DNA content of micronuclei in human lymphocytes

We have calculated the distribution of DNA contents in micronuclei (MN) induced by ionizing radiation in human lymphocytes on two assumptions: the MN arise from acentric chromosome fragments (ACF), and the ACF result from the random breakage and rejoining of chromosomes. Measurements show that about 80 per cent of MN have a DNA content in the range of 0.5-6 per cent of the G1 nucleus. This group is consistent with the model and shows little dependence on radiation dose over the dose range of 0.5-4 Gy, or on lymphocyte culture time, varying from 48 to 76 hours. The MN with DNA content from 6 to 20 per cent of the G1 nucleus are probably the result both of spindle defects and of DNA synthesis in MN.     

Radiation induction of micronuclei in human lymphocytes

An examination of the cytokinesis-blocked micronucleus technique confirmed its potential usefulness as a method of biological dosimetry for radiation accidents. Several advantages and disadvantages of the system are discussed. It has been demonstrated that under these conditions of these experiments, the blocking agent, cytochalasin B does not induce micronuclei or unstable chromosome aberrations. The induction of sister-chromatid exchanges proved just significant.Analysis of the dose response for 250 kVp X-rays indicates that although the Y = αD + βD² model fits the data, the relationship does not correspond to that for total aberration induction as might have been expected. The background frequency of micronuclei and the value of the α coefficient are higher than for total aberrations and the β term is lower. This indicates that simple incorporation of acentric chromosome fragments into micronuclei may not wholly account for the phenomenon.     

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.     

Quantitative estimation of nuclear buds and micronuclei in bovine cells transformed by Rous sarcoma and SV 40 viruses

Summary A study of nuclear-budding and micronuclei formation has been performed on bovine fibroblastic cells in serial tissue culture. A comparison was made between control cells and cells morphologically transformed by Rous sarcoma virus and SV 40 virus. It was found that no obvious differences in the frequency of nuclear buds and micronuclei existed between RSV transformed and control cells whereas SV 40 transformed cells showed a high frequency of nuclear buds and micronuclei. There are indications that the frequency of nuclear buds and micronuclei is correlated to chromosomal abnormalities.Supported by grants from the Medical Faculty of the University of Uppsala.     

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.     

Effect of ELF-EMF on antioxidant status and micronuclei in K562 cells and normal lymphocytes

The effect of ELF-EMF on DNA through changes in antioxidative enzyme activities has not been sufficiently explored yet. The aim of this study was to determine ELF-EMF effect on antioxidative enzymes in cancer cell line and genotoxic potential on normal human lymphocytes. K562 cells were exposed to 50 Hz ELF-EMF (40 μT, 100 μT; 3 h, 24 h) and spectrophotometric determination of lipid peroxidation and antioxidative enzyme activities was conducted. Genotoxicity of ELF-EMF (50 Hz, 100 μT) was investigated by cytokinesis-block micronucleus assay in a normal human lymphocytes (exposure 24 h and 48 h). Results demonstrated that ELF-EMF did not alter the process of lipid peroxidation and superoxide dismutase activity. Catalase activity was increased only after application of 100 μT EMF for 24 h. Glutathione-S-transferase and -reductase activities were increased. Treatment with 100 μT ELF-EMF (24 h, 48 h) significantly reduced micronuclei incidence, while cell proliferation was significantly increased. Results indicate that 50 Hz ELF-EMF (40 μT, 100 μT) are week stressors which alone cannot generate enough ROS to induce process of lipid peroxidation in cancer cell line but strong enough to induce response of antioxidative system. Furthermore, 100 μT ELF-EMF in human lymphocytes did not exhibit genotoxic potential during 24 h and 48 h treatment, but stimulated cell proliferation.     

Induction of micronuclei in human lymphocytes exposed in vitro to microwave radiation

Increasing applications of electromagnetic fields are of great concern with regard to public health. Several in vitro studies have been conducted to detect effects of microwave exposure on the genetic material leading to negative or questionable results. The micronucleus (MN) assay which is proved to be a useful tool for the detection of radiation exposure-induced cytogenetic damage was used in the present study to investigate the genotoxic effect of microwaves in human peripheral blood lymphocytes in vitro exposed in G(0) to electromagnetic fields with different frequencies (2.45 and 7.7GHz) and power density (10, 20 and 30mW/cm(2)) for three times (15, 30 and 60min). The results showed for both radiation frequencies an induction of micronuclei as compared to the control cultures at a power density of 30mW/cm(2) and after an exposure of 30 and 60min. Our study would indicate that microwaves are able to cause cytogenetic damage in human lymphocytes mainly for both high power density and long exposure time.     

The induction of micronuclei in human lymphocytes by low doses of radiation

The appearance of micronuclei (MN) is delayed with respect to cell division in populations of irradiated human lymphocytes, so that the length of time in culture, as well as the number of divisions, is a factor in MN assays. Using two assays that control for cell kinetics, we measured the yield of cells with MN exposed to graded doses of 60Co gamma rays and 90KVP X-rays. The yields showed a non-linear increase with dose. They can be represented by two straight lines: the one in the range below 0.15 Gy has a slight slope, the other in the range above 0.15 Gy has a significantly greater slope. The radical scavengers cysteamine and glycerol, which reduced the MN yields sharply at 3 Gy, were less effective at 0.3 Gy, indicating that terminal deletions arising from the direct ionization of DNA are a major source of the MN induced by low radiation doses. It is likely that the non-linear dose response is due to the saturation of a DNA repair process.     

Induction of mitotic micronuclei by X-ray contrast media in human peripheral lymphocytes

In vitro and in vivo cytogenetic effects of X-ray contrast media (CM) were determined by scoring micronuclei (MN) in 72-h cultures of human peripheral lymphocytes. Both ionic (sodium meglumine diatrizoate, methylglucamine diatrizoate, and sodium meglumine ioxaglate and nonionic CM (iosimide, iopromide, iohexol and iotrolan) were able to induce MN in lymphocytes. Based upon their calculated percent probabilities for MN induction, these agents could be ranked in their decreasing order of probability, as iosimide greater than sodium meglumine ioxaglate greater than iohexol greater than sodium meglumine diatrizoate greater than iopromide greater than methylglucamine diatrizoate greater than iotrolan. Stepwise logistic regression analysis of the data indicated that the frequency of MN in CM-exposed lymphocyte cultures was significantly higher than the frequency of MN in control cultures (P less than 0.001). In clinical studies where 14 patients were injected with an ionic CM methylglucamine diatrizoate, lymphocyte cultures from 10 patients showed higher frequencies of MN. The differences between pre- and post-CM counts of MN were significant in a Mann-Whitney U test (P less than 0.05). The effect of X-irradiation on MN formation in lymphocytes was separately determined and was found to be insignificant. These results indicate that irrespective of ionic and osmolality differences, X-ray contrast agents are capable of producing chromosomal damage in peripheral lymphocytes. Further studies are required to establish molecular mechanisms in the observed cytogenetic effects of CM in cell cultures.     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which – although providing antioxidant defence – may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, β-carotene, B₁₂, folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and β-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B₁₂ were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and β-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P < 0.001). There were no significant differences in serum levels of folic acid and Vitamin B₁₂ between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r = 0.62, P = 0.001, N = 24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P = 0.007), as did vegetarian women compared with non-vegetarian women (P = 0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r = 0.62, P < 0.001, N = 48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N = 48, P < 0.001).     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which - although providing antioxidant defence - may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, beta-carotene, B(12), folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and beta-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B(12) were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and beta-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P<0.001). There were no significant differences in serum levels of folic acid and Vitamin B(12) between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r=0.62, P=0.001, N=24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P=0.007), as did vegetarian women compared with non-vegetarian women (P=0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r=0.62, P<0.001, N=48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N=48, P<0.001).     

The effect of aging on micronuclei frequency and proliferation in human peripheral blood lymphocytes

INTRODUCTION:

Increase in the instability of cellular genome with an increasing age is the result of an accumulation of cellular damage and mutations. This instability which might be observed as chromosome damage or chromosome losses can be measured by the micronucleus technique.

AIM:

The aim of this study was to investigate the effect of aging and oxidative stress induced by non-toxic levels of H₂O₂ on micronuclei induction and their relationship to cell proliferation in human peripheral blood lymphocytes.

MATERIALS AND METHODS:

Healthy volunteers with different ages were choosen. Spontaneous and H₂O₂ induced micronuclei frequencies were measured in peripheral blood lymphocytes of 30 volunteers by the micronucleus method.

RESULTS:

Spontaneous micronuclei frequencies increased first then started to decrease after 50 years of age. This biphasic response was significantly higher than micronucleus (MN) frequencies induced by H₂O₂ (P < 0.05), which followed the similar shape of response to increasing ages with lower frequencies. Proliferative capacity of cells either treated with H₂O₂ or not did not differ with an increasing age giving similar responses.

CONCLUSION:

These results indicate biphasic character of chromosome damage; first increase and decrease after 50 years with an increasing age. But this change pattern was not correlated with the steady state of proliferation capacity of cells through an increasing age. Decreases in H₂O₂-induced MN frequencies compared to spontaneous MN frequencies may be inducing an apoptosis by H₂O₂ treatment leading to underscoring damaged cells.     

Protective effects of cimetidine on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes

The radioprotective effects of cimetidine, which has been used clinically as an antagonist of H 2 receptor, on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes (PBL) prepared from healthy donors were studied. Cells were treated with cimetidine before or after X-irradiation, and then cytokinesis-blocked micronucleus assay and flow cytometry for measurement of phosphatidylserine externalization were utilized to evaluate the radiation-induced cytogenetic damage and apoptosis. The protective effect of pre-irradiation treatment of cimetidine on radiation-induced micronuclei was dependent on the concentration. The maximum protection rates of cimetidine (1 mM) on frequencies of micronuclei were 38.8 and 30.2% for cells treated before and after X-irradiation (5 Gy), respectively. Protective effects of pre- and post-irradiation treatment with cimetidine on radiation-induced early apoptosis and decreased activity of caspase-3 were observed. A study of electron paramagnetic resonance-spin trapping with 5,5'-dimethyl-1- N -oxide revealed that the rate constant of cimetidine with radiation-induced OH radicals is about 4.5 ×10 9 l/mol/s. Cimetidine did not significantly increase the intracellular concentration of glutathione. These results suggest that cimetidine suppresses radiation-induced micronuclei and apoptosis via OH radical scavenging and an intracellular antioxidation mechanism. Cimetidine appears to be a useful candidate for the future development of post-irradiation radioprotectors.     

Mechanism of the formation of micronuclei in PHA-stimulated lymphocytes from human peripheral blood

The micronuclei of PHA-stimulated human lymphocytes were assayed by comparison of electron microscopy and light microscopy data. The length of the cell cycle was checked by the flow cytofluorimetry method. Both the variability of the apoptosis stages and the increase in (G2+M) period in gamma-irradiated cultures were found. It is concluded that at least part of the micronuclei results from the cell nucleus "partition" during the apoptosis type cell death.     

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.     

Glucocorticoid receptors in normal human lymphocytes

Glucocorticoid (GC) receptors were studied in intact lymphocytes from 11 donors. GC binding parameters were found to be highly reproducible in repeated experiments with lymphocytes. It was shown that GC receptors in donors' lymphocytes could be distributed into two different classes similarly to the pattern seen in skin fibroblasts. Human lymphocytes are an adequate object for studying genetically determined variability of GC receptors and its clinical importance.     

The size of micronuclei in human lymphocytes varies according to inducing agent used

Micronuclei were induced in vitro in human lymphocytes by mitomycin C, X-rays, vincristine, and colcemid and analyzed in cells with preserved cytoplasm. The micronucleus/cell nucleus ratio was measured. It was found that micronuclei induced by mitomycin C and X-rays were significantly smaller than those formed by vincristine and colcemid. Thus, in spite of the wide size span of human chromosomes, it could be shown that it is possible to differentiate between micronuclei formed by spindle-damaging agents (vincristine and colcemid) and those induced by agents directly damaging the chromosomes (mitomycin C and X-rays). Mitomycin C-induced micronuclei were smaller than those induced by X-rays, probably because the former agent preferentially produces chromatid fragments and the latter chromosome fragments.