Protective effect of cysteine against X-ray- and bleomycin-induced chromosomal aberrations and cell cycle delay

The protective effect of cysteine was studied in muntjac and human lymphocytes in vitro scoring chromosomal aberrations in harlequin stained first cycle metaphases, induced by X-irradiation at G₀. Its protective efficiency was also studied against the radiomimetic clastogen, bleomycin, in muntjac cells. 30 μg and 1 mg/ml of cysteine were given prior to 2, 3, and 4 Gy, and 2 mg/ml prior to only 4 Gy. 30 μg cysteine protected only against deletions in 4 Gy-treated cells while 1 mg protected against deletions by all three doses of X-rays. However, rearrangements were not reduced significantly in any of these, probably due to their low frequency. But when cysteine was increased to 2 mg, both types of aberrations were reduced significantly. This shows that a sufficient number of aberrations and an optimum concentration of the protector are essential for eliciting the best protective effect. This conclusion is further supported by the results of 2 mg cysteine treatment in human lymphocytes which yielded higher frequencies of rearrangements with 2 and 3 Gy X-rays than 4 Gy in muntjac, but had a relatively lower frequency of deletions. Thus the most abundant categories of aberration, i.e., deletions in muntjac and exchanges in humans, were reduced significantly by 2 mg cysteine, associated with a prominent reduction in the frequency of aberrant metaphases. Therefore, the differential protection observed with a low concentration of the protector and an insufficient yield of aberrations induced only indicates protection provided to the most frequent type of aberration by a protector when present in lower concentration.

Cysteine pretreatment yielded weak protection against the effects of bleomycin, but posttreatment caused a mild potentiation of the clastogenic effect of BLM without altering the cell cycle kinetics. In this context, an action of cysteine as a reducing agent on BLM is suggested. Although cysteine alone caused severe retardation of the cell cycle, when given prior to X-irradiation, not only its delaying effect was not observed, but also it reduced the X-ray induced cell cycle delay. This might be due to the oxidation of cysteine by its radical scavenging action.     

Chromosome and cytome analysis of the somatic cells of nuclear-chemical production workers with incorporated 239Pu

The analysis of plutonium production factors has been carried out by using two methodical approaches: assessment of chromosomal aberrations level in routine and G-banded metaphases and molecular-cytogenetic investigation of aneugenic/clastogenic damages in cytokinesis-block binuclear lymphocytes by FISH with centromere specific DNA probes. The obtaining data point out for the first time about both aneugenic and clastogenic influences of incorporated 239Pu with activity range from 0.37 to 6.95 kBq. Correlation analysis of chromosome aberrations with cytome abnormalities allowed finding significant connection between number parameters of metaphase and interphase approaches. The results of this study support the suggestion that aberrant chromosomes are involved preferable in aneugenic events. The FISH technique in binucleated cytokinesis-blocked lymphocytes allows extending of detecting spectrum of chromosome damages and glance of aneugenic mechanisms. Correlations between metaphase and interphase-FISH results point out a high sensitivity of FISH cytome assay, which could be used as an independent test for detection both clastogenic and aneugenic environment influences.     

Blood lymphocyte culture system: quantitative analysis of X-ray-induced chromosome aberrations in man, muntjac and cattle

Peripheral blood lymphocytes of 3 mammalian species, man, muntjac and cattle, which have various amounts of DNA and divergent karyotypes, were exposed to 100-400 rad of X-rays, and frequencies of dicentrics and other aberrations were analysed at first post-irradiation metaphases. During experiments, various preparative or physical and biological factors that could influence the yield of chromosome aberrations were taken into account. The frequency of dicentrics scored at first post-irradiation metaphases showed best fit to both linear and quadratic dose-response curves, y = a + bD and y = bD + cD2 with a high correlation coefficient of 0.98 (P less than 0.001). The frequency of dicentrics obtained at different post-irradiation fixation times did not show significant variation, indicating a homogeneous sensitivity of peripheral lymphocytes to X-irradiation. BrdU incorporation following X-irradiation showed no increase in the frequency of chromosome aberrations. The frequency of dicentrics in man, muntjac and cattle showed a close correlation with their DNA content, but no meaningful correlation was found between the yield of dicentrics and the chromosome arm number or the nuclear volume. The ratio of dicentric yields, 1.00:0.67:1.04 obtained in man, muntjac and cattle were comparable to the ratio of their DNA contents, 1.00: 0.65: 1.07. The base-line frequency of SCEs was similar in the 3 species and no significant variation in SCE frequency was noticed even after administration of 400 rad of X-rays.     

Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.     

Aberrant and multiaberrant (rogue) cells in peripheral lymphocytes of Hodgkin's lymphoma patients after chemotherapy

We analyzed spontaneous chromosome lesions in peripheral lymphocytes cultured from Hodgkin's lymphoma (HL) patients before and after cytostatic chemotherapy. The mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20+/-0.58 per 100 metaphases) than in non-treated HL patients (4.80+/-0.54), and in non-treated patients than in healthy subjects (2.12+/-0.13). In lymphocytes of HL patients, who received chemotherapy, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (or rogue) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. Rogue cells were found in 15 out of 18 chemotherapeutically treated HL patients (in total, 60 rogue cells per 5,568 scored cells), whereas in 30 non-treated patients only 1 rogue cell was found (per 4,988 scored cells). No correlation was found between the yield of rogue cells and the aberration frequency in ordinary aberrant cells. Aberration spectra (ratios of chromatid- to chromosome-type aberrations and of breaks to exchanges) were essentially different in ordinary aberrant and multiaberrant cells. These data, as well as analysis of cellular distributions of aberrations, implied independent induction of chromosome damage in ordinary aberrant and rogue cells. Analysis of aberration patterns in diploid and polyploid rogue metaphases belonging to the first, second, and third in vitro division indicated that rogue cells could be formed both in vivo and in vitro, and could survive at least two rounds of in vitro replication, given blocked chromosome segregation. These results suggested that formation of rogue cells, unlike ordinary aberrant cells, was triggered by events other than direct DNA and/or chromosome lesions. A hypothesis regarding disrupted apoptosis as a candidate mechanism for rogue cell formation seems to be most suitable for interpretation of our data. Cultured lymphocytes of chemotherapeutically treated HL patients may represent a model system for further examination of the multiaberrancy phenomenon.     

Chromosome aberrations in human lymphocytes at a various duration of cultivation after irradiation

Human peripheral blood lymphocytes were exposed to 60Co gamma-rays (a dose of 3 Gy) and cultivated during seven days in the presence of PHA and BrdU. It was shown that the metaphases of the first and second mitosises occurred during cultivation of the irradiated and unirradiated lymphocytes, being evidence about of irregularity of the coming into division of various fractions of lymphocytes. The time of cultivation did not influence a rate of aberrations in metaphases of the first and second mitosises of the irradiated lymphocytes. During the first and the subsequent mitosises the number of exchange chromosome aberrations decreased and reached a control level in metaphases of the fourth and fifth mitosises. The number of paired fragments at second and third mitosises increased a little and started to decrease only in metaphases of the fourth and fifth mitosises. The decrease in chromosome aberrations with prolongation of the cultivation of lymphocytes after irradiating is a consequence of elimination of cells with chromosome damages during sequential mitotic divisions.     

Aphidicolin and bleomycin induced chromosome damage as biomarker of mutagen sensitivity: a twin study

The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of sensitivity to mutagens. To assess their validity, we have investigated whether the individual expression of induced chromosome damage has a genetic rather than an environmental basis. Metaphase analysis for chromosomal aberrations (CA) and micronucleus (MN) assay in cytokinesis-blocked cells have been performed in peripheral blood lymphocytes from 19 healthy male twins (9 monozygotic and 10 dizygotic pairs), aged 70-78 years, after APC, BLM and APC+BLM treatments.Concordance between twins revealed a high genetic component in the sensitivity towards clastogenic action of APC both as percentages of CA and MN. The micronucleus assay demonstrated a genetic basis also in the expression of chromosome damage induced by BLM and APC+BLM treatments. Since twins were elderly people, to investigate the possible role of age, CA and MN frequencies were compared with those found in lymphocytes from 11 young male donors. Basal and APC-induced chromosome damage were clearly increased in the former. Following BLM and APC+BLM treatments, age significantly increased mitotic delay, as shown by the mitotic indexes (MI) and by the ratios between binucleated and mononucleated (B/M) cells.     

Severe chromosomal damage in mammalian cells induced by pulse labeling with small amounts of [3H]thymidine

Pulse labeling of cultured mammalian cells with tritiated thymidine ([3H]TdR; different specific activities, 2-47 Ci/mmole) for 30 min at low concentrations of radioactivity (0.2-2 microCi/ml) causes an unusually high degree of chromosomal aberrations which becomes fully evident in the cohort of cells analyzed 8 h later. The effect depends on the total amount of radioactivity. In HeLa cells up to 93% aberrant metaphases with up to 3.6 aberrations per metaphase were observed; in CHO cells the corresponding figures are 62% aberrant metaphases with 1 aberration per metaphase. The results may be valuable (i) for users of the pulse-labeling technique for analysis of cell-cycle kinetics, as this shows the technique as such may delay the labeled cohort of cells and therefore interferes with the matter under study and (ii) for risk assessment.     

G0 chromosomal radiosensitivity in ataxia telangiectasia lymphocytes

Contrary to an earlier report, peripheral lymphocytes from 4 AT patients were not found to exhibit higher yields of unequivocal chromosome type aberrations following irradiation in the G₀ phase of the cell cycle, providing that only first post-irradiation metaphases were included in the samples (ensured by 5-bromodeoxyuridine (BrdU) incorporation and differential fluorescence or Giemsa staining). We were able, however, to confirm the earlier-reported increase in chromatid-type aberrations in the G₀-irradiated cells. AT lymphocytes were found to experience more cell-cycle delay following G₀ irradiation than normal cells. These observations appear consistent with the damaged base excision DNA-repair defect reported for AT cells.     

The influence of pretreatment with exogenous DNA on the frequencies of aberrations induced by X-Rays

Pretreatment with calf thymus DNA decreases the frequency of chromosomal aberrations in metaphases and anaphases induced by X-rays in barley root meristem cella. This effect is manifested in a wide range of concentrations (5% -0.001%) without any great dependence on the concentration. Calf thymus DNA alone does not induce any chromosomal aberrations in barley root meristem cells.     

Possible role of the T antigen in inducing chromosome aberrations in SV40 virus-transformed cells

A possible role of the simian virus 40 T antigen in chromosome damages in transformed cells was examined. Two lines of Golden hamster embryonal fibroblasts, transformed by SV40 tsA30 and ts239 mutants (He30 and He239, respectively), were incubated at nonpermissive (40.5-41 degrees C) or permissive (33 degrees C) temperatures. Chromosome aberrations were registered in either subline after 3, 6, 9 and 12 weeks of cultivation under the above conditions. In the both cell lines kept at 33 degrees the frequency of aberrant metaphases and the number of chromosome breaks per cell increased drastically by week 3 of cultivation, and such a state was preserved up to week 12. The frequency of aberrant metaphases in cells cultivated at 41 degrees was maintained at the constant level (He239) or at slightly higher than that in the original culture (He30). The sublines He239, originally incubated at 33 or 40.5 degrees, were then shifted to 40.5 and 33 degrees, respectively. As a result the number of chromosome aberrations either decreased (33----40.5 degrees) or increased (40.5----33 degrees) as early as on day 2, and these patterns were stabilized at the level corresponding to the new conditions. We assayed the induction of DNA breaks in cells, grown at the permissive or nonpermissive temperatures, by using DNA sedimentation in the alkaline sucrose gradient. The DNA sedimentation peaks of cells cultured at 37 and 41 degrees coincided, whereas the DNA of cells cultured at 33 degrees was represented by shorter fragments.     

The analysis of chromosome lesions in lymphocytes of Hodgkin's lymphoma patients after chemotherapy and radiation therapy

The analysis of chromosome lesions in peripheral blood lymphocytes of Hodgkin's lymphoma (HL) patients after chemotherapy and chemotherapy with the subsequent course of radiation therapy is carried out. Is shown, that the mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20 +/- 0.58 per 100 metaphases) than in non-treated HL patients (4.80 +/- 0.54, p < 0.01). The subsequent carrying out of radiation therapy enlarges number of chromosome aberrations on 100 metaphases up to 46.7 +/- 10.7 (p < 0.05), of which chromosome-type aberrations (43.2 +/- 10.3 on 100 metaphases) averaged 92.5%. In lymphocytes of 37 out of 43 HL antitumoral treatment patients, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (MA-cells) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. In 30 non-treated HL patients only one MA-cell was found. From 171 MA-cells which were in 43 HL patients after antitumoral treatment, 114 MA-cells were found at inspection of 9766 diploid metaphases, and the remaining 57 MA-cells were found at inspection of 196 polyploid metaphases. The carrying out after chemotherapy of radiation therapy enlarges in lymphocytes frequency of appearance of MA-cells. The analysis of MA-cells in diploid and polyploid metaphases shown, that the MA-cells could be formed both in vivo, and in vitro in absence of influence of clastogenic factors, and could survive at least two rounds of in vitro replication.     

The number of aberrations in aberrant cells as a parameter of chromosomal instability. 1. Characterization of dose dependency

Analysis of chromosome instability (CI) is of great importance in view of pollution of the environment by genotoxic factors. Frequency of aberrant cells, spectrum of chromosome aberrations, damages of aberrant cell and distribution of aberrations in the cells are the most conventional parameters of CI. We have carried out the comparative analysis of the frequency of aberrant cells and the dynamics of aberrant cell damages induced by different mutagenic factors (alpha-irradiation from 241Am, gamma-irradiation from 60Co and tioTEPA) in Allium-test. This comparative analysis denotes that the studied parameters have different dynamics characterizing different mechanisms of CI in Allium cepa L.     

Liquid holding effects on methylmercury genotoxicity in human lymphocytes

Methylmercury chloride (MMC) treatment of resting (GO) human lymphocytes resulted in the formation of chromosome and chromatid aberrations. This treatment also induced aneuploidy in second metaphases, suggesting that MMC produces stable damage involved in chromosome segregation errors. The storage of treated cells (liquid holding for 48 h before cell proliferation) did not result in an important recovery from induced cell toxicity or chromosome damage. Therefore, MMC seems to be an X-ray-like agent, able to produce long-lasting damages giving rise to both structural and numerical chromosome abnormalities.     

Radiosensitivity of somatic cells and imaginal disks of male and female Drosohila]

Primary chromosome damages as well as the frequency of spontaneous and X-rays induced chromosome aberrations in Drosophila melanogaster males and females are studied. It is found using cytofluorimetric method that primary chromosome damages in ganglia cells of females and males are the same. In these cells as well as in cells of imaginal discs the frequency of induced chromosome aberrations, except gaps, is considerably higher for females. Ganglia cells of females and males of Drosophila are found not to differ from each other in their proliferation activity. The frequency of morphoses for both sexes is also the same. The assumption is made concerning the role of the non-identical repair in the increased mutability of female somatic cells, which is more intensive in this sex, as well as concerning more intensive identical repair in imaginal discs of females.     

Quantitative characteristics of the frequency of chromosomal aberrations in a group of residents of a large western Siberian industrial region

The results of a 15-year study of chromosome aberration frequency in cultured peripheral blood lymphocytes from subjects living in the Kuzbass industrial region are presented. The database for the analysis of the main parameters of chromosome aberrations contains data on 925 subjects, with the total number of cells examined being 92,900. It has been found that the total frequency of aberrant metaphases in the database is 3.73 +/- 0.1%, whereas this frequency for the sample of subjects from industrial areas of this region (the basic control group) is significantly lower (2.86 +/- 0.26%). It has been demonstrated that the sex and age of the subjects do not affect substantially the frequency of any type of chromosome aberrations. Tobacco smoking is associated with a slight increase in chromosome damage frequency; however, the difference between smokers and nonsmokers is insignificant even among subjects exposed to occupational hazards. The possible causes that have determined the increased basic and background chromosome aberration frequencies in the population of the Kemerovo oblast compared to these parameters for the European part of Russia and the CIS are discussed.     

The effect of 3-aminobenzamide on X-ray induction of chromosome aberrations in Down syndrome lymphocytes

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.     

Mechanism of the chromosome damaging effect of ftorafur]

Chromosome damage induced by three antineoplastic drugs -- ftuorafur (Ft), 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUdR) hase been studied in Djungarian hamsters cell line after 24 hours exposition with these agents before the fixation. Ft at a dose of 10 micron/ml induced aberrations in 56.7% of metaphases. 60.0% of aberrant metaphases were obtained in experiments with 0.1 micron/ml of 5 FU. FUdr at the same dose induced 24.0% of aberrant metaphases. The high frequency of chromatid breaks and gaps was typical for the mutagenic action of these fluorinated pyrimidines. The addition of Ft or 5-FU to the cell cultures 1--12 hours before fixation did not produce any significant chrosome damage, while further exposition with the drugs for 15--24 hours caused breaks in more than 50% of metaphases. Thymidine at a concentration of 1.0 micron/ml suppressed the chromosome breaking effect of Ft and 5-FU. The results obtained are in accordance with the idea that fluorodeoxyruidinemonophosphate is the ultimate mutagen for both Ft and 5-FU and that the aberrations observed are due to the lack of thymidine nucelotides caused by the former agents while DNA replication.     

The database for analysis of quantitative characteristics of chromosome aberration frequencies in the culture of human peripheral blood lymphocytes]

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330,000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 +/- 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.     

The Database for Analysis of Quantitative Characteristics of Chromosome Aberration Frequencies in the Culture of Human Peripheral Blood Lymphocytes

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330 000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 ± 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.