Acentromeric micronuclei are increased in peripheral blood lymphocytes of untreated cancer patients

Increased micronucleated cell rates, dicentric chromosomes, and other chromosomal damages have been reported in lymphocytes of cancer patients prior to the initiation of chemotherapy, and/or radiotherapy. The cause of these chromosomal damages in these lymphocytes remains unclear. In the present work, we investigated whether these micronuclei mainly reflect structural or numerical chromosomal aberrations by applying the cytokinesis-blocked micronucleus (CBMN) assay in combination with fluorescent in situ hybridization (FISH) of a DNA centromeric probe on blood samples of 10 untreated cancer patients (UCPs), and 10 healthy subjects (HSs). Micronucleated binucleated lymphocyte rate was significantly increased in patients (mean+/-S.D.: 19.0 per thousand +/-14.1 versus 9.2 per thousand +/-4.6 in controls). Trinucleated cytokinesis-blocked cells were not significantly higher in patients than in controls. Acentromeric, centromeric, and multicentromeric micronucleus levels were two-fold higher in patients than in controls, but the difference was significant only with acentromeric micronuclei. The percentage of micronuclei containing one or more centromeres averaged 69.2, and 71.5% in patients, and controls, respectively. The percentage of micronuclei containing several centromeres was 44.7% in patients, and 54.6% in controls. Among centromere-positive micronuclei, the percentage of micronuclei containing several centromeres averaged 59.7% in patients, and 75.4% in controls. These results indicate that genetic instability in peripheral blood lymphocytes of UCPs occurs because of enhanced chromosome breakage. However, a substantial proportion of this genetic instability occurs because of defects in chromosome segregation.     

Cytogenetic monitoring of industrial radiographers using the micronucleus assay

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years±11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv±49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7‰±5.2 versus 6.6‰±3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C−MN) in exposed subjects than in controls (8.5‰±4.9 versus 2.2‰±1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C−MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.     

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium, cobalt, and nickel

Dental laboratory technicians may be exposed to metal alloys that are used in the production of crowns, bridges and removable partial dentures. These alloys consist of 35–65% cobalt, 20–30% chromium, 0–30% nickel, and small amounts of molybdenum, silica, beryllium, boron and carbon. The aim of this study was to assess whether dental technicians are occupationally exposed to chromium, cobalt and nickel, by analyzing urinary excretion levels of these metals and to investigate the genotoxic effects of occupational exposure associated with dental prostheses production operations by analyzing cytokinesis-blocked micronucleus (CB-MN) frequencies in peripheral lymphocytes and micronucleus (MN) frequencies in exfoliated nasal cells from 27 dental laboratory technicians and 15 control subjects. The differences in the urinary excretion of metals between technicians and controls were statistically significant. The mean (±S.D.) CB-MN frequencies (‰) in peripheral lymphocytes were 4.00 (±2.98) among the dental technicians and 1.40 (±1.30) among the controls, a statistically significant difference (P<0.005). The mean (±S.D.) MN frequencies (‰) in nasal cells were 3.50 (±1.80) among the dental technicians and 1.19 (±0.53) among the controls, which was also a statistically significant difference (P<0.005). There was a significant correlation between duration of exposure and MN frequencies in lymphocytes (r=0.642, P<0.01), but not in nasal cells of technicians. Our data reveal that in vivo exposure to chromium, nickel and cobalt metals is evident and that this occupational exposure may contribute to the observed genotoxic damage in two types of cells, e.g. lymphocytes and exfoliated nasal cells. However, it cannot be determined which compound(s) are responsible for the genotoxic damage observed in this study.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods; the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed a genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer.     

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.     

Micronucleus frequency in peripheral blood lymphocytes and exfoliated buccal cells of untreated cancer patients

Some genetic diseases may increase the cellular instability. Since most human tumors have some genetic base, this study was undertaken for the genetic instability in cancer patients by micronucleus analysis, a mutation-screening test, which is more practical and economic technique than metaphase analysis carried out for chromosomal aberrations. Genetic changes were assessed in untreated cancer patients (lung, stomach and colon cancer) by different genotoxical screening methods: the cytokinesis-block micronucleus test and the buccal mucosa cell micronucleus test. The evaluation of micronuclei number in peripheral blood lymphocytes and buccal cells showed genomic instability in somatic cells. There was a significant increase in the number of micronuclei in cancer patients prior to the initiation of chemotherapy, and/or radiotherapy compared with healthy human subjects. Furthermore, there was no significant difference between smokers and non-smoking groups or male and female groups. These results suggest that cancer in humans is characterized by an increase of chromosomal damage and thus, the micronucleus assay carried out here may be useful in routine cytogenetic studies of cancer. The text was submitted by the authors in English.     

Frequency of micronuclei in peripheral blood lymphocytes from subjects occupationally exposed to low levels of ionizing radiation

The aim of this study was to evaluate the genotoxic effect of low dose of occupational radiation exposure in Nuclear Medicine Department employees, by using cytokinesis-blocked micronucleus assay in peripheral blood lymphocytes. The study included 46 exposed individuals together with 27 from the same area without occupational exposure to radiation which served as controls. The results obtained were evaluated with respect to age, gender, smoking habits, pathological condition and the occupational exposure to radiation of the individuals. The frequency of micronuclei increased significantly with the age of the subjects (P = 0.007). However there were no significant differences in micronucleus frequency with gender, smoking habits and occupational exposure. The frequency of micronuclei was significantly higher in individuals with presence of pathological condition (P < 0.0001) in comparison to healthy population irrespective of their exposure status.     

Chromosomal instability in a woman with infertility and two unaffected brothers: a new familial chromosomal breakage syndrome?

Repeated chromosomal analysis of peripheral blood lymphocytes and skin fibroblasts from a woman referred for amenorrhoea, streak gonads, hyperthyroidism, adiposity and elevated α-fetoprotein levels but no other manifestations of known chromosomal breakage syndromes demonstrated an increased spontaneous chromosomal breakage rate (ISCBR). Chromatid and chromosomal breaks were more numerous than sporadic rearrangements and dicentric chromosomes. Exposure of the cells to mitomycin C, diepoxybutane, X-rays or UV irradiation induced an increase in chromosomal and chromatid abnormalities over that in controls. A micronucleus assay demonstrated an increase in the incidence of formation of micronuclei and the population doubling time of the fibroblasts of the proposita was delayed. Chromosomal analysis was performed on lymphocytes of the parents and of five sibs of the proposita. Two brothers had chromosomal abnormalities identical to those of the patient and elevated α-fetoprotein levels, however, without any clinical abnormalities. The parents were affected by only a moderate ISCBR whereas two brothers and one sister were chromosomally normal. The clinical, chromosomal and biochemical findings in this family represent a novel chromosomal instability syndrome. Received: 30 October 1996 / Accepted: 27 March 1997     

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 study on differences between radiation-induced micronuclei and apoptosis of lymphocytes in breast cancer patients after radiotherapy

Cancer patients' responses to radiotherapy vary in severity. It has been suggested that it may be due to differences in intrinsic cellular radiosensitivity. Prediction of tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Towards this goal the micronucleus and apoptosis tests have been proposed as methods for measurement of chromosomal damage in peripheral blood lymphocytes. In this study, gamma-ray sensitivity of cultured lymphocytes of 26 breast cancer patients with early or late reactions was investigated. After irradiation with 4 Gy gamma radiation in G0, the frequency of micronuclei for patients with early reactions was significantly higher (P < 0.05) than for patients with late reactions. In the contrary the frequency of apoptosis for patients with early reactions was significantly lower (P < 0.05) than in the other group. It could be suggested that such a reduced amount of micronuclei in the late effects group is due to the presence of some residual DNA damages which are not completely repaired and lesions show increasing severity when the patients' cells are irradiated again. These induced damages, probably are high enough to stimulate other endpoints like apoptosis instead of micronuclei.     

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.     

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.     

Micronuclei in human lymphocytes: the Co-60 gamma-ray dose-response

Dose-response for micronuclei in cytokinesis-blocked lymphocytes after in vitro irradiation of whole blood from 3 donors with Co-60 gamma-rays in the range 0–5.0 Gy was established. The numerical relationship between radiation induced chromosomal aberrations, and micronuclei is also examined. An increased frequency of micronuclei following low doses of gamma-irradiation is reported from a study of 41 radiation workers.     

Induction of micronuclei by CsCl in vivo and in vitro

In the present study, we report the results of an investigation of the potential of nonradioactive CsCl for the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow and in human lymphocytes cultured and blocked with cytochalasin-B. No significant increase in micronucleus frequency was observed in the polychromatic erythrocytes of mice which received 500 mg/kg of CsCl. In vitro experiments with human lymphocytes cultured in medium containing 250 and 500 μg/ml CsCl also showed no increase in micronucleus frequency compared to untreated controls. These same experiments, however, demonstrated a reduction in mitotic activity with increasing CsCl concentration in the culture medium. This report is the first to describe studies on the possible induction of micronuclei in vitro and in vivo by nonradioactive CsCl.     

Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes

A study was conducted to evaluate the genotoxic effect of occupational exposure to formaldehyde on pathology and anatomy laboratory workers. The level of exposure to formaldehyde was determined by use of passive air-monitoring badges clipped near the breathing zone of 59 workers for a total sampling time of 15min or 8h. To estimate DNA damage, a chemiluminescence microplate assay was performed on 57 workers before and after a 1-day exposure. Assessment of chromosomal damage was carried out by use of the cytokinesis-blocked micronucleus assay (CBMN) in peripheral lymphocytes of 59 exposed subjects in comparison with 37 controls matched for gender, age, and smoking habits. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 18 exposed subjects and 18 control subjects randomized from the initial populations. Mean concentrations of formaldehyde were 2.0 (range <0.1-20.4ppm) and 0.1ppm (range <0.1-0.7ppm) for the sampling times of 15min and 8h, respectively. No increase in DNA damage was detected in lymphocytes after a one-workday exposure. However, the frequency of binucleated micronucleated cells was significantly higher in pathologists/anatomists than in controls (16.9 per thousand+/-9.3 versus 11.1 per thousand+/-6.0, P=0.001). The frequency of centromeric micronuclei was higher in exposed subjects than in controls (17.3 per thousand+/-11.5 versus 10.3 per thousand+/-7.1) but the difference was not significant. The frequency of monocentromeric micronuclei was significantly higher in exposed subjects than in controls (11.0 per thousand+/-6.2 versus 3.1 per thousand+/-2.4, P<0.001), while that of the acentromeric micronuclei was similar in exposed subjects and controls (3.7 per thousand+/-4.2 and 4.1 per thousand+/-2.7, respectively). The enhanced chromosomal damage (particularly chromosome loss) in peripheral lymphocytes of pathologists/anatomists emphasizes the need to develop safety programs.     

Cytogenetic biomonitoring of a floriculturist population in Italy: micronucleus analysis by fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe

Flower production in greenhouses associated with a heavy use of pesticides is very wide-spread in the western part of the Ligurian region (Italy). The formation of micronuclei in peripheral blood lymphocytes is a valuable cytogenetic biomarker in human populations occupationally exposed to genotoxic compounds. In the present study we investigated the micronucleus frequency in peripheral blood lymphocytes of 52 floriculturists and 24 control subjects by use of the cytokinesis-block methodology associated with fluorescence in situ hybridization with a pan-centromeric probe that allowed to distinguish centromere-positive (C+) and centromere-negative (C-) micronuclei. The comparison between floriculturists and controls did not reveal any statistically significant difference in micronucleus frequency, although an increase was observed with increasing pesticide use, number of genotoxic pesticides used and duration of exposure. An increase in C+ as well as in C- micronuclei and in the percentage of C+ micronuclei with respect to the total number of micronuclei was detected in floriculturists, suggesting a higher contribution of C+ micronuclei in the total number scored. The percentage C+ micronuclei was not related to the duration of exposure or to the number of genotoxic pesticides used, but a higher percentage (66.52% versus 63.78%) was observed in a subgroup of subjects using benzimidazolic compounds, compared with the floriculturist population exposed to a complex pesticide mixture not including benzimidazolics. These results suggest a potential human hazard associated with the exposure to this class of aneuploidy-inducing carcinogens.     

Radioprotective effects of hawthorn against genotoxicity induced by gamma irradiation in human blood lymphocytes

The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract was investigated in cultured blood lymphocytes from human volunteers. Peripheral blood samples were collected from five human volunteers 10 min before and 1, 2 and 3 h after a single oral ingestion of 500 mg hawthorn powder extract. At each time point, the whole blood was exposed in vitro to 150 cGy of cobalt-60 gamma irradiation, and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cell. The lymphocytes in the blood samples collected after extract ingestion exhibited a significant decrease in the incidence of binucleated cells containing micronuclei as compared to similarly irradiated lymphocytes collected prior to extract ingestion. The maximum decrease in the frequency of micronuclei-containing cells was observed at 1 h after ingestion of Hawthorn extract (on average a 44% decrease). These data suggest that it may be possible to use Hawthorn extracts in personnel exposed to radiation in order to protect lymphocytes from radiation effects.     

Micronucleus induction and FISH analysis in buccal cells and lymphocytes of nurses administering antineoplastic drugs

A genotoxic effect for antineoplastic drugs, in particular micronucleus induction, has been shown in several studies. The aim of our study was to assess genotoxic effects in nurses administering different mixtures of antineoplastic drugs in an oncology hospital by evaluating the frequency of micronuclei in exfoliated buccal cells and blood lymphocytes by use of the standard micronucleus (MN) test and by identifying, by means of FISH analysis with centromeric probes, the mechanism of micronucleus induction (clastogenic or aneugenic). The study group comprised 23 nurses, 10 of whom worked in the day-care hospital and 13 in the ward. Twenty healthy subjects were selected as controls. Pan-centromeric FISH analysis was performed on lymphocytes from a selected group of nurses (12/23 subjects) characterized by higher MN frequencies as observed by standard Giemsa staining. A significant increase of micronucleus frequency compared with controls was found in exfoliated buccal cells of both groups of nurses: day-care hospital nurses 0.92 versus 0.45 (p=0.034) and ward nurses 0.94 versus 0.45 (p=0.051). An increase, although not statistically significant, of mean MN frequency was also found by the MN standard test on lymphocytes of the day-care hospital nurses (10.9 versus 7.5; p=0.056), while no differences were found in ward nurses (8.15 versus 7.5; p=0.56). We found that the administration of antineoplastic drugs by nurses in ward units induced a higher frequency of FISH MN+ (43% of subjects) than in the day-care hospital (20%). This was associated with the micronucleus size percentage. This finding could be correlated with the different compositions of administered mixtures of antineoplastic drugs: in ward units the mixtures contained drugs, such as vinorelbine, that were absent in the mixtures administered in the day-care hospital. Our results show genetic damage induced by administration of antineoplastic drugs, particularly in exfoliated buccal cells. This result suggests the useful application of this non-invasive sampling to evaluate genotoxic effects of occupational exposure to mixtures of inhalable chemicals at low doses.