Aging and sister chromatid exchange. IV. Reduced frequencies of mutagen-induced sister chromatid exchanges in vivo in mouse bone marrow cells with aging.

Induction of sister chromatid exchanges (SCE's) was examined in bone marrow cells of young and old C57BL/6J mice exposed to three different DNA-damaging agents (cyclophosphamide, mitomycin C, and doxorubicin). At low concentrations of all three mutagens, the levels of induced SCE's were similar in young and old cell populations. However, at higher mutagen concentrations, SCE induction was significantly reduced in old cell populations. Studies of mice aged 5 to 32 months revealed that induced SCE frequencies remain stable during early adulthood (5 to 12 months) and then begin to decline as a function of age. These results indicate that with aging there exists a gradual alteration of cellular response to DNA damage.     

Effect of mouse age and genotype on the frequency of sister chromatid exchange in bone marrow cells

No differences were found in both the baseline and mitomycin C induced levels of sister chromatid exchanges (SCE) between 101/H and C57BL/6J mice differing in chromosome mutability. An increase with the age of the spontaneous and mutagen induced SCE rates was similar in the strains compared, though instability of chromosomes was much higher in old 101/H than in C57BL/6J mice. Thus, no correlation was observed between chromosomal aberration and SCE levels in these strains. As 101/H mice were recently found to be DNA repair-deficient, possible connection of SCE and repair is discussed.     

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

Summary The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3, under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2′-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanism(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5°C. (b) The concentration limits for BrdU incorporation are 5 to 100 μM; baseline frequency is about 11 SCE/metaphase (constant up to 20 μM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 μM) cause a decrease of SCE rates below that found at 100 μM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division. This work was supported by a grant from the Medizinisch-wissenschaftlicher Fond des Bürgermeisters der Bundeshauptstadt Wien.     

Different baseline sister chromatid exchange levels in density fractionated human lymphocytes

Summary Different activation states of B and T lymphocytes, as manifested by differences in cell density, were obtained by Percoll density centrifugation of unstimulated human lymphocytes. Four different density fractions were defined: B cells with low (1.043 g/ml) and high (1.056) density, and T cells with low (1.067) and high (1.077) density, respectively. Sister chromatid exchange (SCE) conditions and proliferation rates were determined. Total B cells, stimulated by the bacterial mitogen Branhamella, had 4.6 SCE per cell, the lowest mean baseline SCE level recorded among lymphocytes. The growth rate was intermediate between that of low and high density T cells. The two T cell fractions stimulated by phytohemagglutinin (PHA) had different baseline SCE frequencies and different growth characteristics: the low density cells had 5.7 SCEs per cell and a short cell cycle, whereas high density cells had 12.5 SCEs per cell and a longer cell cycle. The differences in baseline SCE frequency and growth characteristics between the two T cell fractions seem to be correlated with the differences in the activation state as reflected by the cell density. Both high and low density T cell are G₀ populations which supposedly differ with respect to previous history in vivo such as age and contact with antigens. The reason why these cells react differently to bromodeoxyuridine (BrdU) is unknown, but differences in intracellular DNA precursor pools and enzyme activities might play a role.     

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3 , under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2'-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0 mg/L, induced a 1.9–3.9-fold increase in MN frequency and a 1.5–1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P < 0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15–80% decrease in mitotic indices (MI), but at the lowest concentration (0.005 mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

Elevated sister chromatid exchange rate in lymphocytes of subjects treated with arsenic

Summary An elevated sister chromatid exchange (SCE) rate was found in the lymphocytes of six patients treated with arsenic. All had stigmata of arsenic use as well as biopsy-proven skin cancers. The arsenic exposed patients had a mean of 14.00 SCE/mitosis while 44 normal controls had a mean of 5.8 SCE/mitosis. Chromosome breakage analysis revealed no difference between the two groups.SCE rate has been shown to be elevated in a variety of systems where cell cultures or experimental animals were exposed to known mutagens and carcinogens. We suggest that the relationship carcinogen exposure-elevated SCE rate-cancer may also be valid in humans treated with arsenic.This paper is supported in part by USPHS Grants No. T01 AM 05 560 (WB) and 5T01 GM 01 156 (KK).     

Ultrasonic induction of sister chromatid exchanges in human lymphocytes

Summary We analyzed sister chromatid exchange (SCE) frequencies as an indicator of DNA damage induced in human lymphocytes by real-time ultrasound. A range of exposure times and intensities was tested in a series of blind, randomized, in vitro experiments under spatial and sonographic conditions simulating exposure of a gravid abdomen and uterus. Our studies showed small but consistent effects of ultrasound on SCE frequencies, for each experiment. Differences between matched control and exposed means were significantly different from zero. X ² tests for homogeneity indicated no significant differences among either the means or the total distributions of the controls, nor among each of the separate dose levels. Consequently, experiments were pooled, and X ² analysis indicated significant differences both among distributions and among means of SCE frequencies for controls versus exposed cells (P(0.001). The pooled control mean was also significantly different from each of the pooled dose means. Correcting for multiple comparisons gave identical results for the paired comparisons of means except for the 20-min level which was borderline (0.025P(0.01). We conclude that the well-established value of clinical ultrasonography warrants its continued use; however, minimizing the numbers and lengths of exposure per patient would seem prudent, pending further information on clinical implications of our results.Supported in part by NIH-HD82855 and HD 11021 and a National Foundation Summer Science Research Grant for Medical Students, 8-80-22     

Correspondent reaction of mitotic recombination in yeast and sister chromatid exchange in human lymphocytes

Summary In the lower eukaryote Saccharomyces cerevisiae, 4,5,6-trichloro-2-(dichlorophenoxy)phenol and acridine orange cause different specific genetic alterations, either gene mutations or recombinations. These specific effects were used to characterize the mechanism of sister chromatid exchange (SCE) formation in human lymphocytes. Assuming that genetically active substances have comparable effects in lower and higher eukaryotes, the observations provide indirect evidence for a connection between induced mitotic recombination in yeast and SCEs in human lymphocytes and suggest that SCEs may be the consequence of a repair process.     

Elevated rates of sister chromatid exchange at chromosome ends

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Baseline sister chromatid exchange in human cell lines with different levels of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.     

A decrease in sister chromatid exchange frequency during the prolonged cultivation of human lymphocytes

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Increased frequency of sister chromatid exchange in persons occupationally exposed to ethylene oxide

Mutagenic effects of ethylene oxide have been demonstrated by short-term testing in vitro and in vivo in several organisms. Its oncogenic activity for man has been suspected and recently supported by experiments in mice. Exposure can occur during ethylene oxide gas sterilization of medical materials. We have tested effects on chromosomes by estimating sister chromatids exchange (SCE) frequency. Our results, reported here, show that exposure to this substance during work is followed by a very highly significant increase of SCE frequency as compared with controls. Thus, the SCE test may, under particular conditions, represent a reliable test for exposure to certain toxic agents.     

Statistical and image analysis of sister chromatid exchange in maize

The present study reports the use of the fluorescence plus Giemsa (FPG) technique, image analysis and statistical methods to assess the sister chromatid exchanges (SCEs) frequency in maize. Roots derived from germinated maize seeds were treated with BrdU solution and fixed. The slides were prepared by enzymatic cellular dissociation, air-drying technique, stained with Hoechst 33258 fluorochrome, and incubated in salt solution. The chromosomes were irradiated with ultraviolet light and stained with Giemsa solution. The FPG technique associated with digital analysis system was used to measure the length of 597 BrdU-incorporated maize chromosomes and to identify 0.5243 SCE per chromosome. A range from 0 to 4 SCE events were classified and the chi-square test (chi2=1.586, P=0.662) showed a good fit to the hypothesis that the SCEs are independent and random events that follow Poisson distribution. The SCE frequencies in long and short chromosome arms corresponded to a mean value of 0.876 SCE microm(-1). Considering that the maize line used in this study contains 5.78 picogram (pg) DNA (2C value) in interphasic G0/G1 nuclei or 11.56 pg DNA (4C value) in metaphase, and that the DNA mean value corresponds to 0.578 pg/metaphasic chromosome, the analysis suggests an occurrence of approximately 0.9 SCE/pg DNA.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0mg/L, induced a 1.9-3.9-fold increase in MN frequency and a 1.5-1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P<0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15-80% decrease in mitotic indices (MI), but at the lowest concentration (0.005mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange

Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro. To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination. In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids. Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences. This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases. In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis. Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.