Thiocyclam does not induce structural chromosome aberrations in human lymphocytes in vitro

Thiocyclam (trade name Evisect) is a broad-spectrum nereistoxin analogue insecticide used widely for agricultural applications. The aim of this investigation was to determine its genotoxic effects in the chromosome aberration (CA) test and determining of mitotic index (MI), using lymphocytes from peripheral blood samples of healthy human donors. A negative and a positive control (MMC) were also included. Chromosomal analyses of the metaphase plates of the samples treated with 14 different concentrations (from 0.1 to 120 μg/ml) of thiocyclam, indicating the lack effect on chromosomes. Thus thiocyclam is not genotoxic but highly toxic on cell proliferation in human lymphocytes.     

A comparative study of the micronucleus test and chromosome aberrations in PHA-stimulated human lymphocytes

A dose dependence of the number of cells with chromosome aberrations was studied in PHA-stimulated donor's peripheral blood lymphocytes irradiated in vitro with doses of 10-400 cGy. In studying the number of chromosome aberrations and percentage of cells with micronuclei in parallel cultures no correlation was found between these indices within the groups exposed to a similar radiation dose.     

Frequency and distribution of mitomycin C-induced structural chromosome aberrations in lymphocytes from non-Hodgkin lymphoma patients

Lymphocytes from 15 untreated patients with non-Hodgkin lymphoma (NHL) and 15 controls were exposed to 0.08 micrograms/ml mitomycin C, and the frequency and distribution of structural chromosome aberrations (chromatid and chromosome gaps, breaks, and exchanges) were analyzed in 100 mitoses per subject. The mean frequencies of aberrant cells, and gap, break, and gap + break events were 8.7, 0.9, 9.7, and 10.6 in the NHL group and 11.6, 1.1, 12.7, and 13.8 in the control group. None of the differences between the two groups was significant (P greater than 0.05). The distribution of breakpoints was nonrandom (P less than 0.001) in both groups, with a particularly marked excess of breaks in 9q11. The other breakage-prone bands were 1q11 and 1q21 in the NHL group and 1p11, 1q11, 2q31, and 16q11 in the control group. None of these hot spots coincided with any of the 60 bands known to be involved in primary chromosome abnormalities in NHL.     

Induction of structural chromosome aberrations and sister chromatid exchanges in human lymphocytes in vitro by aristolochic acid

The medicinal use of Aristolochia clematitis has been known for some time. The main active agent of this medicinal plant is aristolochic acid, a nitrophenanthrenecarbonic acid. Very recently, however, the Federal Health Office withdrew the licence for all drugs containing aristolochic acid, because of the well-founded suspicion that aristolochic acid may be a very potent carcinogen. We investigated the induction of structural chromosome aberrations and sister chromatid exchanges (SCEs) by aristolochic acid in human lymphocytes in vitro. Cells were treated with the agent tested throughout culture time and during the G0 phase of the cell cycle. We tested concentrations over a range of 1 to 20 micrograms/ml. Both treatment conditions resulted in an increased aberration frequency. The induction of gaps and breaks as well as the induction of SCEs showed a dose-dependent increase. The number of SCEs per metaphase was enhanced by a factor of 2 to 3. If conventional cytogenetic methods had been applied in time, one would have recognized the mutagenic risk of aristolochic acid earlier.     

Significance of structural chromosome aberrations in human sperm: analysis of induced aberrations

Summary A significant increase in the incidence of structural chromosome anomalies has been observed in the sperm of patients treated with radio and/or chemotherapy for different types of cancer when analyzed by the interspecific fertilization of hamster eggs. The analysis of these aberrations shows that while in controls only 9.4% of structural abnormalities are of the stable type, in treated patients this figure increases to 39.3%, thus indicating that the anomalies have not been produced during the fertilization of the hamster egg. However, it is possible that part, or even most, of the breaks appear as a result of a reduced repair capacity of sperm chromosomes in the cytoplasm of the hamster egg.     

Detection of structural chromosome aberrations in immunophenotyped mitoses.

The recently developed MAC (morphology-antibody-chromosome) method allows simultaneous immunophenotype and karyotype analysis in the same cell. To date, application of this new method has been hampered by the poor quality of chromosome banding. In this paper, we describe a modified simultaneous immunofluorescence and Q-banding technique, as well as a new combination of immunohistochemical and fluorescent R-banding methods. By further modifying the MAC method, we were able not only to achieve unequivocal results with weakly expressed antigens but also to improve the quality of the banding techniques, so that even structural chromosome abnormalities were well defined.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa

An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H(2)O(2) or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G(1) population was pointed out by flow cytometry. Sub-G(1) cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.     

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     

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01–0.5 mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.     

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01-0.5mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.     

Persistence of drug-induced chromosome aberrations in peripheral blood lymphocytes of the rat

We have studied the persistence of pre-clastogenic lesions, detected as induced chromosomal aberrations, in rat peripheral lymphocytes at various time intervals after acute treatment with 3 different antineoplastic drugs: cyclophosphamide (CPA), 5-fluorouracil (5-FU) and adriamycin (AM). Single i.p. doses were administered to groups of rats and heart blood samples from each group were taken after 3, 12, 24 or 48 h or weekly up to 20 weeks later. The cytogenetic analysis was performed on lymphocytes cultured for 33 h after sampling. The results for CPA exposure (10 mg/kg) show that the yield of chromosome aberrations is maximal 3 h after the treatment (20 times the control level). For up to 8 weeks the values remain about 6 times the baseline; afterwards a decrease is observed and the control level is reached after 20 weeks. For 5-FU (50 mg/kg) a remarkable increase (13-fold) in chromosomal damage is observed at the first sampling time. Within 48 h the effect is drastically reduced but persistent (3 times the control level), and the level returns to spontaneous values 1 week later. AM treatment (2 mg/kg) induced an increase of about 8 times the control level at 3 h post exposure. The clastogenic effects remained at a detectable level for 1 week (about 6 times the control level at all sampling times); 2 weeks after the treatment the control level was found. A parallel analysis was performed on bone marrow cells. In this tissue the clastogenic effects of the treatments were maximal, as in lymphocytes, at the first sampling time (20-25 times the control level) and were no longer detectable within 72 h after exposure, irrespective of the administered drug.     

RBE of thermal neutrons for induction of chromosome aberrations in human lymphocytes

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with slow neutrons was examined to assess the maximum low-dose RBE (RBE_M) relative to ⁶⁰Co γ-rays. For the blood irradiations, cold neutron beam available at the prompt gamma activation analysis facility at the Munich research reactor FRM II was used. The given flux of cold neutrons can be converted into a thermally equivalent one. Since blood was taken from the same donor whose blood had been used for previous irradiation experiments using widely varying neutron energies, the greatest possible accuracy was available for such an estimation of the RBE_M avoiding the inter-individual variations or differences in methodology usually associated with inter-laboratory comparisons. The magnitude of the coefficient α of the linear dose–response relationship (α = 0.400 ± 0.018 Gy^?1) and the derived RBE_M of 36.4 ± 13.3 obtained for the production of dicentrics by thermal neutrons confirm our earlier observations of a strong decrease in α and RBE_M with decreasing neutron energy lower than 0.385 MeV (RBE_M = 94.4 ± 38.9). The magnitude of the presently estimated RBE_M of thermal neutrons is—with some restrictions—not significantly different to previously reported RBE_M values of two laboratories.     

Regularities in forming chromosome aberrations in cattle lymphocytes from irradiation in vitro

Aberrations in lymphocytes of cattle blood exposed to gamma-radiation with doses from 1 to 7 Gy were studied. The rate of variable cells depended linearly on the irradiation dose, whereas the total frequency of aberrations, as well as that of dicentric and annular chromosomes followed a linear-quadratic dependence.     

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 x-ray-induced chromosome aberrations in stimulated and unstimulated human lymphocytes

The repair time for chromosome breaks induced by X-irradiation of unstimulated (G₀) and stimulated (G₁) human lymphocytes has been determined by dose fractionation studies. In both types of cells repair time was approx. 4–5 h. Treatment with hydroxyurea, a DNA synthesis inhibitor, did not prevent or delay the rejoining of broken chromosomes, whereas treatment with cycloheximide, a potent protein synthesis inhibitor, did. Thus, the repair of radiation-induced chromosome breaks in human lymphocytes is similar to the repair observed with plant cells.     

Trenimon-induced SCEs and structural chromosomal aberrations in early- and late-dividing lymphocytes

In cultures of human peripheral lymphocytes the frequencies of Trenimon-induced SCEs in second post-treatment metaphases and of structural chromosomal aberrations in first, second and third post-treatment metaphases were clearly lower at late as compared with early fixation times. These results, which are discussed, indicate that T cells (early dividing) are more sensitive with respect to the induction of SCEs and structural chromosomal aberrations by Trenimon as compared with B cells (late dividing).