Flow cytometric analysis of micronuclei found in cells after irradiation

Exposure of mammalian cells to either ionizing radiation or mutagenic and carcinogenic substances can induce chromosome aberrations. These aberrations in turn may give rise to micronuclei which can be found in cells during the interphase after division. A two-step method is presented that allows separation of micronuclei from cell nuclei. They can then be measured and analysed according to their DNA content in a flow cytometer. The method involves an initial detergent treatment of cells followed by a second treatment with sucrose and citric acid. Micronuclei with DNA content larger than 2% of the G1-nuclei can be measured. The method is tested and compared with microscopic observations of micronucleated cells in irradiated, asynchronous, and synchronized Ehrlich ascites tumour cells growing in vitro. The agreement between the flow cytometric technique and microscopic observations is excellent when the dose-dependent number of micronuclei per cell is taken into consideration.     

Persistence of micronuclei in lymphocytes of cancer patients after radiotherapy

To verify the applicability of the micronucleus (MN) yield in peripheral blood lymphocytes (PBLs) as a quantitative biodosimeter for monitoring in vivo ionizing radiation damage, we applied the cytokinesis-blocked micronucleus assay in PBLs of cancer patients treated with partial-body radiotherapy. Dosimetric information on these 13 patients represented a wide range in the number of fractions, cumulative tumor dose, total integral dose, and equivalent total-body absorbed dose. We found in PBLs of these patients that (1) the MN yield increased linearly with the equivalent total-body absorbed dose (r = 0.8, P = 0.002), (2) the distributions of the MN yields deviated significantly from Poisson, and (3) there was a general decline in MN yields with increasing length of follow-up, but with considerable variation between individuals. The average rate of decline was found to be linear and was correlated with the equivalent total-body absorbed dose (r = 0.7, P = 0.007). Further, at 19-75 months of follow-up time, seven patients showed higher MN yields than their respective levels before radiotherapy, indicating the persistence of radiation-induced residual cytogenetic damage. Our findings suggest that the MN yield in human PBLs offers a reliable acute and perhaps chronic biodosimeter for in vivo radiation dose estimation. After the completion of radiotherapy, the persistence of elevated MN yield in PBLs is a reflection of the surviving population of radiation-induced genetically aberrant cells.     

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.     

The effect of electron and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human blood lymphocytes

The effect of electrons and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human peripheral blood lymphocytes was investigated to understand the relative biological effectiveness (RBE) of electrons compared with gamma rays. Blood samples were irradiated with an 8 MeV pulsed electron beam, at a mean instantaneous dose rate of 2.6 × 10⁵ Gy s⁻¹. Gamma irradiation was carried out at a dose rate of 1.98 Gy min⁻¹ using ⁶⁰Co gamma source. A dose-dependent increase in micronuclei yield was observed. The dose–response relationships for induction of micronuclei fitted well to a linear–quadratic relationship and the coefficients α and β of the dose–response curve were estimated by fitting the data using error-weighted minimum χ ² method. The RBE of 8 MeV electrons were found to be near unity as compared with gamma rays.     

Induction of micronuclei in plant cells after exposure to accelerated ion irradiation

The root apex cells ofPisum sativum were irradiated in an U-240 isochronous cyclotron at the Institute for Nuclear Research of the Ukrainian National Academy of Sciences, Kyiv. Several types of micronuclei, differing in structural peculiarities, were observed in the 84 h following exposure to beams of accelerated¹H,⁴He, and¹⁴N ions with linear energy transfer (LET) of 0.95, 9.34, and 221 keV/µym, respectively. The maximum micronucleus induction was observed after irradiation with helium ions. Results obtained show that the micronucleus assay is a responsive test for investigations of cytogenetic damage produced by high LET beams in dividing cell systems in vivo.     

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.     

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.     

Meta-analysis of increases in micronuclei in peripheral blood lymphocytes after angiography or excretory urography

Norman, A., Cochran, S. T. and Sayre, J. W. Meta-analysis of Increases in Micronuclei in Peripheral Blood Lymphocytes after Angiography or Excretory Urography. Radiat. Res. 155, 740-743 (2001). Meta-analysis of 10 studies confirms a significant increase in the frequency of micronuclei in peripheral blood lymphocytes after angiography or excretory urography; the weighted average increase is 4.2 (95% confidence interval 2.8-5.6) per 1000 binucleate lymphocytes, about the same increase in micronuclei as that produced in vitro by a diagnostic X-ray dose of 4 cGy. The analysis failed to reveal a significant effect of the specific contrast medium used in the X-ray examinations on the increased frequency of micronuclei. These results are consistent with the hypothesis that the effect of the contrast media is limited to the enhancement, by the photoelectric effect, of the X-ray dose absorbed by the lymphocytes irradiated while suspended in the contrast medium. Therefore, an estimate of increased cancer risk based on elevated frequencies of micronuclei or chromosome aberrations in peripheral blood lymphocytes may be greatly exaggerated whenever the radiation damage is largely confined to the cells circulating in the blood, as it is in people who have recently had X-ray examinations that use intravenous injections of contrast medium. Such examinations include angiography, excretory urography and CT scans, which are received annually by millions of people.     

Activation of transcription function in lymphocytes after irradiation with He-Ne-laser

The influence of He-Ne-laser irradiation (lambda = 632.8 nm) in dose 56 J/m2 on the ultrastructure of the nucleolus from human peripheral lymphocytes was studied electronmicroscopically. After 1 h irradiation a well-expressed reaction of the nucleolus was observed in 70% of the lymphocytes under examination. Changes consist in the appearance of a wrong-shaped fibrillar center or in its fragmentation, the increase of RNP-containing fibrillar and granular components, and also in expansion of vacuoli. In a number of irradiated lymphocytes nucleoli with several fibrillar centres and with a strand-like organization of RNP part were observed. The size of these nucleoli increases. Following the accepted functional interpretations the observed changes can be connected with the intensification of RNA metabolism including the synthesis, processing of pre-rRNA and preribosome transport from the nucleolus. Similar rearrangements of the nucleoli were revealed in parallel experiments with phytohemagglutinin-treated lymphocytes. They were observed 1 h after the stimulation of lymphocytes. Taking into account the absence of mitogenic action of He-Ne-laser irradiation on lymphocytes, the ultrastructural changes of nucleoli under the action of irradiation are considered as functional activation of rRNA synthesis in the Go-period.     

Diaziquone-induced micronuclei in cytochalasin B-blocked mouse peripheral blood lymphocytes

A mouse peripheral blood lymphocyte (PBL) micronucleus (MN) test was developed using a modification of the technique for assessing MN in human PBLs described by Fenech and Morley (1985). Male C57Bl/6 mice (5/dose) were injected i.p. with either 0, 2.5, 5.0, 7.5, or 10.0 mg diaziquone (AZQ)/kg. After 24 h the mice were bled by cardiac puncture, PBLs were isolated on a Ficoll-density gradient and then cultured in RPMI 1640 medium using 8 micrograms phytohemagglutinin/ml. In some cultures cytochalasin B (CYB) was added at 21 h during the medium change to block cytokinesis. In other cultures, CYB was omitted to compare the sensitivity of analyzing MN in binucleate versus unblocked mononucleate cells. All doses of AZQ yielded significant increases in MN-containing binucleated PBLs. The use of CYB in the mouse PBL MN test increased the sensitivity approximately 3-fold. The MN test in mouse PBLs should be useful in comparative cytogenetic studies of mice and humans.     

Ascorbic acid 2-glucocide reduces micronucleus induction in distant splenic T lymphocytes following head irradiation

PurposeEvidence from in vivo studies suggests there are enhanced radiation effects in abscopal regions after local head gamma ray irradiation. Splenocyte apoptosis and T lymphocyte micronuclei were induced at higher rates than what would be estimated given the dose at a shielded, distant position. In addition, we evaluated the radio-protective effects of ascorbic acid, acting as a radical scavenger on enhanced radiation effects in the shielded spleen following local head irradiation.Methods and materialsThe heads of C3H mice were exposed to γ-rays (10–20 Gy), while the other parts of the body were shielded with a 5 cm-thick lead block. The effective dose for the spleen was calculated at 1.0–2.0 Gy. Splenocytes were isolated 24 h after cranial irradiation and their apoptosis was measured with an Elisa kit (Roche). The induction of T lymphocyte micronuclei was studied using the cytokinesis-block micronucleus assay. The ascorbic acid glucoside, 2-O-alpha-d-glucopyranosyl-l-ascorbic acid (AA-2G), was orally administered to mice 1 h before whole body irradiation. The radio protective effects of AA-2G were estimated by comparing the induction of splenocyte damage (by apoptosis) and micronucleus induction.ResultsThe splenocyte damage, as measured by the above two methods, was more excessive than what would be expected given exposure to 1.0–2.0 Gy of radiation. Our results suggest that the effects were enhanced in a distant, non-irradiated organ after localized irradiation. Plasma ascorbic acid concentrations were increased 8–10× over control. Treatment with ascorbic acid slightly protected mouse splenocytes from the induction of apoptosis by the enhanced effects of radiation in the abscopal region. However, ascorbic acid significantly inhibited micronucleus induction in splenic T lymphocytes following local head irradiation.ConclusionsOur results suggest that ascorbic acid effectively scavenged radiation-induced radicals and protected against the enhanced effects of radiation in an abscopal region after local head gamma ray irradiation.     

Increased radiosensitivity after irradiation of lymphocytes low adaptive doses

The variability of blood lymphocyte reaction on the adaptive irradiation (0.05 Gy at first, then 1.0 Gy 5 h later) was investigated by micronuclei assay. Blood samples were obtained from 700 children. It was shown that in all groups studied there were children with enhanced radiosensitivity ("radiosensitivity syndrome"-RS) after exposure to adaptive low dose of radiation. The radiosensitivity syndrome occurred more often in groups of ill children; part of them was characterized by the enhanced blood content of immunoglobulin E, enhanced level of T helpers and T suppressors. A high spontaneous level of lymphocytes with micronucleus is a factor of radiosensitivity formation. The possible factors resulted in radiosensitivity syndrome are discussed.     

Evaluation of micronuclei frequency in the cultured peripheral blood lymphocytes of cancer patients before and after radiation treatment

The frequency of micronucleated binucleate lymphocyte (MNBNC) was determined in the peripheral blood lymphocytes of patients suffering from various types of cancer before the onset of radiation treatment, middle (mid-) of the treatment and after completion of the treatment (post-treatment). The frequency of micronuclei increased significantly in the pretreatment sample of cancer patients when compared with the normal untreated healthy volunteers. During the middle of the radiotherapy an approximate two or > two-fold increase was observed in the micronuclei frequency in most of the patients when compared with the concurrent pretreatment samples. Immediately after the completion of treatment, the frequency of micronuclei further increased, and this increase was significantly higher than that of pretreatment and mid-treatment samples. Out of 27 patients analyzed, only nine patients did not have any history of smoking, tobacco chewing or alcohol consumption, while the remaining 18 patients had a history of either smoking, tobacco chewing or alcohol consumption or combination of two or all habits at the time of blood collection.     

Frequency of micronuclei induced in peripheral lymphocytes by trimethyltin chloride

The clastogenicity of trimethyltin chloride was evaluated in human peripheral blood lymphocytes with micronucleus counts (MNC) as the endpoint. Two concentrations (0.5 micrograms and 1.0 microgram) of trimethyltin chloride were added to lymphocytes of healthy male and female subjects of different age groups, in mitogen-stimulated and serum-supplemented culture medium (RPMI 1640, Gibco) for 48 h at 37 degrees C. A significant increase in micronucleus counts was observed with both doses, which was more pronounced with the lower dose. ANOVA in male and female donors revealed significant differences between age groups (P less than 0.001), chemical concentrations (P less than 0.001) and for the interaction of these 2 factors (P less than 0.05 in females only). However, no regular increase or decrease in MNC frequencies was observed with the donor's age. Higher frequencies of MNC enhancement were observed in male individuals than in females.     

Increased frequencies of micronuclei in T8 lymphocytes of smokers

Micronucleus frequencies and mitotic indices were analyzed in B, T4, and T8 lymphocytes from 40 smokers and 42 non-smoking referents. The highest level of micronuclei was found in T4 cells followed by T8 and B cells. These differences were statistically significant. There were statistically significant linear correlations between the micronucleus frequencies of all three subsets. There was a statistically significant effect of smoking only in the T8 cells. Smoking also increased the number of neutrophilic granulocytes and lymphocytes in the peripheral blood. There was a statistically significant effect of age on the micronucleus frequencies in T4 and T8 lymphocytes. The mitotic indices did not have any effect on the micronucleus frequencies and they were not influenced by smoking, age or sex.     

Residual activation events functional after irradiation of mouse splenic lymphocytes

The radioresistance of lymphocytes increases after mitogenic stimulation, suggesting that a radiosensitive activation event contributes to the overall radiosensitivity of lymphocytes. We have sought to identify this activation event by determining the extent of activation of mitogen-stimulated lymphocytes previously exposed to growth-inhibiting doses of radiation. Mouse splenic lymphocytes were exposed to 0-15 Gy 137Cs radiation, and structural and functional damage were assayed. Although damage to cellular thiols and nonprotein thiols was modest, there was a significant loss of viability by 6 h as determined by uptake of propidium iodide (PI). Since cells did not die immediately after irradiation, the activation events which remained were evaluated. Growth-inhibiting doses of radiation left cells partially responsive to mitogen, in that cells were able to exit G0 phase, but they could progress no further into the cell cycle than G1a phase. It is important to note that assessment of viability by uptake of PI indicated substantial cell death after 15 Gy (45%, 6 h; 90%, 24 h); however, cell cycle analysis at 24 h indicated no significant decrease in progression from G0 to G1a phase. The LPS-stimulated response of B cells was more radiosensitive than the Con A-stimulated response of T cells. Further analysis of the Con A response indicated that production of interleukin-2 (IL-2) was unaffected, but expression of the IL-2 receptor was inhibited. Inhibition of poly-ADP-ribosylation and damage to lipids did not prevent the lack of mitogen responsiveness, since neither the ADP-ribose transferase inhibitor 3-aminobenzamide nor lipid radical scavengers had restorative effects on the mitogenic response. Nor was Con A-stimulated incorporation of [3H]thymidine restored with inhibitors of prostaglandin or leukotriene synthesis, suggesting that inhibition was due to direct effects on the Con A responders, and not indirect effects mediated by arachidonate metabolites. These results indicate that growth-inhibiting doses of radiation trigger the process in lymphocytes that culminates in apoptosis, yet leave the cells partially responsive to mitogenic stimuli.