Modeling, using nucleases, of the effect of radiation on Chinese hamster fibroblasts. On the role of single-stranded DNA breaks, induced by DNAase I, in the initiation of chromosome aberrations

DNAase I injected to Chinese hamster fibroblast cells resulted in the chromosome aberration induction at all stages of the cell cycle and death of cells. Comparison of the effects of DNAase I and gamma-radiation on Chinese hamster cells showed that with close values of the induced DNA breaks there were close values of the cytogenetic damage and the number of DNA damages per aberrant cell.     

The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro

The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid-type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone.     

Chromosome aberrations in individuals occupationally exposed to ethylene oxide, and in a large control population

Chromosome aberration frequencies in 61 employees potentially exposed to ethylene oxide (ETO) were compared with those in unexposed control groups. We studied 3 worksites with differing historical ambient levels of ETO. Within worksites, groups were classified as high potential exposed, low potential exposed, or controls. Further control groups including an off-site community control group were added to give a total of 304 control individuals. Blood samples were drawn several times over a 24-month period. Aberrations were analyzed in 100 cells per sample after culture for 48-51 h. Worksites I, II and III respectively represented increasing levels of potential ETO exposure. At worksites I and II, no consistent differences in aberration frequencies were found among groups. At worksite III aberration frequencies in potentially exposed individuals were significantly increased compared with controls. The frequencies of cells with aberrations were 5.6% for the 2 individuals in the high potential exposure category and 2.6% for 23 persons in the low potential exposure group. The overall frequency of cells with aberrations in the matched control individuals was 1.4%. In the total control group of 304 individuals we found significant increases in aberrations associated with smoking and with increasing age. We have also reported previously an association between sister-chromatid exchange (SCE) frequency and ETO exposure (Stolley et al., 1984). When aberration frequencies were compared with levels of SCEs there was only a weak overall association. The correlation was found in potentially exposed but not in control groups, and for any individual, one observation could not be used to predict the other.     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. I. Cell cycle inhibition and aberration induction in diploid and Down's fibroblasts.

The effect of N-methyl-N-nitrosourea (MNU) on the cell cycle, DNA synthesis and chromosomal sensitivity of cultivated diploid fibroblasts and fibroblasts with trisomy 21 was investigated in vitro. With the exception of the inhibition of G2, Down's cells proved to be more sensitive than diploid cells with respect to the decrease of the mitotic and labelling index, the inhibition of the progression of cells through the early and middle S and the frequency of induced chromosomal aberrations. The chromosomal sensitivity was dependent on the position of cells in the cell cycle during treatment with MNU. If treated during late S no differences concerning the S block and aberration frequencies were found between diploid and Down's cells. However, if MNU treatment took place in the middle and early S, Down's cells were more sensitive. The higher aberration frequencies in Down's cells resulted from elevated levels of chromatid breaks, multiple fragmentations and chromatid translocations. Possible reasons for the increased sensitivity of Down's cells are discussed.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of DNA double-strand breaks (DSBs] by restriction endonucleases (REs) and inhibitors of DNA topoisomerase II known to induce DNA strand breaks. Five different types of REs, namely CfoI, EcoRI, HpaII (which induce cohesive DSBs), HaeIII and AluI (which induce blunt DSBs) were employed. REs that induce blunt-end DNA DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks. xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type CHO-K1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant CHO cells, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type cells. We also studied the effect of inhibitors of DNA topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type CHO-K1 cells respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G1 treatment of CHO cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than CHO-K1 cells. The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of DNA topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.     

Effect of heat treatment on chromosomal aberrations induced by the alkylating agent trenimon or the restriction endonuclease Alu I in Chinese hamster ovary (CHO) cells

Heat treatment of CHO cells in the G1-phase of the cell cycle leads to chromatid-type aberrations in first posttreatment metaphases. Posttreatment of heat-treated cells with the alkylating agent trenimon leads to a synergistic effect on the production of chromatid-type exchanges. These results indicate that heat induces lesions which like the lesions produced by trenimon give rise to chromatid-type aberrations during the first posttreatment S-phase, and that these lesions can interact with each other to produce chromatid-type exchanges. Treatment of CHO cells in the G1-phase of the cell cycle with the restriction endonuclease Alu I induces chromosomal aberrations. Pretreatment of cells with heat leads to a reduction of Alu I induced chromosome-type aberrations. When cells are allowed to recover after heat treatment for 22 h, the aberration frequencies produced by Alu I are the same as in cells not treated with heat. These findings can be explained by assuming that heat-induced accumulation of accessory proteins in the chromatin protects the DNA from being cut by Alu I, and that the cells recovered from the heat-induced protein accumulation after 22 h.     

Biological monitoring of workers in the rubber industry. I. Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of vulcanizers

To evaluate the possible genetic consequences of the industrial exposure among the vulcanizers of a rubber plant we measured the in vivo levels of chromosomal aberrations and sister-chromatid exchanges in peripheral lymphocytes of 34 vulcanizers and in an adequate control population. The observed chromosomal aberration frequencies were 1.9 +/- 1.4 aberrations/100 cells in the exposed group and 2.1 +/- 1.5 aberrations/100 cells in the controls. No difference was found between the two groups for the mean value of sister-chromatid exchanges (5.2 +/- 1.3 in the exposed, 5.2 +/- 0.7 in the control group). Cigarette-smoking was clearly associated with increased sister-chromatid exchange frequencies both in the exposed and in the control groups, while chromosomal aberration frequencies were not correlated with smoking habits.     

Genotoxicity of 'gudakhu', a tobacco preparation. I. In mice in vivo.

'Gudakhu' is a paste-like tobacco preparation used widely in Orissa and neighbouring states of India. During use it is rubbed over the teeth and gum with a finger tip. Besides tobacco, it contains molasses, lime, red soil and water. The genotoxic potential of acetone extract of gudakhu was evaluated in mice in vivo using the chromosome aberration assay, micronucleus test and SCE analysis following single as well as long-term repeated treatment. The animals received an aqueous suspension of the extract via the oral route. Gudakhu extract induced significantly high frequencies, compared to controls, of chromosome aberrations, micronuclei (MN) and SCEs. Single treatment with different doses clearly revealed a distinct dose-dependent increase of the effects in all the assays. Analysis of MN in regenerated hepatocytes also indicated a significant positive correlation between time-course of chronic treatment and frequencies of micronucleated cells. But incidences of chromosome aberrations, MN and SCEs in bone marrow cells following repeated treatment for different periods did not differ greatly from each other; and these repeated treatment data, particularly in the MNT in bone marrow cells and the SCE assay, also did not differ markedly from the respective single treatment data for the same dose. This was probably due to the proliferative nature of the bone marrow cells.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.     

Role of maternal exposures and newborn genotypes on newborn chromosome aberration frequencies

Maternal exposures may induce chromosome damage and birth defects in the fetus. Polymorphic variation in genes coding for enzymes involved in metabolic activation and detoxification of environmental procarcinogens may account for some of the differences in chromosome aberration frequencies in newborns. In this study, 40 mothers completed questionnaires regarding exposures they received during their pregnancy. Umbilical cord blood samples were analyzed for chromosome aberrations. An average of 1020 metaphase cell equivalents (equal to 1020 G-banded cells) were examined from each newborn. In 26 of the newborns, genotyping analysis was performed for genes functioning in metabolic activation and detoxification (cytochrome P450 genes: CYP2D6 and CYP1A1, and phase II genes: NAT1, NAT2, GSTT1, GSTM1, GSTP1, and epoxide hydrolase). A significant association between the CYP1A1 MspI polymorphism and chromosome aberration frequencies was observed in the newborns (p=0.02), with heterozygotes showing higher aberration frequencies than the wild type homozygotes. Some large differences in chromosome aberration frequencies for other genotypes were also noted, but these were not statistically significant. Exposure to tobacco smoke in utero also appeared to increase translocation frequencies. The mean frequency of translocations per 100 cell equivalents from newborns of mothers who smoked during pregnancy was significantly higher than that of newborns whose mothers did not smoke (0.21 vs. 0.11, respectively, p=0.045).     

Cytogenetic effects of separate and combined action of 232Th and Cd nitrates on Allum cepa root meristem cells

Effects of separate and combined action of 232Th and Cd on Allium cepa root tip cells were investigated. It has been shown that cytogenetic effects of 232Th results mainly from its ability to induce genome damages. Cadmium in the examined concentration does not induce chromosomal vagrants, and this effect is not intensified by the combined action of 232Th and Cd (with 232Th). Cd induced increased aberrations of chromosome type frequency (1.5 times more than in control group), and fragment frequence, while mitotic index was significantly decreased. While taking into consideration the frequency of anaphase--telophase damages, and the number of aberrations per cell, the combined effects of 232Th and Cd may be considered as synergic. The aberration type ratio, frequencies of bridges and fragments, and also the level of proliferative activity of 232Th and Cd in the presence of both ions were similar with the corresponding parameters obtained when Cd acted alone. Possible mechanisms of formation of 232Th and Cd synergic effects are discussed.     

Efficient mutation induction by 125I and 131I decays in DNA of human cells

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.     

Comparative in vivo and in vitro study of the cytogenetic effects of 153Sm-EDTMP in lymphocytes of patients with bone metastases.

153Sm-EDTMP is a radiopharmaceutical used in nuclear medicine for relief of metastatic bone pain with promising results, but there are few studies about the effects of 153Sm-EDTMP in human cells. This study was conducted for the evaluation of the cytogenetic effects of 153Sm-EDTMP in blood lymphocytes from patients with bone metastases (without previous radio or chemotherapy), using the chromosome aberration technique. The degree of cytological damage found in in vivo blood cells of patients was compared with those found in in vitro in an adjusted dose-response curve. Blood samples were collected before and 1 hr after the administration of 153Sm-EDTMP(about 42.31 MBq/kg). The frequency of structural chromosome aberration per cell observed in 1 hr samples (0.054+/-0.035 CA/cell) was higher than basal ones (0.031+/-0.026 CA/cell), although this difference was not statistically significant (p= 0.101). For in vitro assay, blood samples were exposed to different concentrations of 153Sm-EDTMP, during 1 hr (0.37-1.11 MBq/ml). An increase in the frequency of chromosome aberration per cell as a function of the radioactive concentration was found. The data were adjusted by linear regression model (Y= 3.52+/-2.24 x 10(-2) + 11.15+/-3.46 x 10(-2) X). The frequency of aberration/cell found in vivo was 0.054 and for the same activity in vitro was 0.098, this difference being statistically significant (p = 0.02). This result may be related to blood clearance, osteoblastic activity and individual variability. For a more accurate analysis, the study of more donors is necessary.     

Delayed Formation of Chromosome Aberrations in Mouse Pachytene Spermatocytes Treated with Triethylenemelamine (Tem)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis–metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis–metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed—a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Development of a rodent lung macrophage chromosome aberration assay

Lung macrophages are the first line of defense against inhaled xenobiotics. They are able to accumulate airborne particulates as well as having metabolic capability. They may thus be sensitive indicator cells for detecting inhalation exposure to environmental mutagens. Their usefulness as a short-term in vivo genotoxic assay has not, however, been adequately explored. We have systematically investigated the feasibility of developing a lung macrophage chromosome-aberration assay. It was found that with different types of spindle-binding chemicals (vinblastine and vincristine), and with improved harvesting procedures, an adequate number of metaphase cells can be collected from mice and Chinese hamsters. The chromosome aberration frequencies in macrophages from control mice and Chinese hamsters were found to be 1.2 +/- 2.3 and 0.75 +/- 2.2 per 100 cells respectively. These frequencies are within normal ranges for other somatic cells. After inhalation exposure to an occupational-exposure level of benzene (0, 0.1 and 1 ppm), significant dose-dependent induction of aberrations (1.2 +/- 2.3, 5.7 +/- 6.3 and 6.8 +/- 6.2 chromatid deletions per 100 cells resp.) were observed in the macrophages. Thus, these cells can be used as one of a battery of in vivo assays for inhalation exposure studies.     

Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets

The p53 tetramer recognizes specifically a 20-bp DNA element. Here, we examined symmetries encoded in p53 response elements (p53REs). We analyzed base inversion correlations within the half-site, as well as in the full-site palindrome. We found that p53REs are not only direct repeats of half-sites; rather, two p53 half-sites couple to form a higher order 20bp palindrome. The palindrome couplings between the half-sites are stronger for the human than for the mouse genome. The full-site palindrome and half-site palindrome are controlled by insertions between the two half-sites. The most notable feature is that the full-site palindrome with coupling between quarter-sites one and four (H14 coupling) dominates the p53REs without insertions. The most frequently observed insertion in human p53REs of 3bp enhances the half-site palindrome. The statistical frequencies of the coupling between the half-sites in the human genome correlate with grouped experimental p53 affinities with p53REs. Examination of known p53REs indicates the H14 couplings are stronger for positive regulation than for negatively regulated p53REs, with repressors having the lowest H14 couplings. We propose that the palindromic sequence couplings may encode such potential preferred multiple binding modes of the p53 tetramer to DNA.