Induction of sister chromatid exchanges by inhibitors of topoisomerases

To investigate the role of topoisomerases in the production of sister chromatid exchanges, the effects of inhibitors of type I and II topoisomerases on baseline and mutagen-induced sister chromatid exchanges were compared. V79 cells were treated with VM-26 and m-AMSA, known inhibitors of type II topoisomerase, or with camptothecin, the only known inhibitor of type I topoisomerase. We observed that inhibitors of both type I and II topoisomerases induced high levels of sister chromatid exchanges at 10^–6 M, and that the dose-response curves of these drugs were very similar. A clear heterogeneity in the distribution patterns of exchanges induced by inhibitors of topoisomerases was observed. We believe that this heterogeneity in response to these compounds is due to variation in sensitivity within the cell cycle. We also studied interactions of these agents with mitomycin-C and with PUVA (8-methoxypsoralen + UVA), both cross-linking agents and potent sister chromatid exchange inducers, and with x-rays, an agent that induces high levels of DNA strand breaks. No significant change in exchange levels was observed in interactions between topoisomerase inhibition and the levels induced by the agents studied. We conclude that double-strand break prevalence, known to be increased through inhibition of type II topoisomerase, is not the primary mechanism for induction of sister chromatid exchanges. We further conclude that acute inhibition of type I and type II topoisomerases does not influence substantially the induction of exchanges by other agents.Abbreviations MMC mitomycin C - 8-MOP 8-methoxypsoralen - SCE sister chromatid exchange - SFM serum-free medium     

Induction of chromatid breaks by carbon K-shell ultrasoft X rays

Chromatid breaks have previously been shown to be induced in G2-phase cells after exposure to ionizing radiation (X and gamma rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for 60Co gamma rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.     

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     

Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide

Styrene and styrene oxide mutagenicity was tested in cultured human lymphocytes treated in vitro with various concentrations of test agents. Styrene alone was found mutagenic at the highest concentration used (5 X 10(-4) mol. l-1, combined with the alkylating agent THIO-TEPA it did not affect the chromosome aberration yield. Exposure to styrene oxide gave a positive result showing a clear-cut dose-effect relationship within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1. In combination with THIO-TEPA its effect on chromosome aberration yields was additive. Styrene oxide proved also to be a very potent inducer of sister chromatid exchanges (SCE) within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1 tested. Combined with THIO-TEPA it exhibited a distinct additive effect in the production of SCEs.     

The human leukocyte test system. VI. The use of sister chromatid exchanges as possible indicators for mutagenic activities.

The trifunctional alkylating chemical mutagen trenimon increases the frequency of sister chromatid exchanges in human leukocyte chromosomes in vitro, as revealed by a BUdR-Giemsa method. Treatment with lead acetate exhibited negative results in this respect. The use of sister chromatid exchanges as possible indicators for mutagenic activities is discussed.     

High incidence of sister chromatid exchanges and chromatid interchanges in the conditions of lowered activity of poly(ADP-ribose)polymerase

The effect of lowering the activity of poly(ADP-ribose)polymerase on chromosome stability has been examined. Chinese hamster ovary cells, CHO-Kl grown in a nicotinamide-free medium exhibited an increased frequency of sister chromatid exchanges in a time-dependent manner. Furthermore, addition of m-aminobenzamide which is known to be a strong inhibitor of poly(ADP-ribose)polymerase caused a manyfold increase in the frequency of both sister chromatid exchanges and non-sister chromatid interchanges. These results suggest that appropriate levels of NAD and the activity of poly(ADP-ribose)polymerase are required for maintaining chromosome stability.     

Selective differential staining of sister chromatids of the facultative heterochromatic X chromosome in the female mouse

Selective differential staining of sister chromatids for the facultative heterochromatic X chromosome in the female mouse has been achieved by the combination of two differential staining techniques; one for the heterochromatic X chromosome and the other for sister chromatids. Thermal hypotonic treatment moderately destroyed the chromosome structure except for the heterochromatic X in BrdU labelled metaphase cells, resulting in the selective sister chromatid differentiation of this X with Giemsa stain. This technique enables us to know the exact frequency of the spontaneous sister chromatid exchanges in the heterochromatic X without using ³H-TdR labelling for detecting the late DNA replication. The results indicate that the sister chromatid exchange frequency of the heterochromatic X chromosome is not affected by its late DNA replication during S phase, or by the genetic inactivation and the resulting heterochromatinization.     

Sister chromatid exchange frequency in human lymphocytes exposed to ionizing radiation in vivo and in vitro

This paper reports statistically significant elevations in peripheral blood lymphocyte sister chromatid exchange frequencies in persons occupationally exposed to low levels of ionizing radiation when compared with unexposed persons. Low doses of X or gamma rays administered in vitro also produce significant elevations in sister chromatid exchange frequencies, though the magnitude of the increases is dependent upon culture medium and other factors.     

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.     

Genotoxic effect of benzene hexachloride in cultured human lymphocytes

Summary Chromosomal aberrations, sister chromatid exchanges, mitotic index and cell kinetics were observed in human peripheral lymphocytes after treatment with four different concentrations (0.0125, 0.025, 0.05 and 0.1 g/ml) of benzene hexachloride (BHC), an organochlorine pesticide. Cells were treated with BHC for 24, 48 and 72h. There was a dose-dependent increase in the frequency of chromosomal aberrations and sister chromatid exchanges. A significant decrease in mitotic index was observed at all concentrations and times of exposure. BHC did not show a significant effect on cell kinetics.     

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.     

The fragile site (16)(q22)

Summary In the lymphocytes of heterozygous carriers of the rare autosomal fragile site (16)(q22) an exceptionally high frequency of sister chromatid exchanges was demonstrated at the induced fragile site by means of simultaneous berenil and BrdU treatment of the cultures. The rate of sister chromatid exchanges at q22 is also increased in the fragile chromosome 16 by treating the cells with BrdU alone. The possible reasons for the preferential occurrence of induced and spontaneous sister chromatid exchanges at fra (16)(q22) are discussed.     

Sister chromatid exchange induced by anti-herpes drugs.

The rate of sister chromatid exchange induced by several anti-herpes agents was measured to assess their potential mutagenicity. The agents--5-iodo-deoxyuridine (IDU), 5-trifluoromethyl-deoxyuridine (TFT), and [E]-5-(2-bromovinyl)-deoxyuridine (BVDU)--were incubated at various concentrations with human lymphocytes and fibroblasts, and that rate of sister chromatid exchanges was measured. In lymphocytes and fibroblasts BVDU and IDU did not induce exchange except at concentrations of 50 mg/l, while TFT increased the rate of exchange at a concentration of 0.5 mg/l. The rate of sister chromatid exchange is a sensitive index of chromosomal damage, and these findings provide information on the safety of some of the antiherpes agents tested. TFT increased the rate of exchange at a concentration that coincides with its minimal antiviral concentration, but BVDU did not induce exchange at therapeutic concentrations.     

Evaluation of genotoxic effects in human peripheral blood leukocytes following an acute in vitro exposure to 900 MHz radiofrequency fields

Human peripheral blood leukocytes from healthy volunteers have been employed to investigate the induction of genotoxic effects following 2 h exposure to 900 MHz radiofrequency radiation. The GSM signal has been studied at specific absorption rates (SAR) of 0.3 and 1 W/kg. The exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and temperature. The same temperature conditions (37.0 +/- 0.1 degrees C) were realized in a second waveguide, employed to perform sham exposures. The induction of DNA damage was evaluated in leukocytes by applying the alkaline single cell gel electrophoresis (SCGE)/comet assay, while structural chromosome aberrations and sister chromatid exchanges were evaluated in lymphocytes stimulated with phytohemagglutinin. Alterations in kinetics of cell proliferation were determined by calculating the mitotic index. Positive controls were also provided by using methyl methanesulfonate (MMS) for comet assay and mitomycin-C (MMC), for chromosome aberration, or sister chromatid exchange tests. No statistically significant differences were detected in exposed samples in comparison with sham exposed ones for all the parameters investigated. On the contrary, the positive controls gave a statistically significant increase in DNA damage in all cases, as expected. Thus the results obtained in our experimental conditions do not support the hypothesis that 900 MHz radiofrequency field exposure induces DNA damage in human peripheral blood leukocytes in this range of SAR.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro

Fasinex (triclabendazole) has been reported to be an active fasciolocidal agent used in humans and in farm animals. The clastogenic effects of fasinex were tested in lymphocyte cultures of the river buffalo at three final concentrations: 25, 50 and 100 microg/ml. Chromosomal aberrations, sister chromatid exchanges and micronucleus formation are the three cytogenetic parameters used in this study.The results demonstrated that the number of cells with different types of chromosomal aberrations, including chromatid breaks and gaps, isochromatid breaks and gaps and polyploidy, was increased significantly in cultures treated with different doses of fasinex compared to the control. This increase was dose-dependent where there was a positive correlation between increased drug concentration and induction of chromosomal aberrations.The frequency of sister chromatid exchanges and the formation of micronuclei in all lymphocyte cultures treated with different doses of fasinex were increased significantly compared to the control; these increases were also dose-dependent.In conclusion, the three cytogenetic parameters used to evaluate the effect of fasinex revealed that the drug has a strong clastogenic effect on river buffalo lymphocytes in vitro.     

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

THE FREQUENCY OF SISTER CHROMATID EXCHANGES FOLLOWING EXPOSURE TO VARYING DOSES OF H3-THYMIDINE OR X-RAYS

In the Chinese hamster cell line CHEF-125, sister chromatid exchanges occurred at a rate of a little higher than one per three chromosomes for each cell cycle. The exchanges were detectable by labeling with H³-thymidine and autoradiographic analyses of chromosomes at the second and subsequent metaphases after labeling had occurred. To test the hypothesis that sister chromatid exchanges are caused by radiation, cells were incubated in media with different amounts of H³-thymidine. No statistically significant change in the exchange rate was detected over 100-fold range of variation in the amount of incorporated H³-thymidine (determined by grain counts of autoradiographs). We have concluded that sister chromatid exchanges are not caused by tritium radiation and therefore are spontaneous events. Cultures were also irradiated with acute doses of x-rays up to 200 r and scored for sister chromatid exchanges. Between zero and 50 r there was a statistically significant increase in the rate of exchanges. This is interpreted as evidence that x-rays can induce some exchanges, although the majority of these events are probably spontaneous.