Inhibition of condensation in the late-replicating X chromosome induced by 5-azadeoxycytidine in human lymphocyte cultures

Summary The cytidine analogue 5-azadeoxycytidine (5-aza-dC) induces a very distinct inhibition of condensation in the genetically inactive, late-replicating X chromosome (X_L) when applied to human lymphocyte cultures. One of the two X chromosomes in cytogenetically normal female cells becomes dramatically longer than its homologous partner. The highest rate of metaphases with an undercondensed X_L chromosome is achieved when 5-aza-dC is added at a final concentration of 10^-5 M 2 h before cell harvesting. The interactions between 5-aza-dC and chromosomal DNA as well as the factors involved in X chromosome inactivation are discussed.     

Analysis of visible light-induced sister chromatid exchanges in 5-bromodeoxyuridine-substituted chromosomes

SCEs were studied in the chromosomes of Allium cepa L. stained by the FPG technique at the second and third divisions after BrdU-substitution during only the first replication round or the three consecutive cycles, respectively. Cells were cultured in the dark and exposed to visible light at different moments throughout the three cycles. The results obtained show that visible light-illumination, which has no apparent effect upon native DNA, is able to increase the frequency of SCEs in BrdU-substituted chromosomes. The comparison of expected and observed figures clearly reveals that BrdU-substituted DNA is the target of visible light. Finally, the formation of visible light-induced SCEs in BrdU-substituted chromosomes appears to be an S-dependent process, even though a post-replicational mechanism closely associated with semi-conservative S phase replication might be responsible.Abbreviations SCE sister chromatid exchange - BrdU 5-bromodeoxyuridine - Thd thymidine - FdU 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa - UV ultraviolet - CHO Chinese hamster ovary     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Differential condensation of human chromosomes 9 and Y

Some differences were observed in the mitotic condensation of regions composing human chromosomes 9 and Y: regions 9p, 9h and Y nf are characterized by an intense condensation by the end of the spiralization interval studied (the length of the repair chromosome 3 varying from 5.4 to 2.9 mkm). At the same time, the condensation of regions 9q-h (region 9q without heterochromatic block) is slowing in the initial spiralization interval (the length of chromosome 3 varying from 16.6 to 5.5 mkm). The Yf-block of Y-chromosome is condensing faster than nf-region. The condensation parameters of Q-heterochromatic blocks are most variable while the euchromatic regions are most stable. The dynamics of 9h and of f-block condensation are independent within one karyotype. Based on the data obtained we doubt the correctness of studies on linear dimensions of the constitutive heterochromatin blocks for the evaluation of its quantity in the karyotype. A possible association of differential mitotic condensation with the chromosome segregation disturbances is discussed.     

Mitomycin C-induced sister chromatid exchange in X chromosomes of Bovidae

Sister chromatid exchanges (SCEs) in early- and late-replicating X chromosomes of seven female cattle (Bos taurus L.) and five female river buffalo (Bubalus bubalis L.) were studied in untreated lymphocytes and lymphocytes treated with mitomycin C (MMC). In the experiment, 577 SCEs on X chromosomes of MMC-untreated cells and 825 SCEs on X chromosomes of MMC-treated cells from both species were observed. No significant differences between the number of SCEs in early- and late-replicating X chromosomes were found even when singular species and subjects were considered.     

DNA-protein cross-links and sister chromatid exchanges induced by dimethylarsinic acid in human fibroblasts cells

Biotransformation of inorganic arsenic to form both methylarsinic acid (MA) and dimethylarsinic acid (DMA) has traditionally been considered as a mechanism to facilitate the detoxification and excretion of arsenic. However, the methylation of inorganic arsenic as a detoxification mechanism has been questioned due to recent studies revealing an important role of organic arsenic in the induction of genetic damage. In a previous report a reduction of DNA migration after treatment of cells with DMA was described. In order to further evaluate the possible induction of protein-DNA adducts, an experiment was performed taking into account other parameters and modifications of the standard alkaline comet assay. In addition, the results obtained with the comet assay were compared with those obtained by analyzing the induction of sister chromatid exchanges (SCEs). SCE frequencies were significantly increased in treated cells in relation to controls (p<0.001). Furthermore, in the standard alkaline comet assay, as well as in the control assay for proteinase K treatment, a significant dose-dependent reduction in tail moment was observed. Nevertheless, the post-treatment with proteinase K induced the release of proteins joined to the DNA and consequently, a dose-dependent increment in DNA migration was observed (p<0.001). These results suggest that DNA-protein cross-links may be an important genotoxic effect induced by dimethylarsinic acid in human MRC-5 cells.     

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes

Summary When [³H]dC was added with a high dose (4x10^-1 mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [³H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block.BrdC 5x10^-2 mM added with a high dose of dT (4x10^-1 to 1.0 mM) to the cultures did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT.The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.     

Frequency and sites of sister chromatid exchanges in rat kangaroo chromosomes

The frequency of sister chromatid exchanges in the chromosomes of a cell line from the Tasmanian rat kangaroo was determined to be 0.79 exchanges per chromosome for two cell cycles. Twenty-five percent of these exchanges occurred at the kinetochore. The mean frequency of exchanges per chromosomal arm was roughly proportional to the length of the chromosome, with the exception of a mean frequency of 0.20 exchanges per chromosome found at the kinetochore of all chromosomes, regardless of length. Thus, the kinetochore is a highly preferential site for sister chromatid exchanges. Compared to the main portion of the chromosomal arms the exchange frequency was somewhat lower adjacent to the kinetochore and at chromosome ends. The number of exchanges per unit length also tended to be lower for the short arm of chromosome 1. No correlation was found between the frequency of exchanges and late-replicating DNA.     

Sister chromatid exchanges induced in human lymphocyte chromosomes by trifluralin, atrazine and simazine

Many epidemiological and experimental "in vivo" studies have proved in recent years the carcinogenic properties of herbicides. In order to evaluate the "in vitro" action on the human DNA of Trifluralin, Atrazine and Simazine (active principles of herbicides Treflan and Fogard S respectively) the authors have studied the rates of SCE in cultures of human lymphocytes exposed to different concentrations of a solution 1 ppm of the substances. Trifluralin and Simazine, but not Atrazine, increase SCE per cell, with statistical significance, in the cultures with the highest concentrations of these substances. (SCE per cell: Trifluralin 5.27 +/- 1.38, Simazine 5.09 +/- 1.19, Control 3.51 +/- 1.14).     

Induction of sister chromatid exchanges by benzidine in rat and human hepatoma cell lines and inhibition by indomethacin

The genotoxic activity of benzidine was studied in two cell lines derived from rat (H4) and human (HepG2) hepatomas which have been shown to be capable of activating certain promutagens. The responses were compared to results in two lung-derived fibroblast lines (IMR-90 and V79) which appear to have little or no metabolizing capability. Benzidine was found to induce sister chromatid exchanges in the two liver-derived cell lines in a dose-dependent fashion but failed to induce sister chromatid exchanges in the fibroblast lines. Since one proposed pathway for benzidine activation involves prostaglandin-mediated metabolism, we tested the effect of pretreatment with indomethacin, an inhibitor of this metabolic pathway. Indomethacin was highly effective in inhibiting benzidine-induced sister chromatid exchanges in both H4 and HepG2 cells. These results suggest that some DNA damage may occur in the livers of fast acetylating species such as the rat without prior N-acetylation and that some amount of DNA damage may occur in the livers of slow acetylating species, even when the liver is not the target organ for carcinogenesis.Abbreviations RI replication index - SCE sister chromatid exchanges     

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.     

Induction of sister chromatid exchanges by UV light and its inhibition by caffeine

Sister chromatid exchanges in Chinese hamster chromosomes were studied after pulse-labeling cells with ³H-thymidine at various concentrations. Whereas the frequency of chromatid aberrations varied widely, depending upon tritium dose, there was no significant change in the sister chromatid exchange frequency, even with a 40-fold range of variation in the tritium concentration in the medium. When cells were exposed immediately after labeling to UV light at 40 erg/mm² and examined at the second mitosis, the frequency of sister chromatid exchanges was found to be 4 times higher than that of the unirradiated controls. A synchronization treatment utilizing 2 mM thymidine also caused a two-fold rise in the exchange frequency above the control level. Furthermore, when synchronized cells were irradiated with UV light at a dose of 40 erg/mm², the exchange frequency exceeded 5 times that of the untreated controls. However, this effect was detectable only when cells were irradiated at the earlier part of the S phase, while no change was detected when irradiated at the late S or G2 phase. A post-treatment of irradiated cells with caffeine caused a remarkable decrease in the frequency of sister chromatid exchanges. On the other hand, the frequency of chromatid aberrations of the deletion type increased strikingly after the same treatment. The results appear to suggest a certain correlation between the mechanism involved in the induction of sister chromatid exchanges and a post-replication repair of DNA damage.     

Unequal mitotic sister chromatid exchange and different length of Y chromosomes

Summary There is wide variation in the length of the Y chromosome. In the same individual the length varies continuously and is normally distributed. We describe a boy with borderline mental retardation, gross and fine motor coordination difficulty, muscle rigidity, ptosis, clinodactyly, and a Y chromosome of different lengths in two separate cell populations. The most probable explanation of the cytogenetic finding is a mitotic unequal sister chromatid exchange of the Y chromosome.     

Differential rates of sister chromatid exchanges between euchromatin and heterochromatin

In two rodent species, the Chinese hamster and the montane vole (Microtus montanus), the rate of sister chromatid exchange was lower in constitutive heterochromatin than in euchromatin.     

Increased rates of sister chromatid exchanges induced by the herbicide 2,4-D

The potential for genetic damage from widely used hormonic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), continues to be of serious concern. The mutagenic effect as reflected by the rates of sister chromatid exchanges (SCE) was determined in cultured human lymphocytes. Data were based on the analysis of 50 cells for the control and each of the three treatments. A 50 micrograms/ml dosage caused a highly significant increase in SCE. Dosages of 100 and 250 micrograms/ml elevated the rate of SCE, but not significantly. Since 2,4-D biodegrades rapidly in soil and water, its continued use is not in serious question until safer compounds are available. However, the results of this study suggest that the danger of genetic damage from direct exposure to commercial samples of 2,4-D should not be ignored.     

Segmentation of human chromosomes induced by 5-ACR (5-azacytidine)

Summary The 5-ACR (5-azacytidine) introduced in human lymphocyte cultures induces a lack or a delay of condensation of some chromosome segements corresponding to the G-bands. The resulting R-banding is very similar to that obtained with a 7-h treatment by BrdU, although the segmentation may be much stronger (pulverization) with high doses. However, the 5-ACR does not induce chromatid asymmetry, as BrdU does. This constitutes a new argument for considering that the segmentation and the asymmetry of chromatids depend, at least partly, on two different mechanisms, where proteins are probably involved. Another effect of 5-ACR is to increase chromosome associations by satellites, secondary constrictions, and telomeric regions.