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.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

The fate of micronucleated cells post X-irradiation detected by live cell imaging

Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B. Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about 70% of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38 stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38 immunofluorescence staining. Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.     

Correlation between cell survival and micronuclei formation in V79 cells treated with vindesine before exposure to different doses of gamma-radiation

Effect of 20 nM vindesine sulphate (VDS) treatment was studied on cell survival, growth kinetics and micronuclei induction in V79 cells exposed to 0-300 cGy of gamma-radiation at 16, 22 and 28 h post-irradiation. Treatment of V79 cells with VDS before exposure to different doses of gamma radiation resulted in a significant decline in cell survival and growth kinetic when compared with the concurrent PBS+irradiation group. The decline in cell survival and growth kinetics was dose related. Similarly, the cell proliferation indices also declined in a dose dependent manner in both PBS+irradiation and VDS+irradiation groups and this decline was higher in VDS+irradiation group in comparison with the PBS+irradiation group. In contrast, the frequency of micronuclei increased in a dose related manner in both PBS+irradiation and VDS+irradiation groups. However, the frequency of micronuclei was significantly greater in the VDS+irradiation group when compared to the PBS+irradiation group at all the post-irradiation time periods studied and the dose response for both groups was linear for all the scoring time periods. The biological response was determined by plotting surviving fraction and micronuclei frequencies on X- and Y-axes, respectively. The plot between surviving fraction and micronuclei induction showed a close correlation. The surviving fraction of V79 cells reduced with the increasing frequency of micronuclei in both groups and the relationship between micronuclei induction and cell survival could be fitted on a linear quadratic model.     

Changes in micronucleus frequency resulting from preirradiation of cell culture surfaces

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.     

The effect of 835.62 MHz FDMA or 847.74 MHz CDMA modulated radiofrequency radiation on the induction of micronuclei in C3H 10T(1/2) cells

To determine if radiofrequency (RF) radiation induces the formation of micronuclei, C3H 10T(1/2) cells were exposed to 835.62 MHz frequency division multiple access (FDMA) or 847.74 MHz code division multiple access (CDMA) modulated RF radiation. After the exposure to RF radiation, the micronucleus assay was performed by the cytokinesis block method using cytochalasin B treatment. The micronuclei appearing after mitosis were scored in binucleated cells using acridine orange staining. The frequency of micronuclei was scored both as the percentage of binucleated cells with micronuclei and as the number of micronuclei per 100 binucleated cells. Treatment of cells with cytochalasin B at a concentration of 2 microg/ml for 22 h was found to yield the maximum number of binucleated cells in C3H 10T(1/2) cells. The method used for the micronucleus assay in the present study detected a highly significant dose response for both indices of micronucleus production in the dose range of 0.1-1.2 Gy and it was sensitive enough to detect a significant (P > 0.05) increase in micronuclei after doses of 0.3 Gy in exponentially growing cells and after 0.9 Gy in plateau-phase cells. Exponentially growing cells or plateau-phase cells were exposed to CDMA (3.2 or 4.8 W/kg) or FDMA (3.2 or 5.1 W/kg) RF radiation for 3, 8, 16 or 24 h. In three repeat experiments, no exposure condition was found by analysis of variance to result in a significant increase relative to sham-exposed cells either in the percentage of binucleated cells with micronuclei or in the number of micronuclei per 100 binucleated cells. In this study, data from cells exposed to different RF signals at two SARs were compared to a common sham-exposed sample. We used the Dunnett's test, which is specifically designed for this purpose, and found no significant exposure-related differences for either plateau-phase cells or exponentially growing cells. Thus the results of this study are not consistent with the possibility that these RF radiations induce micronuclei.     

Tumor radiosensitivity prediction by the cytokinesis-block micronucleus assay

An in vivo to in vitro cytokinesis-block micronucleus assay technique using cytochalasin B (Cyt-B) was established in xenografted human and murine tumors, and the correlation between radiosensitivity measured by this assay and that measured by a colony-forming assay was investigated. Tumors were irradiated in situ, excised immediately, and disaggregated to single cells that were plated for the micronucleus and colony-forming assays. Some of the tumor cells were irradiated in vitro rather than in vivo. For the micronucleus assay, Cyt-B (0.5-3 micrograms/ml) was added to dishes soon after plating or in vitro irradiation and the cells were subsequently fixed and stained at intervals (12-144 h). The micronucleus frequency in binucleate cells was evaluated under conditions of maximum yield of the binucleate cells. The micronucleus frequency after irradiation was quite variable depending on the tumor type and the average number of micronuclei per single binucleate cell after 4 Gy ranged from 0.2 to 1.4. The results of in vitro irradiation were not significantly different from those of in vivo irradiation for all tumors. A good correlation was found between the radiosensitivity determined by the micronucleus assay and that found with the colony-forming assay in six human tumors (r = 0.94 approximately 0.98) but not in four murine tumors because of one exceptional tumor. When this tumor was excluded, a correlation was also found for the remaining nine tumors (r = 0.62 approximately 0.96). These results indicated that the cytokinesis-block micronucleus assay has some promise as a rapid predictive assay of radiosensitivity.     

Micronucleus induction in mammalian cell cultures treated with ionizing radiations

Summary Exponentially growing and plateau phase cultures of Ehrlich ascites tumor cells (suspension strain) were treated with either fast electrons, X-rays, fast neutrons or Am-241-alpha-particles in a dose range from about 0.02 Gy to 1 Gy and for comparison also at higher doses. After the first post-irradiation division, cells were scored for the presence of micronuclei and the micronucleus fraction as well as the number of micronuclei/cell was determined. Micronuclei were counted using the DNA specific stain H 33258 in a fluorescence microscope. A comparison with cytofluorometric measurements established that microscopic detection accounted for up to 90% of all micronuclei present within a sample, the rest probably being hidden in direct observation by the main nucleus.Dose response curves based on the micronucleus fraction as well as on the number of micronuclei/cell were found to be linear in the whole dose range tested at low and at high ionization density. Linearity was maintained also when repair of primary lesions was promoted or suppressed. The RBE of alpha-particles compared with X-rays was dependent on the time of fixation and was at a maximum immediately after the first division (RBE = 4.8 ± 0.5). Micronucleus distribution showed overdispersion relative to Poissonian statistics with every radiation quality used, in accordance with earlier observations on the distribution of acentric fragments in irradiated cultures.     

Preimplantation growth delay and micronucleus formation after in vivo exposure of mouse zygotes to fast neutrons.

Mouse zygotes were irradiated with fast neutrons (0.06 to 1.00 Gy) 1 h after conception and examined at various intervals (24 to 100 h after conception) for embryonic development and micronucleus formation. The frequency of micronuclei per cell increased linearly with dose in 2-cell embryos observed at 24 h after conception and in 4-cell and 8-cell embryos at 48 h after conception. Compared with X rays, the relative biological effectiveness of neutrons for the induction of micronuclei per embryo was 2.5 at 24 h after conception and 3.5 at 48 h after conception. Neutron-induced micronucleus formation was accompanied by morphological growth delay and a significant decrease in the number of cells in the embryos. An inverse relationship was found between the number of cells in embryos and the number of micronuclei when observed at 48 h after conception following irradiation with 0.12 to 1.00 Gy and at 78 h after conception following exposure to 0.50 Gy. The effect of neutron irradiation on embryonic development was likely to be mediated by cell death, as suggested by a significantly increased dead cell index in blastocysts following irradiation of zygotes.     

The effect of gamma-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes.

There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of gamma-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cells, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.     

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.     

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation

The frequency of micronuclei in cultured mouse splenocytes increased positively and in a dose-related manner to exposure to ferrous ions and ultraviolet irradiation, but not to hydrogen peroxide. Combined treatments, especially when ferrous ions were present with hydrogen peroxide or with ultraviolet irradiation, led to a synergistic enhancement in micronucleus frequency. The results indicate that a significant level of chromosome damage is associated with exposure to ultraviolet light and to general cellular pro-oxidative stress, and indicate that under these conditions the micronucleus assay can provide an effective in vitro model for the study of genotoxicity in relation to oxygen-derived free radicals.     

Bystander effects induced by direct and scattered radiation generated during penetration of medium inside a water phantom

Background

The biological effects of ionizing radiation have long been thought to results from direct targeting of the nucleus leading to DNA damage. Over the years, a number of non-targeted or epigenetic effects of radiation exposure have been reported where genetic damage occurs in cells that are not directly irradiated but respond to signals transmitted from irradiated cells, a phenomenon termed the “bystander effects”.

Aim

We compared the direct and bystander responses of human A 549, BEAS-2-B and NHDF cell lines exposed to both photon (6 MV) and electron (22 MeV) radiation inside a water phantom. The cultures were directly irradiated or exposed to scattered radiation 4 cm outside the field. In parallel, non-irradiated cells (termed bystander cells) were incubated in ICM (irradiation conditioned medium) collected from another pool of irradiated cells (termed donor cells).

Materials and methods

In directly irradiated cells as well as ICM-treated cells, the frequency of micronuclei and condensation of chromatin characteristic for the apoptotic process were estimated using the cytokinesis-block micronucleus test.

Results

In all tested cell lines, radiation induced apoptosis and formation of micronuclei. A549 and BEAS-2B cells cultured in ICM showed increased levels of micronuclei and apoptosis, whereas normal human fibroblasts (NHDF line) were resistant to bystander response. In A549 and BEAS-2B cells placed outside the radiation field and exposed to scattered radiation the formation of micronuclei and induction of apoptosis were similar to that after ICM-treatment.

Conclusion

Results suggest that the genetic damage in cells exposed to scattered radiation is caused by factors released by irradiated cells into the medium rather than by DNA damage induced directly by X rays. It seems that bystander effects may have important clinical implications for health risk after low level radiation exposure of cells lying outside the radiation field during clinical treatment.     

Characterization of accelerated iron-ion-induced damage in gap junction-competent and -incompetent thyroid follicular cells

Early- and late-passage cultures of Fischer rat thyroid cells differ in their growth properties and gap junction competency. Previous studies comparing early- and late-passage cultures exposed to gamma rays and proton beams revealed that differences in growth rate did not influence their responses; however, the presence of connexin 32 gap junctions conferred resistance to gamma radiation. To further assess differences in radiation quality, suspension cultures of early- and late-passage cells were exposed to accelerated iron ions, and their comparative biological responses were measured. The iron-ion-irradiated cells displayed sustained levels of incorporated dUTP, reflecting persistent DNA damage. These results were supported by the frequency of chromosomal damage measured by micronucleus formation. Iron-ion irradiation induced micronuclei at a rate of eight per gray per 100 binucleated cells scored in early-passage cells and nine per gray per 100 binucleated cells scored in late-passage cells. Relative to photons, the calculated radiobiological effectiveness for frequency of micronuclei was 5.7 and 6.4 for the early- and late-passage cultures, respectively (P > 0.05). Levels of apoptosis fluctuated as a function of dose, and modest increases above basal levels persisted throughout the 48-h period. The comparison of retained follicular structures revealed differences in the alpha components of the linear-quadratic dose-response curves (0.60 Gy(-1) for early-passage and 0.71 Gy(-1) for late-passage cultures, P < 0.014). Cell cycle phase redistribution resulted in a G2 arrest (P < 0.001) for both early- and late-passage cultures. In conclusion, the response of thyroid follicular cells to high-LET radiation was not influenced by the presence of gap junctions or the proliferative status of the target cells.     

The time dependence of bystander responses induced by iron-ion radiation in normal human skin fibroblasts

Although bystander effects have been shown for some high-LET radiations, few studies have been done on bystander effects induced by heavy-ion radiation. In this study, using a Transwell insert co-culture system, we have demonstrated that irradiation with 1 GeV/nucleon iron ions can induce medium-mediated bystander effects in normal AG01522 human fibroblasts. When irradiated and unirradiated bystander cells were combined in shared medium immediately after irradiation, a two- to threefold increase in the percentage of bystander cells with gamma-H2AX foci occurred as early as 1 h after irradiation and lasted at least 24 h. There was a twofold increase in the formation of micronuclei in bystander cells when they were co-cultured with irradiated cells immediately or 1 or 3 h after irradiation, but there was no bystander effect when the cells were co-cultured 6 h or later after irradiation. In addition, bystander micronucleus formation was observed even when the bystander cells were co-cultured with irradiated cells for only 1 h. This indicates that the crucial signaling to bystander cells from irradiated cells occurs shortly after irradiation. Moreover, both gamma-H2AX focus formation and micronucleus formation in bystander cells were inhibited by the ROS scavengers SOD or catalase or the NO scavenger PTIO. This suggests that ROS and NO play important roles in the initiation of bystander effects. The results with iron ions were similar to those with X rays, suggesting that the bystander responses in this system are independent of LET.     

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

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

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.     

Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients

BackgroundPelvic organs morbidity after irradiation of cancer patients remains a major problem although new technologies have been developed and implemented. A relatively simple and suitable method for routine clinical practice is needed for preliminary assessment of normal tissue intrinsic radiosensitivity. The micronucleus test (MNT) determines the frequency of the radiation induced micronuclei (MN) in peripheral blood lymphocytes, which could serve as an indicator of intrinsic cell radiosensitivity.AimTo investigate a possible use of the micronucleus test (MNT) for acute radiation morbidity prediction in gynecological cancer patients.Materials and methodsForty gynecological cancer patients received 50 Gy conventional external pelvic irradiation after radical surgery. A four-field “box” technique was applied with 2D planning. The control group included 10 healthy females.Acute normal tissue reactions were graded according to NCI CTCAE v.3.0. From all reaction scores, the highest score named “summarized clinical radiosensitivity” was selected for a statistical analysis.MNT was performed before and after in vitro irradiation with 1.5 Gy. The mean radiation induced frequency of micronuclei per 1000 binucleated cells (MN/1000) and lymphocytes containing micronuclei per 1000 binucleated cells (cells with MN/1000) were evaluated for both patients and controls.An arbitrary cut off value was created to pick up a radiosensitive individual: the mean value of spontaneous frequency of cells with MN/1000 ± 2SD, found in the control group.ResultsBoth mean spontaneous frequency of cells with MN/1000 and MN/1000 were registered to be significantly higher in cancer patients compared to the control group (t = 2.46, p = 0.02 and t = 2.51, p = 0.02). No statistical difference was registered when comparing radiation induced MN frequencies between those groups.Eighty percent (32) of patients developed grade 2 summarized clinical radiosensitivity, with great variations in MNT parameters. Only three patients with grade 2 “summarized clinical radiosensitivity” had values of cells with MN/1000 above the chosen radiosensitivity threshold.ConclusionThe present study was not able to confirm in vitro MNT applicability for radiosensitivity prediction in pelvic irradiation.     

Apoptosis and appearance of Trp53-positive micronuclei in murine tumors with different radioresponses in vivo.

The purpose of this paper is to determine the relationship between the response to radiation and the appearance of apoptosis and micronuclei with Trp53 protein in murine tumors after irradiation. Two murine tumors, EL4, which was derived from a mouse lymphoma, and FM3A, which was derived from a mouse mammary carcinoma, were locally irradiated with 15 Gy and sections were stained with H&E and an anti-Trp53 antibody. The response to radiation was greater in EL4 tumors than in FM3A tumors. The frequency of apoptotic cells in EL4 tumors was 6.1 +/- 1.2% at time zero, reached a peak of 36.3 +/- 3. 8% at 6 h, and then decreased with time through 72 h to 2.5 +/- 1.5% after 15 Gy irradiation. In FM3A tumors, no apoptotic cells were detected at 0, 1, 3, 6 or 24 h after exposure. At 48 and 72 h, the frequency was only 3.0 +/- 0.6% and 1.3 +/- 0.3%. Apoptotic cells increased significantly at 3, 6 and 24 h after irradiation in EL4 tumors (P < 0.008) and at 48 and 72 h in FM3A tumors (P < 0.006). The frequency of Trp53-positive cells was 17.9 +/- 2.2 and 15.2 +/- 2.3% at time zero in EL4 and FM3A tumors, respectively, increased to 74.5 +/- 4.5% in EL4 cells (P = 0.001), and increased to 33.9 +/- 1. 1% in FM3A cells (P = 0.005) 1 h after irradiation. Trp53-positive micronuclei appeared in cells in both tumors from 24 to 72 h after irradiation. The frequency of Trp53-positive micronuclei was 3.8 +/- 0.5 and 13.5 +/- 1.3% at 24 h in EL4 and FM3A tumors, respectively, and gradually decreased by 72 h. After exposure to 15 Gy, Trp53-positive micronuclei increased significantly in FM3A tumors compared to EL4 tumors at both 24 and 48 h (P < 0.02). The frequency of these micronuclei increased with increasing dose in FM3A tumors, and the difference between these percentages after 3 Gy and after 5, 10 and 15 Gy was significant (P < 0.02). Many apoptotic cells were observed in the radiosensitive EL4 tumor after irradiation. Death by apoptosis may be related to an early response to radiation in these tumors. The appearance of micronuclei may be an important mechanism of cell death in FM3A tumors in which no apoptosis was induced.