and its dependence on age, sex and smoking status were studied. Analysis of lymphocytes of 102 healthy Kyiv residents showed that the spontaneous frequency of micronuclei in individuals at the age of 21 to 67 (mean age of 42.6) was 10.5 +/- 0.5@1000. The frequency of micronuclei depends on individual age and increases by 3% per year, and also depends on smoking habits (the micronucleus frequency in smokers was 1.3 times higher then nonsmokers). There is no dependence of the micronucleus frequency on the sex of persons.     

Kinetochore immunofluorescence in micronuclei: a rapid method for the in situ detection of aneuploidy and chromosome breakage in human fibroblasts

We have developed a rapid and simple immunodetection assay for the in situ identification of aneuploidy in mitotic fibroblasts. Kinetochore (centromere)-containing micronuclei can be detected easily and rapidly by immunofluorescence. The action of colchicine and its derivatives on the mitotic spindle apparatus of mammalian cells induces chromosome lag and aneuploidy. The treatment of normal human fibroblasts with Colcemid resulted in increased levels of micronuclei. Using an immunofluorescence stain (scleroderma CREST antiserum, biotinylated goat antihuman IgG and streptavidin-Texas Red) to detect the presence of kinetochores, it was observed that 90% of the Colcemid-induced micronuclei contained one or more fluorescent bodies (kinetochores). Cultured skin fibroblasts from a patient with ataxia telangiectasia (AT), which is a chromosome breakage syndrome, were used as a control. The AT fibroblasts exhibited elevated levels of spontaneous micronuclei when compared with normal fibroblasts, and 85% of these micronuclei were kinetochore-negative. This finding supports the hypothesis that the majority of spontaneous micronuclei in AT cells arise from chromosome breakage. The spontaneous micronucleus frequencies for 8 strains of human fibroblasts were in the order of 0.5-2%. Spontaneous levels of kinetochore-positive micronuclei were measured for these 8 strains; in 5 of the strains, about 25% of the micronuclei were kinetochore-positive, and in the other 3 strains approximately 50% of the micronuclei were kinetochore-positive. These data suggest that genetic factors may play a role in the control of the spontaneous levels of chromosome breakage and/or segregation errors which result in aneuploidy.     

Sex chromosomes,micronuclei and aging in women

Several studies on aneuploidy and aging have shown a significant increase in the loss of chromosomes in both males and females with age. Others have observed a significant increase in micronucleus formation in lymphocytes with age. The objectives of this investigation were to determine the relationship between sex chromosome loss and increased micronucleus frequencies with age, to establish sex chromosome loss frequencies unbiased by cellular survival factors or slide preparation, and to determine the effect of smoking on sex chromosome loss. Blood samples were obtained from 8 newborn females and 38 adult females ranging in age from 19 to 77. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Two thousand binucleated cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. Slides were then hybridized with a 2.0 kb centromeric X chromosome-specific probe labeled with biotinylated dUTP, and detected with fluorescein-conjugated avidin. All micronucleated cells were relocated and their X chromosome status was determined. We found the X chromosome to be present in 72.2% of the micronuclei scored; additionally our results show a significant increase with age in the number of micronuclei containing an X chromosome.     

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C

We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.     

Age-dependent inclusion of sex chromosomes in lymphocyte micronuclei of man.

Two-color centromeric FISH was used to study the inclusion of the X and Y chromosomes in micronuclei of cultured lymphocytes from 10 men representing two age groups (21-29 years and 51-55 years). In addition, pancentromeric FISH was separately performed to identify any human chromosomes in micronuclei. One hundred micronuclei per probe were examined from each donor. A higher mean frequency of Y-positive micronuclei was observed in the older men than in the younger men. In both age groups, the X chromosome was micronucleated clearly more often than expected by chance, and the Y chromosome was overrepresented in micronuclei among the older men but not among the younger men. In lymphocytes of four women, X-positive micronuclei were more frequent than they were in men, even after the fact that women have two X chromosomes was taken into account. Similar results were obtained in first-division lymphocytes identified by cytochalasin-B-induced cytokinesis block. In comparison with normal cells, these binucleate cells showed a higher frequency (per 1,000 nuclei) of X-positive micronuclei (in the older men) but a lower frequency of micronuclei harboring autosomes or acentric fragments. In conclusion, the results show that both the X chromosome and the Y chromosome are preferentially micronucleated in male lymphocytes, the Y chromosome only in older subjects. Although the X chromosome has a general tendency to be included in micronuclei, it is micronucleated much more often in women than in men, which is probably the main reason for the high micronucleus frequency in women that has been documented in many previous studies.     

Micronuclei frequency induced by bleomycin in human peripheral lymphocytes: correlating BLHX polymorphism with mutagen sensitivity

Mutagen sensitivity assay, by measuring chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been proposed as a biomarker for assessing cancer susceptibility. Recently, a single nucleotide polymorphism (SNP A1450G) of the gene for bleomycin hydrolase (BLHX), a specific neutral cysteine protease able to metabolise bleomycin, was proposed as a plausible candidate to variation in mutagen sensitivity. To shed more light on the effect of BLHX genotype on the expression of chromosome damage induced in vitro by bleomycin, we determined mutagen sensitivity for 45 non-smoker healthy volunteers. The level of bleomycin-induced chromosome damage was assessed as frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes. The subjects were genotyped for the BLHX gene, to determine the possible effect of this polymorphism on mutagen sensitivity. No difference in the spontaneous value of MN was detected between the homozygotes wild-type (A/A) and the carriers of variant alleles A/G heterozygotes or G/G homozygotes (MN/1000 binucleated (BN) cells: 6.69+/-2.53 and 6.37+/-4.87, respectively). A substantial effect of BLHX polymorphism in predetermining individual mutagen sensitivity status was observed: subjects with the BLHX A/A genotype displayed significantly lower mean levels of bleomycin-induced MN frequency than the carriers of A/G or G/G variant alleles combined (12.00+/-3.76 MN/1000 BN vs. 16.37+/-8.86 MN/1000 BN, respectively; P=0.029). The multiple regression analysis, including BLHX genotype and age, confirmed the significant effect of BLHX variant alleles (A/G, G/G) on the chromosome damage induced by bleomycin (P=0.01), whereas age correlated only with the spontaneous MN frequency.     

The human lymphocyte micronucleus assay: Response of cord blood lymphocytes to γ-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with γ-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 μg/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

Screening for interindividual differences in radiosensitivity by means of the micronucleus assay in human lymphocytes

The response of unstimulated peripheral lymphocytes to a single dose of 3 Gy of 137Cs gamma rays was analysed in blood samples from 30 donors by a conventional micronucleus assay and from 14 donors by the cytokinesis-block (CB) method. Significant interindividual variations could be detected for the baseline levels and for induced levels of micronuclei. An age effect could be demonstrated with the conventional method for the number of spontaneous MN, but not with the CB method. The corresponding numerical estimate was 3.4 +/- 1.3% increase per year. No such increase was apparent for induced frequencies. Provided that cell proliferation kinetics is reliably taken into account the micronucleus assay could be helpful for diagnosing potential radiosensitive individuals.     

A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay.

A cytogenetic study was performed in 215 nuclear power plant workers occupationally exposed to radiation using the micronucleus-centromere assay for peripheral blood lymphocytes. As control population served administrative staff with yearly doses below 1 mSv. The increase of the micronucleus frequency with age, observed in the non-smoking control population, is mainly due to an enhanced number of centromere-positive micronuclei, pointing to an increased chromosome loss. No differences in the number of micronuclei, centromere-positive and centromere-negative micronuclei between smokers and non-smokers are observed. An analysis of the micronucleus data vs. the dose accumulated over the 10 years preceding the venepuncture shows no significant clastogenic or aneuploidogenic effects of the exposure in the studied population which is representative for workers in the nuclear industry at present. According to the linear fits to our data an increase of the micronucleus frequency pro rata 0.5 per 1000 binucleated cells per year, related to the centromere-negative micronuclei, may be expected for workers with the maximal tolerable dose of 20 mSv/year.     

The human lymphocyte micronucleus assay. Response of cord blood lymphocytes to gamma-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with gamma-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 micrograms/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.     

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes,oral mucosa and hair root cells

Summary Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.     

A case of elevated spontaneous micronucleus frequency derived from chromosome 2

This work tested the hypothesis that the content of spontaneous micronuclei in lymphocytes in an apparently healthy normal human subject, who exhibited an unusually high micronucleus frequency, was non-random. Several DNA probes were used in fluorescent in-situ hybridization (FISH), beginning with a probe generated from the subject's micronuclei. Micronuclei obtained from peripheral blood lymphocytes by microdissection were subjected to random amplification of polymorphic DNA (RAPD-PCR), and a unique PCR product was then used to isolate a cosmid clone from a human genomic library. This clone hybridized to chromosome 2. Subsequently, commercial probes were included in FISH analyses of micronuclei from the subject and age- and sex-matched controls. No significant differences were found between subject and controls in the percentages of micronuclei hybridizing with a centromere probe for the X chromosome or a painting probe for chromosome 3. However, the subject had a very highly significant increase (p<0.0001) in chromosome 2 in micronuclei over a level that might be expected to be present by chance. Characterization of micronuclei may be a promising tool in studies of mechanisms of inherited or induced chromosome instability. The strength of the strategy employed in this study is that, by characterizing the chromosomes present in micronuclei, this work has advanced from an observation of chromosomal instability to a foundation for study of the mechanism underlying the observation.     

A case of pure red cell aplasia with a high incidence of spontaneous chromosome breakage: A possible X-ray sensitive syndrome

Summary A patient with a pure red cell aplasia (Blackfan-Diamond Anemia), and with many congenital abnormalities and growth retardation, has been found to have a chromosome breakage syndrome. In this patient, the frequencies of spontaneous chromosome aberrations and micronuclei in PHA stimulated peripheral blood lymphocytes are elevated when compared to those in normal individuals. The frequency of sister chromatid exchanges is within normal range. The response to mitomycin C (MMC) in the micronucleus test, using lymphocytes, shows a similar increase in the patient's lymphocytes to that in normal individuals, indicating no increased sensitivity to MMC.The frequencies of X-ray induced dicentric chromosomes and micronuclei in the peripheral blood lymphocytes are elevated in the patient. But as the patient clinically does not have any signs of ataxia telangiectasia, this combination of clinical and laboratory findings of this case does not correspond with any of the other known chromosome breakage syndromes.     

Genome instability is increased in lymphocytes of women with polycystic ovary syndrome and is correlated with insulin resistance

BACKGROUND: Polycystic ovary syndrome (PCOS) is associated with insulin resistance and reproductive and metabolic abnormalities. The potential genetic contributors to PCOS are unclear. We tested the hypothesis that genomic instability (chromosome malsegregation and DNA damage) is increased in PCOS. METHODS: Overweight age, weight and BMI-matched women with (n=14) and without (n=16) PCOS (age 34.2+/-6.0 years, weight 90.7+/-14.5 kg, BMI 34.0+/-5.6 kg/m(2), mean+/-S.D.) were assessed for chromosome malsegregation (assessed by X chromosome chromogenic in situ hybridisation) and micronucleus frequency (assessed by the cytokinesis block micronucleus index) in lymphocytes. RESULTS: Women with PCOS had significantly elevated genomic instability as demonstrated by a significantly higher number of binucleated lymphocytes containing micronuclei, total number of micronuclei, a higher proportion of aneuploid X chromosome signals (2:1 X and 3:1 X) and a lower proportion of normal X chromosome segregation signals (2:2 X) in binucleated lymphocytes than women without PCOS. Surrogate measures of insulin resistance positively correlated with the proportion of aneuploid cells (2:1; 3:1 X chromosome signals) and inversely with the proportion of normal cells (2:2 X chromosome signals). CONCLUSION: Women with PCOS display increased genomic instability (higher micronuclei and chromosome malsegregation) compared to women without PCOS and this increase may be related to the insulin resistance phenotype.     

Genotype and the cryopreservation process affect the levels of aneuploidy and chromosome breakage in cultured human fibroblasts

Spontaneous micronucleus frequencies were measured in 11 human fibroblast strains, with early-passage cells that had never been frozen and with cells of comparable population doublings that had been cryopreserved in liquid nitrogen. The mean micronucleus frequency of the 11 strains increased from 14.0 +/- 0.7 to 20.4 +/- 1.8/1250 mononucleated cells (P = 0.002) after the freeze-thaw process. The nature of this increase in micronucleus frequency was examined using an immunodetection assay for the in situ identification of kinetochores in micronuclei. The increase in micronucleus frequency occurred primarily in the kinetochore-positive fraction, which is indicative of aneuploidy, but also by an increase in chromosome breakage in several strains. The findings were reproducible in repeat biopsies from two donors. Plating efficiencies of the 11 strains were studied during 1-9 and 10-20 population doublings from primary outgrowth, before freezing and again after freeze-thaw. The mean plating efficiency of frozen-thawed cells before nine doublings was significantly lower than that of cells of similar ages that had never been frozen (P = 0.004). The four strains that had a greater than 25% decrease in plating efficiency post freeze-thaw also had the highest aneuploidy index post freeze-thaw, suggesting that chromosomal imbalance contributes to the observed reduction in growth. We conclude that the genotype and culture manipulations of a fibroblast strain influence the outcome of the micronucleus assay.     

Activation status of the X chromosome in human micronucleated lymphocytes

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon. Received: 5 May 1995 / Revised: 15 July 1995     

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.