Mean Micronucleus Frequency Research Articles

Urinary cyclophosphamide excretion and micronuclei frequencies in peripheral lymphocytes and in exfoliated buccal epithelial cells of nurses handling antineoplastics

In this study, urinary cyclophosphamide (CP) excretion rate, as well as micronuclei (MN) in peripheral lymphocytes and in buccal epithelial cells were determined for 26 nurses handling antineoplastics and 14 referents matched for age and sex. In urine samples of 20 out of 25 exposed nurses CP excretion rate was found in a range of 0.02–9.14 μg CP/24 h. Our results of the analyses of CP in urine demonstrates that when the nurses were handling CP (and other antineoplastic drugs) this particular compound was observed in urine. The mean values (±SD) of MN frequencies (%) in peripheral lymphocytes from the nurses and controls were 0.61 (±0.32) and 0.28 (±0.16), respectively ( p<0.01). The mean value (±SD) of MN frequency (%) in buccal epithelial cells of nurses was 0.16 (±0.19) and also mean MN frequency in buccal epithelial cells for controls was found to be as 0.08 (±0.08), ( p>0.05). Age, sex and smoking habits have not influenced the parameters analyzed in this study. Handling time of antineoplastics, use of protective equipment and handling frequency of drugs have no effect on urinary and cytogenetic parameters analyzed. No correlation was found between the urinary CP excretion and the cytogenetic findings in nurses. Neither could we find any relationship between two cytogenetic endpoints. Our results have identified the possible genotoxic damage of oncology nurses related to occupational exposure to at least one antineoplastic agent, which is used as a marker for drug handling. As a whole, there is concern that the present handling practices of antineoplastic drugs used in the several hospitals in Ankara will not be sufficient to prevent exposure.

Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5).

MCL-5 cells are Epstein Barr virus-transformed human lymphoblasts which have been genetically engineered for use in mutagenicity testing. We have examined the modal chromosome number, karyotype and spontaneous micronucleus (MN) and sister chromatid exchange (SCE) frequencies of the cell line. Replicate experiments were conducted on two different shipments purchased from Gentest Corp. Although the modal chromosome number was 48 (range 40-54, n = 400 metaphases) for both cell shipments, the second stock showed greater variation in chromosome number than the first. A total of 60 G-banded metaphase cells was analyzed and seven karyotypes were prepared. Consistent structural abnormalities (translocations, deletions and isochromosomes) were found involving the X chromosome and seven autosomes (1-3, 5, 6, 9 and 11). The karyotype typical of this cell line was: 48,der(X)t(X;?)(p22.3;?)Y,t(1;2)(q23;p23),del(3)(q12q21), + i(3q),t(5;6) (q31;p23),+i(9p),der(11)t(11;13)(q23;q12). The mean MN frequency was 41.8 MN/1000 binucleate cells (n = 5000). When compared with our historical controls for primary lymphocyte cultures this number (41.8) is significantly (8.4-fold) higher. The mean SCE frequency was 7.3 per metaphase (n = 100). We observed a hyperdiploid chromosome number of 48 in the majority of metaphase spreads, indicating a significant deviation from the normal diploid number characteristic of the parent cells (RPMI 1788) established in 1969. The variation in chromosome number distribution observed between shipments suggests the potential for further changes. The elevated MN frequency suggests that evaluating mutagenicity using this cytogenetic end-point may require excessive dosing to produce a significant response over background. We conclude that careful interpretation of cytogenetic end-points is necessary when using MCL-5 cells in the light of the possibility of clonal evolution presented here.

The micronucleus assay in the zebra mussel, Dreissena polymorpha, to in situ monitor genotoxicity in freshwater environments

Caged zebra mussels, Dreissena polymorpha, were transplanted to 6 monitoring sites receiving industrial effluents suspected of containing genotoxic chemicals. After a residence time of 2 months, the induction of micronuclei (MN) in haemocytes was determined as a criterion for genetic damage. The mean MN frequencies observed in mussels exposed to effluents ranged between 5.0 and 8.8‰. These rates were significantly higher than the baseline level of 2.0‰ recorded in a concurrent control mussel group reintroduced at the reference location. Biological fitness descriptors (mortality, attachment biotest, condition index, gonadic index) revealed no relationship between the general well-being of the mussels exposed under contrasted environmental conditions and the frequency of MN induced in their haemocytes. The biological feasibility of the transfer technique of zebra mussels, together with a moderate, but significant, inducibility of MN, are major features towards the development of a first tool for in situ monitoring of genotoxicity in freshwater environments using a common indicator species.

Biological monitoring of hospital pharmacy personnel occupationally exposed to cytostatic drugs: urinary excretion and cytogenetics studies.

For evaluation of the risk borne by hospital pharmacy personnel exposed to antineoplastic agents, the incorporation of cyclophosphamide, ifosfamide, and platinum-containing drugs was quantified by the determination of urinary concentrations. In addition, the induction of micronuclei (MN) and sister-chromatid-exchange (SCE) rates in peripheral blood lymphocytes were studied for correlation with the urinary excretion of cytostatic drugs. Cyclophosphamide and ifosfamide were determined in 24-h urine samples using gas chromatography with electron capture (detection limit 2.5 micrograms/l). Voltammetric analysis enabled the determination of platinum concentrations of 4 ng/l. Heparinized blood (20 ml) was drawn and lymphocytes were cultured for MN and SCE studies. In all, 13 hospital pharmacists and pharmacy technicians regularly involved in the preparation of cytostatic drugs participated in this investigation (7 persons represent a follow-up group). All subjects applied standard safety precautions, including the use of a vertical laminar air-flow hood, protective gowns, and latex gloves. On the day of urine sampling an average of 4,870 mg cyclophosphamide, 5,580 mg ifosfamide, and 504 mg platinum-containing drugs were handled. The excretion of 5 and 9 micrograms cyclophosphamide/l urine was measured in two samples, respectively. An elevated level of urinary platinum was found in one pharmacist (22.3 ng/g creatinine) in comparison with a nonexposed control group. Mean frequencies of MN and SCE did not differ significantly between the drug exposed group and control group. The employees who had incorporated chemotherapeutic agents were part of the follow-up group and, thus, particularly cautious and sensitive to a possible hazard. The results emphasize the necessity of improving personal protection of hospital pharmacy personnel occupationally exposed to cytostatic drugs and support the importance of biological monitoring. In an ongoing project in our department the sources of contamination are being investigated parallel to biological monitoring so as to determine critical situations and improve personal protection.

Genotoxic effects of 2- trans-hexenal in human buccal mucosa cells in vivo

In 7 non-smoking healthy volunteers, the number of micronuclei (MN) was determined in exfoliated buccal mucosa cells before and after rinsing the mouth with an aqueous 10 ppm solution of 2- trans-hexenal during 3 consecutive days. All individuals showed at least a doubling of the MN frequency during one of the next 4 days. An increase of the mean group MN frequency was observed on the fourth day, becoming significant between the sixth and the seventh day. During the next 2 days, the MN frequency dropped down to nearly the control level. In a second study, 7 other volunteers were examined before and after eating 3–6 bananas per day over a period of 3 days. The bananas contained about 35 ppm of hexenal. Six of the 7 individuals showed at least a doubling of the MN frequency during one of the next 6 days. An increase in the mean MN counts was also observed, but the difference to the control value became non-significant during the test period. The results show for the first time that the flavoring constituent 2- trans-hexenal, which is present in many human foods exerts genotoxic effects on human buccal mucosa cells in vivo.

Analysis of mitomycin C-induced micronuclei in lymphocytes from malnourished infected children.

The purpose of this study was to determine if peripheral blood lymphocytes from malnourished children with gastrointestinal or respiratory bacterial infection show increased frequencies of Mitomycin C (MMC)-induced micronuclei as compared to well-nourished, infected children. The results indicate that cells from malnourished, infected children had greater chromosome damage. This may indicate that such children would be more susceptible to environmental damage and malignant transformation. Micronucleus frequencies were analyzed in binucleate cells produced by the cytokinesis block method; the overall micronucleus frequency was significantly higher in binucleate cells from malnourished, infected children. The mean micronucleus frequency in MMC-free cultures was 4.3/1000 in malnourished infected children and 1.0/1000 in well-nourished infected children. In MMC-exposed cultures the mean induced micronucleus frequency was 32.6 +/- 6.1 vs. 12.9 +/- 2.3; 68.6 +/- 12.1 vs. 21.0 +/- 5.1, and 88.1 +/- 16.2 vs. 41.7 +/- 5.0 for malnourished and well-nourished children at 20, 40, and 60 ng/ml MMC, respectively. The number of binucleated cells with more than one micronucleus was also higher in malnourished, infected children at all doses tested, including cells with two micronuclei in MMC-free cultures from malnourished, infected children. This increase was not found in peripheral blood lymphocytes from well-nourished infected children.

Intrinsic radiosensitivity of healthy donors and cancer patients as determined by the lymphocyte micronucleus assay

. The purpose of the study was to evaluate the usefulness of the cytokinesis-block micronucleus (MN) assay in assessment of radiosensitivity of lymphocytes in cancer patients. Lymphocytes from 15 cervical cancer patients, 21 head and neck cancer patients, seven lung cancer patients and 19 healthy donors were analysed using MN assay. The proportion of binucleate cells (BC) in cancer patients ranged from 22 to 56% and was significantly lower than in the control group (38-68%). MN frequency assessed five times over 6 months in four healthy donors showed that the interindividual variation was significantly higher than intraindividual. Before (0 Gy) and after irradiation (2 and 4 Gy) no statistical differences in the mean number of MN/BC were observed between healthy donors and cancer patient groups. Nevertheless, statistical cluster analysis allowed each group of donors to be divided into radioresistant and radiosensitive subgroups of patients. They showed significantly different dose-response. Separate comparison of the mean MN frequency within all examined radioresistant and radiosensitive subgroups, showed statistically significant differences only after a dose of 4 Gy. At this dose, the lung cancer patients and cervical cancer patients from radiosensitive subgroups presented significantly higher radiosensitivity than the healthy donors. However, healthy donors from radioresistant subgroup did not differ significantly from cancer patients. This work has shown a high variation in interindividual radiosensitivity of donors and suggests the possibility of identifying radiosensitive patients on the basis of MN assay performed on lymphocytes.

Intrinsic radiosensitivity of healthy donors and cancer patients as determined by the lymphocyte micronucleus assay

The purpose of the study was to evaluate the usefulness of the cytokines-block micronucleus (MN) assay in assessment of radiosensitivity of lymphocytes in cancer patients. Lymphocytes from 16 cervical cancer patients, 23 head & neck (H&N) cancer patients, 7 lung cancer patients and 20 healtly donors were analysed using micronucleus assay. Blood sampies from the patients were taken before radiotherapy. Lymphocytes were irradiated in vitro with60 Co gamma rays in doses 1, 2, 3 and 4 Gy. The proportion of binucleated cell (BC) and the micronuclei (MN) frequency in BC only were counted. The propartion of BC in cancer patients ranged from 20 to 55% and was similar in the control group. MN frequency assessed 5 times over 6 months in 4 healthy donors showed that the inter-individual variation was significantly higher than intra-individual. Before (O Gy) and after irradiation (1–4 Gy) no statistical differences in the mean number of MN/BC were observed between healthy donors and cancer patients group, however the highest values were found in lung cancer patients. Nevertheles, statistical cluster analysis allowed to divide each group of donors into radioresistans and radiosensitive subgroups of patients. They showed significantly different (p<0.05) doseresponse relationship. Separate comparison of the mean MN frequency within all examined radioresistant and radiosensitive subgroups, showed statisticaly significant differences only after a dose of 4 Gy. At this dose, the lung cancer patients fram radioresistant subgroups presented significantly lower radioresistance (p<0.05) than the other subgroups, that is control, cervical and H&N cancer patients groups. Within radiosensitive subgroups, lung cancer patients as well as cervical cancer patients show significantly (p<0.05) higher MN frequency in comparison to control and H&N cancer patients. This work has shown that the MN assay performed on lymphocytes can show differences in the intrinsic radiosensitivity of cancer patients and healthy donors.

Induction of micronuclei in haemocytes and gill cells of zebra mussels, Dreissena polymorpha, exposed to clastogens

Zebra mussels, Dreissena polymorpha, were exposed to four directly acting reference clastogens (mitomycin C, bleomycin, dimethylarsinic acid and potassium chromate) under laboratory conditions. The aim was to examine the inducibility of micronuclei (MN) in haemocytes and gill cells. Positive responses were observed in both tissues for all four substances used under the given test conditions. The mean MN frequencies in treated mussels ranged between 3.2 and 6.9% in haemocytes and between 5.4 and 6.7% in gill cells. The spontaneous MN levels averaged 1.2 and 2.8% in haemocytes and gill cells, respectively. The MN induction capacity of the different chemicals was equivalent in both tissues, except for the treatment with dimethylarsinic acid which generated a significantly higher MN rate in gill cells than in haemocytes. Several characteristics suggest that haemolymph is the more appropriate test tissue for environmental genotoxicity assessment: (1) a shorter preparation time of slides, (2) a more accurate identification of unambiguous MN, (3) a lower baseline MN frequency and a higher induction factor.

Determination of radiation-induced damage in lymphocytes using the micronucleus and microgel electrophoresis 'Comet' assays.

DNA damage assays may be useful as rapid predictors of normal tissue radiosensitivity in clinical samples. We measured in vitro radiation-induced (2 Gy) damage to lymphocytes from cancer patients and normal healthy donors using both the micronucleus and microgel electrophoresis (Comet) assays simultaneously. For damage assessment, there was a good correlation (P < 0.001) between the mean comet lengths and the fraction of cells with comets. There was no correlation with initial damage, determined as the proportion of cells within a sample that formed comets, in comparison with the mean frequency of micronuclei per binucleate cell. However, there appeared to be an association between the determination of repair proficiency in the Comet assay and the mean frequency of micronuclei per binucleate cell in lymphocytes from cancer patients.

Evaluation of micronuclei in exfoliated urothelial cells and urinary thioether excretion of smokers

Mutagens are present in large quantities in the urine of cigarette smokers, thus, their urothelial cells may represent a possible target for absorbed and excreted mutagens. Our aim is to validate the micronucleus (MN) test in exfoliated urothelial cells obtained from urine samples of cigarette smokers. The urinary thioether (UT) test is also carried out on the same individuals in order to find out whether there is any correlation between these two end-points. The mean (±SE) MN frequency and UT determination is 1.93 (±0.11)% and 9.71 (±1.61) mmol SH/mol creatinine for 23 smokers, and 0.66 (±0.05)% and 4.20 (±0.56) mmol SH/mol creatinine for 20 nonsmokers. Our results show a higher frequency of micronucleated cells ( p<0.001) and higher excretion of UT's ( p<0.05) in smokers as compared to nonsmokers. Concentrations of UTs and MN frequencies increased with tobacco consumption. The MN frequencies showed only a marginal increase, not significant ( p>0.05), after passive smoking compared to nonsmoking values. There was no significant correlation between MN frequencies and UTs, either in smokers ( r = 0.164, p > 0.05) or in nonsmokers ( r = −0.018, p > 0.05). Our data demonstrate tobacco-induced chromosome damage in bladder tissue consistent with increased risk of cancer at this site among smokers.

Do persons living near a uranium processing site have evidence of increased somatic cell gene mutations? A first study

The objective of this study was to examine if individuals living near a uranium processing site have greater mutagenic damage, as measured by three mutagenicity assays, compared with subjects unexposed to any nuclear facilities. The design was a cross-sectional exploratory analysis of 112 subjects; 56 volunteer residents were from within a 5-mile radius of the Fernald Uranium Processing site and 56 ‘control’ subjects were from a geographically separate area unexposed to any known uranium emissions. The groups were constrained to be similar in age and sex composition. The main outcome measures were three human somatic gene mutation assays consisting of the HPRT T-lymphocyte cloning assay to measure 6-thioguanine resistant lymphocytes; the glycophorin A assay to detect the loss of expression of the M or N allele; and the micronucleus assay as a marker of chromosomal damage. The results showed no statistically significant or quantitatively important differences between groups for all three mutagenicity assays; only the unselected cloning efficiency was statistically significantly different between groups (0.42±0.16 for the Fernald versus 0.35±0.12 for the comparison groups). In both groups, age was significantly related to HPRT mutant frequency, with a 1.25% rate of increase in mutant frequencies for each 1-year gain of age in the Fernald group and a 1.12% rate of increase in mutant frequencies for each 1-year gain in the comparison group. For the micronucleus data, females had a greater mean micronucleus frequency than males. In addition, smokers had an increased mean ln (natural logarithm) HPRT mutant frequency (3.06±0.14 for current smokers compared with a mean of 2.72±0.05 for non-current (i.e. never plus former)smokers). Our results are consistent with the previously reported association between sex type and micronucleus frequency, the known relationship between age and T-lymphocyte cloning efficiency and age and HPRT mutant frequency, and verify the wide inter-subject variability for the latter. Finally, we conclude that at a population level, the relationhips between current cigarette use and HPRT mutant frequency, and sex type and micronucleus frequency, are stronger than is the association between geographic proximity to a uranium processing site and mutagenic abnormalities.

Praziquantel (antischistosomal drug): is it clastogenic, co-clastogenic or anticlastogenic?

Schistosoma haematobium infection is the most common health problem in Egypt. It is strongly associated with the development of urinary bladder carcinoma. The actual cause for the development of cancer is still under investigations, it can be due to mechanical irritation from schistosomiasis ova, the infection itself or the drugs which are used to treat the patients. Praziquantel (PQ) is a commonly used drug to treat schistosomiasis patients. In mice, an in vivo cytogenetic study showed that PQ is not clastogenic in mice. The frequency of micronuclei in all the study groups were insignificantly different from the control group ( p > 0.05). However, it enhanced the clastogenicity of benzene at a very high dose. Results from combined exposure with benzene and PQ showed a significant PQ dose-dependent increase in micronucleus frequency ( p < 0.05). A metabolite study revealed that PQ enhanced the metabolism of benzene to form muconaldehyde which may be responsible for the enhancement effect. In schistosomiasis patients, two cytogenetic studies were carried out before and after treatment with PQ. In one of these studies, peripheral blood lymphocytes were examined from schistosomiasis patients to detect chromosomal aberrations (CAs) and micronuclei (MN) before and after treatment with PQ. There was no significant increase in CAs in patients compared with the controls ( p > 0.05). The frequency of MN was significantly higher in the infected persons (0.59 ± 0.44) than the control individuals (0.23 ± 0.23) ( p < 0.05). After treatment, there was no significant change in both parameters. The other study was conducted to determined whether infection with this parasite resulted in an increase of chromosomal breakage, micronuclei, in exfoliated urothelial cells. Micronucleus frequencies were significantly higher in the infected group (mean frequency, 0.84 ± 0.69%) than among controls (mean frequency, 0.12 ± 0.21%, p < 0.001). Micronucleus frequencies were higher in infected individuals who smoked compared with those who were non-smokers, although this effect was not significant ( p > 0.05). The mean micronucleus frequencies were reduced significantly in the group of patients who were followed up (before treatment, 0.80 ± 0.70%, after treatment, 0.19 ± 0.23%, p < 0.001), thus supporting a direct involvement of the infection in increased chromosomal breakage in the urothelium and provide proof of the role of PQ in decreasing the risk of cancer development. At this stage, we still need to study the cytogenetic effect of human exposure to environmental agents such as pesticides, smoking, etc., together with treatment with PQ.

Reduction in chromosomal damage in schistosomiasis patients after treatment with praziquantel

Schistosoma haematobium infection is the most common health problem in Egypt. It is strongly associated with the development of urinary bladder carcinoma. The aim of this study was to determine whether infection with this parasite resulted in an increase of chromosomal breakage in the exfoliated urothelial cells. Urine samples were collected from 211 Egyptian male farmers and schoolchildren with Schistosoma haematobium ova in their urine. 66 samples were suitable for scoring. Accordingly, 66 non-infected males attending the same village clinic were included in the study as a control group. The urine was centrifuged and the urothelial cells present in the sediment were assayed for the presence of micronuclei, a quantitative indicator of chromosomal breakage. Micronucleus frequencies were significantly higher in the infected group (mean frequency, 0.84 ± 0.69%) than among controls (mean frequency, 0.12 ± 0.21%, p < 0.001). There was a trend towards an increase in micronucleus frequencies in infected individuals who smoked (17 of the 66 subjects) compared with those who were non-smokers, although this effect was not significant ( p > 0.05). Bladder infection was associated with a 6.1-fold increase in micronucleus frequencies among non-smokers. In this study, patients were treated with praziquantel, an anti-schistosomal agent. Out of the 66 studied patients, only 37 gave scoreable samples after treatment. The mean micronucleus frequencies were reduced significantly (before treatment, 0.80 ± 0.70%, after treatment, 0.19 ± 0.23%, p < 0.001), thus supporting a direct involvement of the infection in increased chromosomal breakage in the urothelium.

Chromosomal damage in urothelial cells from egyptians with chronic Schistosoma haematobium infections

The most common cancer in Egypt is urinary-bladder carcinoma, which is strongly associated with infection with Schistosoma haematobium. The object of this study was to determine whether infection with this parasite resulted in an increase in chromosomal breakage in the urothelium. Urine samples were collected from 37 Egyptian farmers with S. haematobium ova in their urine and from 32 non-infected males attending the same village clinic. The urine was centrifuged and the urothelial cells present in the sediment were assayed for the presence of micronuclei, a quantitative indicator of chromosomal breakage. Micronucleus frequencies were significantly higher in the infected group (mean frequency, 0.97 +/- 0.12%) than among controls (mean frequency, 0.12 +/- 0.04%, p less than 0.001). There was a trend towards an increase in micronucleus frequencies in infected individuals who smoked (10 of the 37 subjects) compared with those who were non-smokers, although this effect was not significant (p = 0.12). Bladder infection was associated with a 6.8-fold increase in micronucleus frequencies among non-smokers. In a pilot study, 11 patients were treated with praziquantel, an anti-schistosomal agent. Mean micronucleus frequencies were reduced significantly (before treatment, 1.27 +/- 0.16%; after treatment, 0.30 +/- 0.16%, p less than 0.001), thus supporting a direct involvement of the infection in increased chromosomal breakage in the urothelium.

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclasses of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the dastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for tile detection of the clastogenic potential of promutagenic chemicals.

A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes.

A collaborative study between two laboratories has been carried out in order to investigate the reproducibility of the cytokinesis-block micronucleus assay in human lymphocytes. A modified protocol to obtain good quality slides from whole blood cultures has been developed. The spontaneous frequencies of micronuclei in binucleated lymphocytes from five male subjects were evaluated in each laboratory at two cytochalasin-B concentrations (3 and 6 micrograms/ml). Our data show that 6 micrograms/ml cytochalasin-B was more effective than 3 micrograms/ml to arrest cytokinesis in all the donors analysed. The mean frequency of micronuclei in binucleated cells was significantly lower at 6 micrograms/ml compared to 3 micrograms/ml cytochalasin-B in both laboratories. The results obtained indicate a very high concordance between both sets of data supporting the validity of the proposed protocol.

Factors contributing to chromosome damage in lymphocytes of cigarette smokers

Cigarette smoking is generally believed to be responsible for a substantial number of human health problems. However, the causal relationship between smoking, the induction of biological effects and the extent of health problems among smokers have not been fully documented. Using the recently developed lymphocyte micronucleus (MN) assay, we have evaluated the chromosome aberration frequencies in 67 cigarette smokers and 59 matched non-smoking control subjects. We found that the mean MN frequency (per 100 cells) in the smokers was slightly higher than that found in the non-smokers (0.71 ± 0.23 and 0.58 ± 0.05 respectively; p < 0.08). Factors which contribute to the expression of chromosome aberrations were also investigated. A significant age-dependent increase in MN frequencies was observed in both groups ( p < 0.05). Linear regression analysis showed that the age-dependent effects among smokers ( r = 0.54; p < 0.02) was further enhanced by cigarette consumption ( r = 0.62; p < 0.005). Consumption of low potency ‘one-a-day’ type multivitamins had no effect on MN frequencies in either sex of non-smokers and in the 1 male smoker who took multivitamins but vitamin intake consistently reduced the MN frequencies among female smokers. Using a challenge assay, fidelity of DNA repair was evaluated. Lymphocytes from both smokers and non-smokers were irradiated with single doses of 0 or 100 cGy of X-rays or with double doses of 100 cGy of X-rays each separated by 15 or 60 min (100/15 or 100/60). Chromosome translocation frequencies were consistently higher after irradiation in lymphocytes from smokers than in those from non-smokers. Statistically significant differences were detected when the cells were irradiated with the double doses of 100 cGy X-rays each separated by 60 min ( p < 0.05). These data suggest that lymphocytes from smokers made more mistakes in the repair of DNA damage than cells from non-smokers. Our studies provide new insights into the genotoxic effects of cigarette smoke and new information which may be useful for understanding the mechanisms for induction of health problems from smoking.

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.