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.     

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.     

Radiation-induced micronucleus frequency in peripheral blood lymphocytes is correlated with normal tissue damage in patients with cervical carcinoma undergoing radiotherapy

In an effort to find a test to predict the response of normal tissue to radiotherapy, the lymphocyte micronucleus assay was used on blood samples from patients with cervical carcinoma. Peripheral blood samples from 55 patients with advanced-stage (II B-IV B) cervical carcinoma were obtained before radiotherapy. The patients were treated with external-beam radiotherapy followed by high-dose-rate brachytherapy. Acute and late normal tissue reactions were scored and correlated with the micronucleus frequency in lymphocytes after irradiation with 4 Gy in vitro. Great interindividual variability was observed in the radiation-induced lymphocyte micronucleus frequency, especially at 4 Gy. The mean number of micronuclei per 100 binucleated cells in cells irradiated with 4 Gy in vitro was significantly higher in samples from patients who suffered from acute and/or late normal tissue reactions than in those from patients with no reactions (51.0 +/- 17.7 and 29.6 +/- 10.1, respectively). A significant correlation was also found between the micronucleus frequency at 4 Gy and the severity of acute reactions and late reactions. However, the overlap between the micronucleus frequencies of patients with high-grade late normal tissue reactions and low-grade reactions is too great to recommend the micronucleus assay in its present form for routine clinical application.     

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.     

Azithromycin treatment modulates the extracellular signal-regulated kinase mediated pathway and inhibits inflammatory cytokines and chemokines in epithelial cells from infertile women with recurrent Chlamydia trachomatis infection

Epidemiological and animal model studies suggest that sequelae of genital Chlamydia trachomatis infection are more often associated with second or subsequent infections than with initial infection. Further, in order to establish an acute or long-term persistent infection, C. trachomatis develops several strategies to circumvent host immune responses. Hence, resolution of the C. trachomatis infection may require modulation of host factors especially during persistent or chronic infection. Moreover, azithromycin treatment has been reported to possess anti-inflammatory properties but its mechanism of action is still not elucidated. Therefore, in order to better understand the effect of azithromycin in chronic conditions, our aim was to study changes in expression of key genes associated with inflammatory cytokines and receptors, mitogen-activated protein kinase (MAPK) signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Real-time polymerase chain reaction was performed to study inflammatory cytokines and receptors, MAPK signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Further, effect of azithromycin on activation of extracellular signal-regulated kinase was studied in epithelial cells by western blotting. Chemokine (C-C motif) ligand 2 (CCL2), CCL5, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, CXCL9, interleukin-1B (IL-1B), IL-8, baculoviral IAP repeat-containing 3 (BIRC3), myeloid cell leukemia sequence 1 (MCL1), and MAPK1 were downregualted after azithromycin treatment. In addition, phosphorylation of extracellular signal-regulated kinase was inhibited after azithromycin treatment in epithelial cells obtained from women with recurrent infection. Hence, our data suggest that azithromycin with its properties apart from antibacterial activity may contribute to its therapeutic potential in treatment of chronic recurrent infection in infertile women.     

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.     

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.     

Mutagenicity studies on paracetamol in human volunteers. III. Cytokinesis block micronucleus method

As paracetamol (PC) affected the frequency of chromosome aberrations and unscheduled DNA synthesis in human volunteers, the genotoxic effects of PC were studied in a group of 12 healthy volunteers (9 females; 3 males, aged 37.8 +/- 8.7 years) using the cytokinesis block micronucleus method in human peripheral lymphocytes. As a positive control a group of elderly people was used, 20 females and 10 males, aged 79.9 +/- 9.5 years. PC was administered orally 3 times in a dose of 1000 mg during 8 h. Blood samples were taken at intervals of 0, 24, 72 and 168 h after the first dose of PC. Cytochalasin B was added to the cultures 44 h after the beginning of the 72-h cultivation at a concentration of 3.0 micrograms/ml. The frequency of cells with micronuclei in the group of volunteers was not significantly increased after PC administration. Using the cytokinesis block micronucleus method, the frequency of micronuclei was stable and the interindividual variability was low. The application of the micronucleus technique in genetic monitoring, e.g., for the occupational exposure to mutagens, is questioned.     

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.     

Treatment of Mycoplasma genitalium. Observations from a Swedish STD Clinic

Objectives

To evaluate therapy for Mycoplasma genitalium infection with doxycycline or azithromycin 1 g compared to five days of azithromycin (total dose 1.5 g).

Methods

A retrospective case study was performed among patients attending the STD-clinic in Falun, Sweden 1998–2005. All patients with a positive PCR test for M. genitalium were routinely offered a test of cure (toc). Response to doxycycline for 9 days, azithromycin 1 g single dose and extended azithromycin (500 mg on day 1 followed by 250 mg o.d. for 4 days) was determined. In patients with treatment failure after azithromycin, macrolide resistance was monitored before and after treatment. Furthermore, the rate of macrolide resistance was monitored for positive specimens available from 2006–2011.

Results

The eradication rate after doxycycline was 43% (48% for women and 38% for men), for azithromycin 1 g 91% (96% for women and 88% for men) and for extended azithromycin 99% (100% for women and 93% for men). Macrolide resistance developed in 7/7 examined (100%) of those testing positive after azithromycin 1 g, but in none of those treated with extended azithromycin. Macrolide resistance before treatment increased from 0% in 2006 and 2007 to 18% in 2011.

Conclusions

These findings confirm the results from other studies showing that doxycycline is inefficient in eradicating M. genitalium. Although azithromycin 1 g was not significantly less efficient than extended dosage, it was associated with selection of macrolide resistant M. genitalium strains and should not be used as first line therapy for M. genitalium. Monitoring of M. genitalium macrolide resistance should be encouraged.     

Micronuclei in human lymphocytes irradiated in vitro or in vivo

Venous blood from healthy donors or from patients with various lympho- and myeloproliferative diseases was incubated in vitro in the presence of cytochalasin B for the induction of binucleated lymphocytes. The time at which cytochalasin B was added depended on the proliferation rate of the lymphocytes. Proliferation was monitored using a semiautomatic microscope photometer/computer system. The background level of micronuclei in binucleated lymphocytes of the patients before radiotherapy was statistically indistinguishable from that of healthy persons. Blood from both groups was irradiated in vitro for the study of the dose-response relationship. The dose-response curves were very similar up to 3.75 Gy, and a somewhat lower micronucleus frequency was found in lymphocytes of patients after a 5-Gy exposure. These in vitro results were compared with in vivo exposure after total-body irradiation of leukemic patients. Due to heavy medication that accompanied radiation therapy, only two doses (1.25 and 2.5 Gy) could be checked after in vivo exposure. There was no statistically significant difference between in vitro and in vivo results after 1.25 Gy, but a slightly lower number of micronuclei was observed after in vivo exposure to 2.5 Gy.     

The genotoxic risk of hospital, pharmacy and medical personnel occupationally exposed to cytostatic drugs--evaluation by the micronucleus assay

The aim of this study was to evaluate the genotoxicity of cytostatic drugs in hospital and pharmacy employees (n=100), occupationally exposed. The micronucleus assay was used to study lymphocytes in 247 peripheral blood samples. Samples were collected at "baseline level" without any cytostatic drugs exposure before recruiting or after at least 3 weeks without cytostatic drugs contact and at three times (cycle 1-3) post-exposure. Samples from 60 office employees served as controls. Furthermore, our results were compared to urinary analyses of cytostatic drugs (oxazaphosporines, anthracyclines, platinum) which were collected in parallel to the cytogenetic investigation. Statistical analyses were performed under consideration of age, gender and X-ray exposure. The frequency of micronuclei was significantly related to the age of the subjects (r(Spearman)=0.16; P<0.05). However, there were no significant differences in micronucleus rates between controls and exposed hospital workers. Similarly, micronucleus rates were not significantly different at the various sampling time points and there was no correlation between duration of employment and micronucleus rates. Furthermore, no correlation between current biomonitoring data of exposure (urine tests) and micronuclei frequency was found. Therefore, significantly increased genotoxic damage of the lymphocytes investigated in this study could not be demonstrated.     

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.     

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.     

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     

Effects of age, sex and diagnostic X-rays on chromosome damage

The frequency of micronuclei assayed in lymphocytes obtained from 73 young adults increases significantly with the age, but not the sex, of the donor. The dose of medical X-rays absorbed by the lymphocytes in the 2 years before the examination has no significant effect on micronucleus frequency, provided the data are adjusted for age. However, a small significant increase in frequency is associated with X-ray examinations that involve the injection of contrast media into the blood.     

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.     

Micronucleus frequency is increased in peripheral blood lymphocytes of nuclear power plant workers

Nuclear power plant workers are exposed to ionizing radiation at relatively low doses and for prolonged periods of time. To investigate the extent of genetic damage in these workers, a group of 133 nuclear power plant workers and 39 healthy controls were compared using the cytokinesis-block micronucleus assay. The frequency of micronuclei was significantly increased in peripheral lymphocytes of nuclear power plant workers (20.5 +/- 9.7% compared to 13.7 +/- 5.9%). A significant dose-response relationship was observed between micronucleus (MN) frequency and both the accumulated dose and the duration of employment (P < 0.01 for both variables after adjusting for age, gender and cigarette smoking) with an evident leveling off for exposures over 200 mSv. Accumulated dose and duration of employment were significantly correlated but exerted independent effects on MN frequency. For non-occupational parameters, age was significantly associated with the frequency of micronuclei, while gender was not. Smoking habit showed no overall effect, whereas increased chromosome damage was evident in smokers of more than 20 cigarettes per day. In conclusion, a dose-related association between MN frequency and exposure to ionizing radiation was evident in nuclear power plant workers, encouraging the application of the cytokinesis-block micronucleus assay in biomonitoring studies of human populations with prolonged exposure to ionizing radiation.     

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.