The distribution of chromosome aberrations after gamma and neutron irradiation among cells in various stages of the mitotic cycle in a human lymphocyte culture

Distribution of chromosome aberrations among cells depending on radiation dose, stage of mitotic cycle of human lymphocyte culture, time of cell fixation under the effect of 6 MeV neutrons and their combination with postirradiation hyperthermia has been investigated. The regularities in the distribution of aberrations within cells are similar with both neutron- and gamma-radiation. In experiments with both types of radiation delivered at the S stage and fixation 52 h after the onset of incubation, distribution of aberrations follows Poisson formula whereas with 62-hour fixation, the correlation is disturbed. This is due to the presence of cells dividing for the second time after irradiation which makes the data obtained with delayed fixation scantily informative. Additional hyperthermia does not affect substantially the structure of cell population with both radiation types.     

Comparative frequency of chromosome aberrations in a human lymphocyte culture depending on the mode of neutron irradiation

The study of the dose dependence of the incidence of chromosome aberrations in a human lymphocyte culture exposed at the G0 stage to pulsed neutrons of 0.7 MeV revealed no significant distinctions in the cytogenetic effect at the pulse frequency of 1 and 5 Hz, but at 100 Hz it was somewhat increased. As to the biological effectiveness, pulsed neutrons of 0.7 MeV occupied an intermediate position between 0.35 and 0.85 MeV static neutrons.     

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.     

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.     

Early changes in the lymphocyte surface membrane of rats after whole-body irradiation with neutrons and gamma rays

Biochemical changes in lymphocyte plasma membranes were studied 3 and 18 h after whole-body exposure of rats to neutrons and gamma-rays at doses from 2 to 6 Gy. It was shown that fast neutrons, with an average energy of 1.5-2.0 MeV, increased the rate of lipid peroxidation more markedly than gamma-rays did. In addition, there was an increase in the number of free aminogroups on the thymocyte surface. Dose- and time-dependent parameters of changes in the aminogroup content on the cellular surface were quantitatively different after the effect of radiation with different LET.     

Nuclei with protrusions--"tailed" nuclei--and radiation cytogenetic markers in a lymphocyte culture after x-ray irradiation

Frequency of the appearance of binuclear cells with nuclei having outgrowth into the cytoplasmic space and arise after first mitosis in human lymphocyte culture is linear-square dependent on the X-irradiation at doses from 0.0 to 4.0 Gy. Positive correlation between frequency of cells with "tailed" nuclei and frequency of metaphases of first mitosis having dicentrics and rings was established. Apparently, formation such "tailed" nuclei is connected with dicentrics and rings.     

DNA synthesis in the mono- and binuclear cells of regenerating rat liver after x-ray irradiation

The effect of X-irradiation on the dynamics of DNA synthesis during the S-period in bi- and mononucleated of regenerating rat liver was studied autoradiographically and microphotometrically. Rats were treated with X-rays at doses 3.84 X 10(-2), 15.48 X 10(-2), and 30.96 X 10(-2) Kl/kg 23 hours after a partial hepatectomy, and were sacrificed one hour after irradiation. In the control liver the rate of DNA synthesis was the lowest at the beginning of the S-period and the highest at the last quarter of this period in both mono- and binucleated cells. The irradiation results in the inhibition of DNA synthesis mainly at the end of the S-period depending on doses employed. This inhibition was the same in bi- and mononucleated cells. In addition, the increase of correlation of the 3H-thymidine incorporation rate and DNA content was found between nuclei of binucleated cells after irradiation.     

Alterations in the baseline sister-chromatid exchange frequency in human lymphocyte culture following a number of cell divisions

A significant decrease in the baseline of sister-chromatid exchanges (SCEs) was observed in cultured human lymphocytes, if 5-bromodeoxyuridine (BrdU) was added after 60 h of culture, and the cells were harvested at least 24–30 h after BrdU exposure. This decrease is supposed to occur if at least one cell division takes place before the addition of BrdU. For cytogenetic monitoring of mutagenic environmental factors, using human lymphocyte cultures, it is assumed that two time periods are sufficient for comparison.     

Integrity of proteins in human saliva after sterilization by gamma irradiation

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.     

Changes in the structure of lymphocyte chromatin after irradiation with He-Ne-laser

The cytofluorometric method was used to study changes occurring in the chromatin structure of lymphocytes during the first few hours following irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) of 28-112 J/m2. The changes were similar to those caused by PHA that is: the increase in acridine-orange binding to DNA during the first 45-90 min, its fall to the control level in 3-4 h and the subsequent increase.     

Variation in the human lymphocyte sister-chromatid exchange frequency: results of a long-term longitudinal study

The variation in lymphocyte sister-chromatid exchange (SCE) frequency as a function of time was investigated in nonsmokers and smokers. The smokers were divided into 3 groups depending on their smoking status. The group termed 'smokers' participated in a program to stop smoking but did not reduce or eliminate their use of tobacco; 'smoke enders' successfully completed the smokending program and remained free of tobacco for the duration of the study, while the 'variable' group stopped smoking for a limited time but then resumed smoking. 8 or more blood samples per person were obtained over a period of at least 12 months. The SCE frequencies for each of these groups were compared with each other and with those of two previous longitudinal study groups from our laboratory. The proportion of high-frequency cells (HFCs) was also determined for each sample. The results confirm our previous finding that SCE frequencies and the proportion of HFCs observed in separate samples from the same individual are more likely to be different as the time between samples increases. We also show that smokers have significantly more SCEs and HFCs than do nonsmokers, that SCE frequencies in smokers do not decline for at least 12 months when smoking is stopped, and that among smokers, significant seasonal variation in the SCE frequency occurs. These results provide useful information concerning the effects of smoking upon SCE frequencies, and will be helpful in designing and interpreting the results of long-term human population cytogenetic studies.     

Activation of an energy providing response in human keratinocytes after gamma irradiation

We performed a microarray study on human differentiated HaCaT keratinocytes exposed to ionizing radiation (2 or 10 Gy). At 3 h after exposure, more than 150 known and unknown genes were found regulated in irradiated HaCaT keratinocytes. Among the genes regulated at 3 h, those involved in cell energy metabolism appeared to be the most abundant and the most responsive. Two mitochondrial ATP-synthases and several other genes involved in energy producing pathways, such as glucose metabolism, were induced, whereas many genes from energy requiring pathways were shut down. These changes in energy metabolism were confirmed both in normal primary keratinocytes and in HaCaT keratinocytes by RT-PCR and proteins studies. Moreover, measures of intracellular ATP revealed a 50% increase in keratinocytes immediately after irradiation, supporting an energy procurement response. The overall results indicate that irradiation induces an immediate burst of ATP that seems to be a general response of human differentiated keratinocytes to the radiation stress. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/v95.html     

In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.     

Dose-response relationship of the yield of chromosome aberrations in a human lymphocyte culture following gamma-irradiation at high doses

A study was made of the yield of chromosome aberrations in a human lymphocyte culture at the G0 stage after 60Co-gamma-irradiation with doses of 5-12 Gy. It was shown that a linear-quadratic dependence of the aberration frequency observed with median doses became purely linear at high doses.     

Enhancement of micronuclei frequency in the Tradescantia/micronuclei test using a long recovery time

The Tradescantia/micronuclei test (TRAD/MCN) is a well-validated test for monitoring environmental genotoxicants. These pollutants induce at the early meiotic stage of pollen mother cells chromosome fragments which become micronuclei at the tetrad stage. The standard test protocol requires some hours of exposure of the inflorescences and a recovery time of about 24 hours to reach the early tetrad stage. Since the recovery period represents a critical step of the TRAD/MCN, experiments were performed to establish its length in plants of clone #4430 of the hybrid T. hirsutiflora x T. subacaulis which is widely used in environmental monitoring. The aim of the present research was to ascertain the exact duration of recovery time in order to improve the sensitivity of the TRAD/MCN test. First, studies were performed to select the flowers at the beginning of the meiosis, and then anthers were sampled and studied for a period of 48-86 hours. The complete meiosis in the plants examined required about 80 hours. Second, exposure to genotoxic substances followed by different recovery times was carried out to demonstrate that effectiveness of the TRAD/MCN test is closely related to the duration of the recovery time. The test was carried out by exposing inflorescences to known mutagens (sodium azide and maleic hydrazide) for six hours followed by different recovery times (24-72 hours). The results showed that the frequency of micronuclei in the pollen mother cells increased with the length of the recovery time.