Dose-response relationship for chromosome aberrations induced by fecapentaene-12 in human lymphocytes

Chromosome analyses were carried out in human lymphocytes exposed to a synthetic racemic all-trans fecapentaene-12 at 2-24 microM. A dose-dependent increase of the incidences of chromatid-type changes with distinct saturation at higher doses could be observed. The results reveal for the first time that fec-12 is a potent direct-acting mutagen in human lymphocytes.     

Distribution pattern and dose-response-relationship of chromosome aberrations in human lymphocytes induced in vitro by 60Co gamma-rays and 110 kV X-rays.

Peripheral blood lymphocyte culture system was used to construct reference dose-response curves for 60Co gamma-rays and 110 kV X-ray-induced chromosome aberrations at 6 dose points ranging from 0.25 to 4.0 Gy. Qualitative and quantitative differences between these two types of radiation for the yield of induced aberrations and their distribution pattern were analysed. Experimental data of aberration yields were compared after fitting them to five different dose-response models. The yields of chromosome aberrations in particular dicentrics, gave a good fit to linear-quadratic besides linear and power models. In this model, single-track events predominated over double-track events for both the qualities of radiation used. The pattern of distribution was mainly Poisson for dicentrics but gave a conflicting result for acentrics which was in excess.     

Recovery kinetics of radiation-induced chromosome aberrations in human G0 lymphocytes

Summary Declining yields of radiation-induced dicentric chromosomes in human G⁰ lymphocytes were observed in split-dose experiments with time intervals varied up to 8 h. In agreement with microdosimetric intratrack-intertrack interaction models, only the dose-squared yield component was reduced and approached an asymptotic value equal to one half of the corresponding single exposure yield. For 150 kV X-rays and 13 MeV electrons, at total doses up to 6 Gy, the time constant of the approximately exponential decline was practically dose- and quality-independent within a range of 100–180 min. For 10 kV X-rays, in the presence of a dominant linear yield component, only a small split-dose effect, but with a consistent-value, was observed for a total dose of 5 Gy. Since can be interpreted as the mean life time of primary lesions in chromatin fibres, its independence from absorbed dose and radiation quality means that radiation damage of the split-dose recovery mechanism can be excluded for doses up to 6 Gy. By correlating the observed split-dose reduction of the acentric fragment yield to the reduction of the dicentric yield, (1.64 ± 0.03) acentrics/dicentric for 150 kV X-rays and (1.51 ± 0.11) acentrics/dicentric for 13 MeV electrons were obtained. Acentrics formed in the course of dicentric formation as well as in other binary interactions of primary lesions are represented in these ratios. Post-irradiation recovery during time intervals between irradiation and cell stimulation up to 24 h did not occur. The relations to comparable results in cell lethality experiments are discussed, and a hypothesis of fast and slow binary interactions of primary lesions is put forward.Dedicated to Prof. Dr. H. Muth on the occasion of his 65th birthdayThis work was supported by the Bundesministerium des Innern, Bonn, FRG     

Enhanced frequency of chromosome aberrations in workers occupationally exposed to diagnostic X-rays

To estimate the level of radiation exposure of personnel handling diagnostic X-ray machines, the yield of chromosomal aberrations was analysed in peripheral blood lymphocyte cultures. These occupationally exposed individuals showed higher frequencies of dicentrics as well as acentrics than normal controls. Absorbed radiation doses calculated by extrapolating reference in vitro dose-response curve for dicentrics ranged between 0.13 and 0.17 Gy. This implies exposure beyond the permissible limit of 0.05 Gy/year for the whole body. However, no obvious trend of increased aberrations as a function of either duration of employment or age was noticed. The increase in the aberration yields in this personnel underscores the need of adopting measures to avoid or minimise such overexposure.     

Analysis of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed in vitro to Hydergine.

Hydergine (dihydroergotoxine mesylate, Sandoz) was examined for its capability to induce chromosome damage and sister-chromatid exchanges (SCEs) in human lymphocyte in vitro. For the chromosome-aberration study, cultures set up from 6 individuals were divided into 5 groups: negative control, positive control (caffeine, 0.5 mg/ml), and Hydergine (0.1, 0.25 and 0.5 micrograms/ml). For the SCE examination, which used 8 individuals, 4 cultures were made per person in the following way: negative control, positive control (mitomycin C, 0.1 microgram/ml), and Hydergine (0.1 and 0.5 micrograms/ml). Lymphocytes were cultivated for 72 h, being exposed to the respective treatments during the final 24 h. The results showed that Hydergine induced no chromosome damage in human lymphocytes in vitro.     

The induction by X-rays of chromosome aberrations in male Guinea-pigs, rabbits and golden hamsters. III. Dose-response relationship after single doses of X-rays to spermatogonia.

The induction by X-rays of translocations in spermatogonia was studied by cytological means in spermatocytes derived from them. In the rabbit and guinea-pig hump shaped dose-response curves were obtained, with a linear relationship at the low doses. The shapes of the curves were similar to those reported for the mouse, except that the maximum occurred at 600-700 rad in the mouse as opposed to 300 rad in the guinea-pig and rabbit. Unlike the guinea-pig and rabbit, the golden hamster showed a hump dose-response curve without a definite peak value and with little decrease in yield at high radiation doses. Over the low dose range 100-300 rad, the slopes of the curves of translocation yield were in the order:mouse (highest), rabbit, guinea-pig and hamster. Data on sterile periods suggested that the amount of spermatogonial killing in the rabbit and guinea-pig was as great or greater than in the mouse, and that in the golden hamster it was most severe. It is suggested that the differing shapes of the dose-response curves can be explained by a lower sensitivity to translocation induction in the test species and, also especially in the golden hamster, a greater sensitivity to cell killing. The possibility of extrapolating from these data to other species is discussed.     

No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers.

The putative genetic risk of people occupationally exposed to nitrogen fertilisers was studied using the structural chromosome aberration assay in peripheral blood lymphocytes. The exposed group included 23 subjects working at complex and mixed fertiliser plants. The percent of aberrant cells (Ab.C %) and break to cell ratio (B/C) were 0.95% and 0.01 respectively. The matched control group (20 subjects) was found to have 0.80% Ab.C and a B/C ratio of 0.0085. The results show a lack of detectable genetic damage in exposed people using this cytogenetic approach.     

Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the 1q12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co-gamma-rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.     

Arbutin,an intracellular hydroxyl radical scavenger,protects radiation-induced apoptosis in human lymphoma U937 cells

Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-β-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.     

The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro.

Human whole-blood samples were exposed to continuous microwave radiation, frequency 7.7 GHz, power density 0.5, 10 and 30 mW/cm2 for 10, 30 and 60 min. A correlation between specific chromosomal aberrations and the incidence of micronuclei after in vitro exposure was observed. In all experimental conditions, the frequency of all types of chromosomal aberrations was significantly higher than in the control samples. In the irradiated samples the presence of dicentric and ring chromosomes was established. The incidence of micronuclei was also higher in the exposed samples. The results of the structural chromosome aberration test and of the micronucleus test were comparatively analyzed. The values obtained showed a positive correlation between micronuclei and specific chromosomal aberrations (acentric fragments and dicentric chromosomes). The results of the study indicate that microwave radiation causes changes in the genome of somatic human cells and that the applied tests are equally sensitive for the detection of the genotoxicity of microwaves.     

Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of X-rays

The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.     

The effect of 29 kV X rays on the dose response of chromosome aberrations in human lymphocytes

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with X rays generated at a tube voltage of 29 kV was examined to assess the maximum low-dose RBE (RBE(M)) relative to higher-energy X rays or 60Co gamma rays. Since blood was taken from the same male donor whose blood had been used for previous irradiation experiments using widely varying photon energies, the greatest possible accuracy was available for such an estimation of the RBE(M), avoiding the interindividual variations in sensitivity or differences in methodology usually associated with interlaboratory comparisons. The magnitude of the linear coefficient alpha of the linear-quadratic dose-effect relationship obtained for the production of dicentric chromosomes by 29 kV X rays (alpha = 0.0655 +/- 0.0097 Gy(-1)) confirms earlier observations of a strong increase in alpha with decreasing photon energy. Relating this value to previously published values of alpha for the dose-effect curves for dicentrics obtained in our own laboratory, RBE(M) values of 1.6 +/- 0.3 in comparison with weakly filtered 220 kV X rays, 3.0 +/- 0.7 compared to heavily filtered 220 kV X rays, and 6.1 +/- 2.5 compared to 60Co gamma rays have been obtained. These data emphasize that the choice of the reference radiation is of fundamental importance for the RBE(M) obtained. A special survey of the RBE(M) values obtained by different investigators in the narrow quality range from about 30 to 350 kV X rays indicates that the present RBE is in fairly good agreement with previously published findings for the induction of chromosome aberrations or micronuclei in human lymphocytes but differs from recently published findings for neoplastic transformation in a human hybrid cell line.     

Induction of complete and incomplete chromosome aberrations by bleomycin in human lymphocytes

Bleomycin (BLM) is a clastogenic compound, which due to the overdispersion in the cell distribution of induced dicentrics has been compared to the effect of high-LET radiation. Recently, it has been described that in fibroblast derived cell lines BLM induces incomplete chromosome elements more efficiently than any type of ionizing radiation. The objective of the present study was to evaluate in human lymphocytes the induction of dicentrics and incomplete chromosome elements by BLM. Peripheral blood samples have been treated with different concentrations of BLM. Two cytogenetic techniques were applied, fluorescence plus Giemsa (FPG) and FISH using pan-centromeric and pan-telomeric probes. The observed frequency of dicentric equivalents increases linearly with the BLM concentration, and for all BLM concentrations the distribution of dicentric equivalents was overdispersed. In the FISH study the ratio between total incomplete elements and multicentrics was 0.27. The overdispersion in the dicentric cell distribution, and the linear BLM-concentration dependence of dicentrics can be compared to the effect of high-LET radiation, on the contrary the ratio of incomplete elements and multicentrics is similar to the one induced by low-LET radiation (~0.40). The elevated proportion of interstitial deletions in relation to total acentric fragments, higher than any type of ionizing radiation could be a characteristic signature of the clastogenic effect of BLM.     

The repair of chromosome aberrations in human lymphocytes after combined irradiation with UV-radiation (254 nm) and X-rays

Human lymphocytes were exposed to UV-radiation and X-rays. The previously reported synergistic effect on the frequency of chromosome aberrations (Holmberg and Jonasson, 1974) was measured as a function of the time between the 2 irradiations to study the effect of repair processes in cells in PBS at 20 degrees C. The synergistic effect was found to be rather constant as a function of time (in the interval up to 90 min) when the UV-radiation is delivered first. The synergistic effect decreases with a half-life of about 20 min when the cells are first X-irradiated and after various times are given a UV-treatment. This is not in accordance with findings from dose-fractionation experiments with X-rays, in which lesions interact with each other for several hours. It is proposed that the enhanced aberration frequency in the combined irradiations originates from interactions between short-lived, X-ray-induced DNA-lesions in close spatial proximity (mainly lesions in the same ionization track), and the repair of these lesions are affected by the UV-treatment. In contrast, the aberrations studied in dose-fractionation experiments, by definition, are due to interactions between (long-lived) lesions in different tracks. Further details of this model for aberration production are discussed.     

Studies on radiation-induced chromosome aberrations in mouse spermatocytes. II. Dose-response relationships of chromosome aberrations induced at zygotene stage in mouse primary spermatocytes following fast neutron- and 60Co gamma-irradiations

The chromosome aberrations induced at zygotene stage in mouse spermatocytes following exposures to fast neutrons and 60Co gamma-rays were examined at diakinesis-metaphase I. The dose-response relationships were well fitted to linear equation for deletion-type aberrations and to linear-quadratic equation for exchange-type aberrations in 60Co gamma-irradiation group. In fast neutron-irradiation group, the dose-response relationships were well fitted to linear equations for deletion- and exchange-type aberrations. The rate of deletion-type aberrations was remarkably high for fast neutrons, about 6 times higher than that after 60Co gamma-irradiation. The main types of chromosome aberrations observed were iso-chromatid breaks or fragments and chromatid exchanges in both irradiation groups as well as X-irradiation. These results indicate that there is a possibility that two double-strand breaks are induced simultaneously at iso-locus position in sister chromatids by a single track of radiations. Production of such single-track-induced two double-strand breaks in iso-chromatids may be very frequently expressed as iso-chromatid-type deletions in the high LET fast neutron-irradiation group. On the contrary, in the low LET 60Co gamma- or X-irradiation group, the above-mentioned mechanism may not be so effective for contribution to chromosome aberration induction in mouse spermatocytes. This mechanism was discussed in detail.     

Analysis of chromosome aberrations in human peripheral lymphocytes induced by 5.4 keV x-rays

Irradiation of human lymphocytes by x-rays has been seen, in past studies, to produce increasing frequencies of chromosome aberrations at lower x-ray energies. However, in one earlier irradiation experiment with chromium x-rays, the relative biological effectiveness (RBE) did not appear to be larger than that of hard x-rays, especially at higher doses. A possible reason for this unexpected result may have been the irradiation and culture conditions. We have, therefore, in the present study used a technique that has been developed in our laboratory to ensure uniformity of irradiation within lymphocytes and to avoid artefacts due to the cell cycle kinetics. Monolayers of 3-h-stimulated lymphocytes were exposed to 5.4 keV x-rays. A linear-quadratic dose-response was found for dicentrics. The comparison to an earlier finding with 220 kV x-rays shows the expected result of the RBE of the 5.4 keV x-rays to be above that of 220 kV x-rays. The intercellular distribution of dicentrics did not differ significantly from a Poisson distribution. Received: 17 January 1997 / Accepted in revised form: 17 July 1997