Evaluation of chromosome painting to assess the induction and persistence of chromosome aberrations in bone marrow cells of mice treated with benzene

Intact pZ189 DNA was allowed to replicate in FL-FEN-1⁻ cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1. E. coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified. The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored. The frequency of such mutants was 19.1 × 10⁻⁴ (34/17781), significantly higher than those of 2.9 × 10⁻⁴ (4/13668) and 3.0 × 10⁻⁴ (3/9857) in the corresponding controls, respectively. Sequence analysis of the supF genes of these mutants showed that all (37/37) the base substitutions occurred at C:G base pairs; 68% (23/37) of the base substitutions were base transversions, while 32% (12/37) were transitions. Approximately 76% (23/37) of these base substitutions occurred frequently at nine positions; two of these sites contain triple pyrimidine (T or C) repeat upstream to the mutated base; four of these sites consist of 5′-TTN₁N₂ and mutations occurred at N₁ site sequence; another two sites have the characteristics of triple A flanked at both 5′ and 3′ side by TCT, with the base substitution occurring at C in the context sequence. These data suggested that these sites are the hot spot of mutagenesis in plasmid replicated in FEN-1-deficient cells. Besides the mutator phenotype of the FEN-1-deficient cell, it was also demonstrated that FEN-1-deficient cell exhibited an increased N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) sensitive phenotype.     

Effect of preliminary chronic irradiation and alpha-tocopherol on the frequency of chromosome aberrations in mouse bone marrow cells, induced by exposure to acute gamma-irradiation

The incidence of chromosome aberrations in bone marrow cells of femur did not exceed the spontaneous one in CBA mice exposed, during 70 days, to gamma-radiation at dose--rates of 33.7-35.8 nA/kg and cumulative dose of 2.75 Gy. A single acute exposure of intact animals to a dose of 2.98 Gy increased significantly the mutation level. Preirradiation with small doses increased the resistance of hereditary structures to sublethal radiation doses. Exogenous alpha-tocopherol (0.06 mg/20 g mass) protected the genetic apparatus of cells from total-body irradiation and was an additional factor decreasing the mutation level after acute exposure of mice at the background of long-term irradiation with small doses.     

Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations of Chinese hamster bone marrow cells

Summary The in vivo SCE formation and the induction of chromosome aberrations in the bone marrow of Chinese hamsters (Cricetulus griseus) were studied after various concentrations of cyclophosphamide, and the sensitivity of the two test methods was compared. The administration of 1.0, 5.0, 13.3, 25.0, and 40.0 mg/kg body weight induced a dose-dependent increase in SCE. The frequency of chromosome aberration, however, was not increased significantly with doses of 1.0 and 5.0 mg/kg body weight. Only with doses of more than 13.3 mg is a significant induction of chromosome aberrations seen. Therefore the SCE test system seems to be 10 times more sensitive than the induction of chromosome aberrations in the same cell type.This work is a part of the M.D. thesis of G. Roszinsky-Köcher, to whom offprint requests should be sent     

DNA-repair kinetics and the sensitivity of cells to X-ray-induced chromosome aberrations: a mouse myeloid leukemia cell line and normal mouse bone marrow cells

The frequency of X-ray-induced chromosome aberrations in G1 ML-1 mouse myeloid leukemia cells and normal mouse bone marrow cells increased with post-irradiation incubation with the DNA-repair resynthesis inhibitor 1-beta-D-arabinofuranosylcytosine (araC), but the frequency of aberrations in the leukemic cells increased with quite a different time response compared to the normal cells. Irradiated normal mouse bone marrow cells had a rapid increase in the frequency of chromosome exchanges and deletions with increasing araC incubation time, for example, an increase was observed with 0.5 h araC incubation. In contrast, the ML-1 cells did not have a significant increase in aberrations until 1-2 h post-irradiation incubation with araC. These results suggest that the ML-1 cells, per unit time, initially undergo less repair of the X-ray-induced DNA damage that can be converted into chromosome aberrations. We previously showed that the ML-1 cells have a higher frequency of X-ray-induced chromosome aberrations compared to normal cells and the results presented here indicate that a slower rate of repair resynthesis is contributing to the increased sensitivity of the ML-1 cells.     

The frequency of chromosome aberrations in rat bone marrow cells in the late period after a single uptake of tritium

A prolonged self-maintenance of haemopoietic tissue cells with stable chromosome rearrangements following a single intake of tritium oxide in the amount of 24 MBq/g of body weight (absorbed dose of 11 Gy) is shown. Mutant cells revealed long after the radionuclide exposure are descendants of stem-cell precursors, bearing stable chromosome aberrations during the period of formation of radiation injury after the radionuclide administration.     

Investigations on the frequency of chromosome aberrations in bone marrow cells of Chinese hamsters after simultaneous application of caffeine and cyclophosphamide

Summary The potentiating effect of caffeine (1,3,7-trimethylxanthine) on chemically induced chromosome aberrations was studied in bone marrow cells of chinese hamsters, exposed to the alkylating agent cyclophosphamide.Four experimental series were performed: In the first two tests caffeine (200 mg/kg) or cyclophosphamide (40 mg/kg), respectively, were administered. A third and fourth test was performed with caffeine plus cyclophosphamide (200+40 mg/kg and 35+40 mg/kg, respectively) simultaneously.Aberrations induced by cyclophosphamide (40 mg/kg) were strongly potentiated by simultaneous application of caffeine (200 mg/kg) not only additively but even synergistically. This increase of aberrations cannot be found after injection of the lower dose of caffeine (35 mg/kg).     

Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Frequency of chromosome aberrations in locally irradiated rat bone marrow cells

A study was made of the frequency of chromosome aberrations induced by cyclophosphamide (CP) in the bone marrow of preirradiated rats (exposure of crus with the body shielded, and vice versa). A reliable evidence was obtained in favor of a change in the metabolism of CP in the irradiated body which was the cause of the enhancement of its cytogenetic action.     

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate.

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEM.MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.     

Analysis of chromosome aberrations in bone marrow cells of rats after treatment with isoniazid

Summary 100 mg INH/kg body weight was administered to male Wistar rats five times, at 24 h intervals, either intraperitoneally or intravenously. In both experimental series the yield of chromosome aberrations in bone marrow cells was not increased as compared to controls.     

Dynamic analysis of the induction of chromosome aberrations in the bone marrow cells of laboratory mice

A study was made of the incidence of such damages as breaks, gaps, and exchanges occurring in bone marrow cells of CBA mice after irradiation with a dose of 12.9 mC/kg. Males and females exhibited a similar spontaneous chromosome aberration level. Nevertheless, the experimental results obtained indicate that males are more radiosensitive than females.     

Chromosomal aberrations and micronuclei induced in rat and mouse bone marrow cells by sodium nitrate

The genotoxicity of several anthraquinone compounds metabolically related to aflatoxin B1 was examined by means of the hepatocyte primary culture (HPC)/DNA repair test and the Salmonella microsome mutagenesis test, and compared to versicolorins A and B which are potent mutagenic and genotoxic intermediates of the aflatoxin biosynthetic pathway. 6,8-O-Dimethyl-versicolorins A, B and 6-deoxyversicolorin A were found to be strongly mutagenic and genotoxic. Genotoxicity of versicolorin A and 6,8-O-dimethylversicolorin A was stronger than that of versicolorin B and 6,8-O-dimethylversicolorin B, respectively, in the HPC/DNA repair test. Nidurufin and norsolorinic acid, which do not possess a bisfuran ring, exhibited questionable activities for mutagenicity and no genotoxicity. It is suspected that 6,8-O-dimethylversicolorins A, B and 6-deoxyversicolorin A as well as versicolorins A and B are genotoxic carcinogens.     

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

To investigate whether subjects with low-acid states are exposed to increased genetic risk with respect to controls, we evaluated mutagenicity and presence of clastogenic factors (CF) in the gastric juice of chronic atrophic gastritis and omeprazole-treated patients. Mutagenic gastric juice was found in 8/15 (53%) chronic atrophic gastritis patients, 8/11 (73%) omeprazole-treated patients, and 2/13 (15%) healthy control subjects. The mean mutagenicity ratio of omeprazole-treated patients (1.52+/-0.48/0.1 ml gastric juice) was significantly higher than those of either controls (1.07+/-0.15; P<0.01) or chronic atrophic gastritis patients (1.16+/-0.21; P<0.05). Only chronic atrophic gastritis patients showed an increased clastogenic index with respect to healthy controls (2.67+/-2.13 versus 0.38+/-0.51; P<0.001). These findings expand our knowledge of gastric disease risk factors, and indicate that there may well be a risk of mucosal DNA damage arising from the presence of mutagenic and CF in the gastric juice.     

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

Fenvalerate, a synthetic pyrethroid insecticide, is commonly used in agriculture and other domestic applications due to its high insecticidal activity and low mammalian-, avian- and phyto-toxicities. However, the genotoxic effect of fenvalerate is highly equivocal. In the present study the genotoxic effects of fenvalerate was evaluated using structural chromosome aberration (CA) and sister chromatid exchange (SCE) assays in mice. Out of the three doses (5, 10 and 20 mg/kg) tested, statistically significant increase in CA was found following intra peritoneal (i.p.) treatment of 20 mg/kg of fenvalerate for 24 h (P<0.01) and 48 h (P<0.05) only. Neither the acute doses of 5 and 10 mg/kg, nor the sub-acute dose (5×4 mg/kg) of fenvalerate could induce any significant effect. All the three acute doses induced significant increase in the frequency of SCEs (P<0.01) in the bone marrow cells, which showed a significant dose-response correlation (r=0.9541, P<0.05). With certain reservations to possible impurities, from the present findings technical grade fenvalerate may be considered as a weak clastogen and a potent inducer of SCEs in mice.     

Resveratrol reduces radiation-induced chromosome aberration frequencies in mouse bone marrow cells

Resveratrol, a polyphenol compound with reported antioxidant and anticarcinogenic effects, a wide range of molecular targets, and toxicity only at extreme doses, has received considerable attention. We evaluated the radioprotective effect of orally administered resveratrol on the frequencies of chromosome aberrations in irradiated mouse bone marrow cells. CBA/CaJ mice were divided into four groups: (1) no treatment, (2) resveratrol only, (3) radiation only, and (4) resveratrol and radiation. Resveratrol treatment (100 mg/kg daily) was initiated 2 days prior to irradiation. Bone marrow was then harvested at 1 and 30 days after a single dose of 3 Gy whole-body gamma radiation. A statistically significant (P < 0.05) reduction in the mean total chromosome aberration frequency per metaphase at both times postirradiation in the resveratrol and radiation group compared to the radiation-only group was observed. This study is the first to demonstrate that resveratrol has radioprotective effects in vivo. These results support the use of resveratrol as a radioprotector with the potential for widespread application.     

The fate of cells with chromosome aberrations after total-body irradiation and bone marrow transplantation

Cytogenetic studies were done on bone marrow cells and peripheral lymphocytes of four patients (three with acute nonlymphocytic leukemia, one with aplastic anemia) at various intervals up to 861 days after total-body X irradiation (TBI) at doses between 4.5 and 10 Gy (450-1000 rad) followed by syngeneic or allogeneic bone marrow transplantation. Whereas no radiation-induced aberrations could be found in the bone marrow, apart from a transient finding in the patient with the lowest radiation dose, aberrant metaphases were seen in the peripheral lymphocytes of three patients in the range from 2.5 to 46% even at 861 days after the exposure. There were no demonstrable aberrations related to TBI in the only patient developing graft-versus-host disease. The dicentric yield as determined in the aberrant metaphases with 46 centromeres ranged between 3.4 +/- 1.3 and 4.9 +/- 0.4. In one patient it was demonstrated by BUdR-labeling that after 10 Gy (1000 rad) TBI the surviving and heavily damaged lymphocytes can go into cell cycle and reach at least the third mitosis. The percentage of aberrant cells diminished by about 25% at each mitotic division.     

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

Frequency of micronucleated cells in the mouse bone marrow after exposure to various doses of gamma-radiation

The frequency of micronucleated cells was determined in the bone marrow of female BALB/c mice exposed to different doses of gamma-radiation at 1, 3, 10 and 14 days post irradiation. The frequency of micronucleated cells increased with the increase in exposure dose at all time period studied. However, the frequency of micronucleated cells declined with time irrespective of exposure dose. The dose-response curve thus obtained was linear.     

Chromosomal aberrations in the bone marrow cells of mice induced by accelerated (12)C(6+) ions

The whole bodies of 6-week-old male Kun-Ming mice were exposed to different doses of (12)C(6+) ions or X-rays. Chromosomal aberrations of the bone marrow (gaps, terminal deletions and breaks, fragments, inter-chromosomal fusions and sister-chromatid union) were scored in metaphase 9h after exposure, corresponding to cells exposed in the G(2)-phase of the first mitosis cycle. Dose-response relationships for the frequency of chromosomal aberrations were plotted both by linear and linear-quadratic equations. The data showed that there was a dose-related increase in the frequency of chromosomal aberrations in all treated groups compared to controls. Linear-quadratic equations were a good fit for both radiation types. The compound theory of dual radiation action was applied to decipher the bigger curvature (D(2)) of the dose-response curves of X-rays compared to those of (12)C(6+) ions. Different distributions of the five types of aberrations and different degrees of homogeneity were found between (12)C(6+) ion and X-ray irradiation and the possible underlying mechanism for these phenomena were analyzed according to the differences in the spatial energy deposition of both types of radiation.