Sister Chromatid Exchange Formation Research Articles

Homologous recombination as a resistance mechanism to replication-induced double-strand breaks caused by the antileukemia agent CNDAC

AbstractThe nucleoside analog 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosyl-cytosine (CNDAC), currently in clinical trials for hematologic malignancies, has a novel action mechanism of causing a single-strand break after its incorporation into DNA. Double-strand breaks (DSBs) are generated thereafter in vivo and, if not repaired, pose lethal impact on cell survival. This study sought to define the mechanisms by which CNDAC-induced DSBs are formed and repaired. We demonstrated that single-strand breaks induced by CNDAC incorporation into DNA were converted to DSBs when cells progressed into the subsequent S-phase. CNDAC-induced DSBs were products of replication, rather than a consequence of apoptosis. ATM, the activator of homologous recombination (HR), was essential for cell survival after CNDAC treatment in cell lines and in primary acute myeloid leukemia samples, as were the HR components, Rad51, Xrcc3, and Brca2. Furthermore, formation of sister chromatid exchanges, a hallmark of HR, increased significantly after CNDAC-treated cells had progressed into a second replication cycle. In contrast, neither the replication stress sensor ATR nor DNA-PK, the initiator of nonhomologous end-joining of DSB, was involved in repair of CNDAC-induced damage. Together, these results indicate that HR, but not nonhomologous end-joining, is the major repair or survival mechanism for DNA damage caused by CNDAC.

Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the SN2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PKCS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following SN2-alkylating agents.

Relationship between DNA Repair and Formation of Sister Chromatid Exchanges and Chromatid Aberrations under the Influence of Poly(ADP-Ribose) Polymerase Inhibition by 3-Aminobenzamide

The mechanisms of formation of sister chromatid exchanges (SCEs) and chromosome aberrations following inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide were studied in Chinese hamster ovary cell lines deficient in different repair pathways. The results confirm earlier findings that (a) the ‘spontaneous’ SCEs are formed due to the incorporated BrdU in the DNA, (b) ‘spontaneous’ and induced SCEs originate from different mechanisms, and (c) SCEs and chromatid exchanges are formed by different pathways.

The structure of the RMI core complex resembles the RPA interface

RecQ DNA helicases are ubiquitously conserved enzymes with critical roles in genome maintenance. BLM, the protein product of the gene mutated in Bloom's syndrome, is one of five human RecQ helicases. BLM functions as part of the “dissolvasome” complex, which includes TopoIIIα (a type IA topoisomerase), and the RMI (for RecQ‐Mediated Instability) subcomplex (RMI1 and RMI2). The dissolvasome separates double Holliday junction DNA without exchange of genetic material. Here we describe the 1.55 Å resolution structure of the RMI core complex, which includes RMI2 and the C‐terminal OB‐fold of RMI1. The overall structure strongly resembles two‐thirds of the trimerization interface of Replication Protein A (RPA). We examined the RMI interface using co‐immunoprecipitations with RMI2 variants and confirmed two key structural interactions. We probed the effects of complex‐destabilizing mutations on sister chromatid exchange (SCE) formation in chicken DT40 cells and discovered that mutations that disrupt the RMI interface cause an increase in SCE formation. We conclude that the RMI interface is crucial to the stability of the BLM dissolvasome. This research was funded by NIH R01‐GM068061.

Cytoprotective activity of amifostine on cultured human lymphocytes exposed to irinotecan

Irinotecan (camptothecin, CAM) is a topoisomerase-I inhibitor with a well established action in the chemotherapy of colorectal and ovarian cancer. Hematological and intestinal toxicity are commonly noted in patients treated with CAM. In this study, we examined the cytoprotective efficacy of amifostine (ethyol, ETH) against chromosomal damage induced by this drug on cultured peripheral human lymphocytes. Cultured lymphocytes were exposed to CAM (50 and 100 ng/ml of final concentrations) without or with ETH (in concentrations varying between 40 and 800 μg/ml of final culture volume). CAM’s genotoxicity was quantified by counting the Sister Chromatid Exchange (SCEs) rate. The mitotic index (MI) and proliferation rate index (PRI) were also assessed. The SCE rate was increased following incubation with CAM, but the combined treatment of CAM with ETH significantly reduced the SCE formation, especially when ETH added at high concentrations. The MIs and PRIs remained also unaltered in cultures with CAM, but MIs were reduced with the combined treatment at high ETH concentrations. Clinical studies are required to assess the predicted benefits from ETH in patients receiving CAM.

Genotoxic potential of cyfluthrin

Cyfluthrin (CAS no. 68359-37-5), a synthetic fluorinated pyrethroid insecticide, is widely used in the home environment and in agriculture because of its high activity against a broad spectrum of insect pests and its low animal toxicity. There are no adequate data on genotoxic effects of cyfluthrin. The aim of this study was to analyze the potential genotoxic effects of cyfluthrin. The genotoxicity of cyfluthrin was evaluated, in vitro, by assessing the ability of the insecticide to induce gene mutation (evaluated using the Ames/microsome test), chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronucleus (MN) formation in cultured human peripheral blood lymphocytes. Additionally, CAs and cytotoxicity induced by cyfluthrin were investigated in rat ( Rattus norvegicus var. Albinos) bone-marrow cells to assess in vivo genotoxicity of cyfluthrin. The counts of reverse mutations in Salmonella typhimurium were not significantly increased ( P > 0.05). The frequency of CAs in human lymphocytes, treated with any concentration of cyfluthrin (500, 1000 or 2000 μg/ml) for a 24-h period, was not significantly increased ( P > 0.05). In contrast, CA was significantly increased for the highest two concentrations (1000 and 2000 μg/ml) in the 48-h treatment group compared with the control group (dimethyl sulfoxide, DMSO). Micronucleus formation was significantly ( P < 0.05) increased for all doses after the 48-h treatment, although the frequency of SCE did not increase significantly ( P > 0.05). Mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) decreased significantly ( P < 0.05) due to the potential cytotoxicity of cyfluthrin, especially after the 48-h treatment period. The frequency of chromosome aberrations in bone-marrow cells of rats treated with the test substance increased significantly ( P < 0.05) for all doses (250, 500 and 1000 mg/kg body weight) for the two treatment periods (12 and 24 h) and the two administration routes, viz. intraperitoneal injection (i.p.) and oral gavage (gvg). In vivo cytotoxicity of cyfluthrin was detected only after administration by gavage for the 24-h treatment period. All these findings were not dose-dependent.

Increased formation of sister chromatid exchanges, but not of micronuclei, in anaesthetists exposed to low levels of sevoflurane

We have assessed, for the first time, genotoxicity (i.e. sister chromatid exchanges and micronuclei) in anaesthetists exposed to a single volatile anaesthetic (sevoflurane) without nitrous oxide. The anaesthetists were exposed to an 8-h time-weighted average of 0.2 parts per million sevoflurane. Internists served as non-exposed controls. Mean (SD) sister chromatid exchanges per cell were significantly higher in anaesthetists compared to internists (6.6 (0.9) vs 5.1 (0.8); p < 0.001) whereas median (IQR [range]) micronuclei per 1000 binucleated cells did not differ (9.5 (6.3-10.8 [2.0-15.5]) vs 8.5 (6.0-10.5 [3.0-25.5]), respectively). Although the anaesthetists were exposed to rather low concentrations of sevoflurane, this 30% increase of sister chromatid exchanges is in agreement with a recently reported 300% increase with a high level exposure to sevoflurane and nitrous oxide. Omitting nitrous oxide does not normalise increased rates of sister chromatid exchanges.

Effects of boric acid and borax on titanium dioxide genotoxicity

Titanium dioxide (TiO(2)) is a potential carcinogenic/mutagenic agent although it is used in many areas including medical industries and cosmetics. Boron (as boric acid and borax) has also well-described biological effects and therapeutic benefits. In a previous study, sister-chromatid exchanges (SCEs) and micronuclei (MN) rates were assessed in control and TiO(2)-treated (1, 2, 3, 5, 7.5 and 10 microm) human whole blood cultures. The results showed that the rates of SCE (at 2, 3, 5, 7.5 and 10 microm) and MN (at 5, 7.5 and 10 microm) formation in peripheral lymphocytes were increased significantly by TiO(2) compared with the controls. The present study also investigated the genetic effects of boric acid and borax (2.5, 5 and 10 microm) on cultures with and without TiO(2) addition. No significant increase in SCE and MN frequencies were observed at all concentrations of boron compounds. However, TiO(2)-induced SCE and MN could be reduced significantly by the presence of boric acid and borax. In conclusion, this study indicated for the first time that boric acid and borax led to an increased resistance of DNA to damage induced by TiO(2).

Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.

Functional interactions between BLM and XRCC3 in the cell

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIα suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)–induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.

Anti-genotoxic effect of hydrated sodium calcium aluminosilicate on genotoxicity to human lymphocytes induced by aflatoxin B1

The anti-genotoxic potential of hydrated sodium calcium aluminosilicate (HSCAS) was investigated using sister chromatid exchanges (SCEs) induced by aflatoxin B1 (AFB1) as genotoxic endpoint in human lymphocytes. Whole blood samples from two healthy male donors were used for this experiment and the effects of different concentrations of HSCAS (5 x 10(-6) and 1 x 10(-5) M) and AFB1 (1, 5 and 10 microM) were tested. The present results established that the frequencies of SCEs (at doses of 5 and 10 microM except for 1 microM) in lymphocytes were significantly increased by AFB1 compared to controls. When HSCAS was added alone at a molar ratio of 5 x 10(-6) and 1 x 10(-5), lymphocytes did not show significant increases in SCE frequencies, but SCE frequencies induced by the various concentrations of AFB1 could be significantly reduced by the presence of HSCAS (at both doses). In fact, HSCAS at 1 x 10(-5) M completely inhibited SCE formations caused by AFB1. These results suggest for the first time, that HSCAS can antagonize the ability of AFB1 to cause DNA damage that leads to the formation of SCEs.

Protective effect of sodium selenite against the genotoxicity of aflatoxin B1 in human whole blood cultures

This study was designed to investigate the effects of selenium and aflatoxin on human whole blood cultures (WBC) in relation to induction of sister-chromatid exchange (SCE). The results showed that the frequency of SCEs in peripheral lymphocytes was significantly increased by the direct-acting mutagen AFB1 (at doses 5 and 10 µM except for 1µM) compared to controls. When sodium selenite (Na2SeO3) was added alone at a molar ratio of 5x10-7 and 1x10-6, cells did not show significant increase in SCE frequency. Whereas, SCE rates induced by the various AFB1 concentrations could be significantly reduced by the presence of Na2SeO3 in a clear dose-related manner. These results indicated that selenite and AFB1 mutually antagonized their ability to cause DNA damage leading to the formation of SCEs. However, selenium didn't completely inhibit induction of SCEs by AFB1 compared to controls. This is first report describing, the protective ability of selenium againist AFB1 genotoxicity on human WBC.

The Cytogenetic Effects of Food Sweetener Maltitol in Human Peripheral Lymphocytes

The effects of the low-calorie artifical sweetener maltitol (E965), a sugar alcohol (Polyol), on sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus formation (MN) were investigated in human peripheral lymphocytes. Maltitol did not induce SCE at all concentrations (1.25, 2.5, and 5 mg/mL) and treatment periods (24 and 48 h). Maltitol induced CA, although not statistically significantly. Maltitol induced the frequency of MN at 24 and 48 h in a non-dose-dependent manner. In addition, maltitol did not decrease the replication index (RI) and the mitotic index (MI) at all concentrations and treatment periods. Maltitol did not alter the pH and osmolality of the medium. In conclusion, it can be concluded that maltitol has a weak genotoxic potential and it appears non-cytotoxic to human peripheral lymphocytes in vitro.

Protective effect of sodium selenite on genotoxicity to human whole blood cultures induced by aflatoxin B1

The aim of this study was to investigate the effects of selenium and aflatoxin on human whole blood cultures (WBC) in relation to induction of sister-chromatid exchange (SCE). Results showed that the frequency of SCEs in peripheral lymphocytes was significantly increased by the direct-acting mutagen AFB1 (at doses 5 and 10 µM except for 1µM) compared with controls. When sodium selenite (Na2SeO3) was added at a molar ratio of 5x10-7 and 1x10-6, cells did not show significant increase in SCE frequency. Whereas, SCE rates induced by the various AFB1 concentrations could be significantly reduced by the presence of Na2SeO3 in a clear dose-related manner. These results indicated that selenite and AFB1 mutually antagonized their ability to cause DNA damage leading to the formation of SCEs. However, selenium didn't completely inhibit induction of SCEs by AFB1 compared with controls. AFB1 induced oxidative damage contributed to its genotoxicity in human WBC.

Intra-nuclear trafficking of the BLM helicase to DNA damage-induced foci is regulated by SUMO modification

The Bloom syndrome gene, BLM, encodes a RecQ DNA helicase that when absent from the cell results in genomic instability and cancer predisposition. We show here that BLM is a substrate for small ubiquitin-like modifier (SUMO) modification, with lysines at K317, K331, K334 and K347 being preferred sites of modification. Unlike normal BLM, a double mutant BLM protein with lysine to arginine substitutions at residues 317 and 331 was not modified by SUMO, and it failed to localize efficiently to the PML nuclear bodies. Rather, double mutant BLM protein induced the formation of DNA damage-induced foci (DDI) that contained BRCA1 protein and phosphorylated histone H2AX. Double mutant BLM only partially complemented the genomic instability phenotypes of Bloom syndrome cells as assessed by sister-chromatid exchange and micronuclei formation assays. These results constitute evidence that BLM is a DNA damage sensor that signals the formation of DDI, and they establish SUMO modification as a negative regulator of BLM's signaling function.

Perspectives On The Genotoxic Risk Of Styrene

Styrene is a highly reactive monomer widely used in the plastics industry. The potential for styrene to produce genotoxic effects has been studied extensively in experimental systems. Styrene can induce sister chromatid exchanges (SCE) and chromosome aberrations (CA) in vitro under test conditions that enhance metabolism of styrene to styrene 7,8-oxide (SO) or reduce detoxification of SO by epoxide hydrolase. The in vivo animal data indicate that styrene is not clastogenic at concentrations (doses) likely encountered by humans under ambient or occupational exposure conditions. DNA binding studies with styrene in rats and mice demonstrated no increased adducts in mice compared to rats or in mouse lung compared to liver. As a result, DNA adducts in the lungs are unlikely to be the sole explanation of the development of lung tumors in mice exposed to styrene for 2 yr. Some epidemiological studies reported that DNA and/or protein adducts and DNA strand breaks result from occupational exposure to styrene and/or SO. Results of some of these studies, however, are difficult to interpret, given that the statistical significance of reported effects (SCE, CA, and micronucleus formation) was often near or at p values of .05; dose and/or temporal response relationships often were missing; confounding variables could not be excluded; and, concomitant exposures to other industrial chemicals that are potentially genotoxic may also have occurred. These studies suggest that styrene, through metabolism to SO, could be clastogenic in humans at workplace levels in excess of 125 mg/m3. However, results from controlled animal studies involving in vivo exposure to styrene alone do not show clastogenic effects at exposures of up to 1500 mg/m3/d. In any event, these studies show that there is an apparent threshold for styrene-mediated effects.

Spontaneous and γ-ray-induced sister chromatid exchanges in patients with carcinoma of cervix uteri

The aim was to investigate whether there are differences in the spontaneous and γ-ray-induced genomic instability in peripheral blood lymphocytes between untreated cervical cancer patients and healthy women using the sister chromatid exchange (SCE) assay as an indicator of chromosomal instability. Lymphocyte cultures from whole venous blood of 10 patients with cervical neoplasia and 10 healthy female volunteers were cultivated in vitro and irradiated using a 60Co-gamma source. Slides were prepared using the standard air-drying procedure and stained by the fluorescence-plus Giemsa (FPG) technique. The number of SCE and the number of chromosomes were assessed in second-division metaphases. A radiation dose-dependent increase of SCE/cell and SCE/chromosome values were found in healthy women as well as in patients, while statistical analysis has shown significantly higher SCE frequencies in healthy women as compared with patients. Cellular kinetics expressed as replication indices (RI) calculated from the frequency of cells in first cell division (M1), second cell division (M2) and third cell division (M3) were also significantly different, while observed RI were higher for patients than for control individuals. The results suggest that patients with carcinoma of the cervix uteri have chromosomal stability changes reflected in statistically different levels of spontaneous and induced SCE in comparison with healthy individuals. Despite the unknown mechanisms of SCE formation, it is felt that the changed SCE frequency, especially after mutagen treatment, may be used as a marker of increased cancer risk.

Effect of sulfur dioxide hydrates on cell cycle, sister chromatid exchange, and micronuclei in barley

The effects of sulfur dioxide (SO 2) hydrates exposure on cell cycle, sister chromatid exchange (SCE), and micronuclei (MN) were investigated in barley ( Hordeum vulgare) roots. A mixture of sodium bisulfite and sodium sulfite (1:3), at various concentrations from 1×10 −5 to 3×10 −2 M, was used for the treatments. The results showed that the mixture induced the formation of SCE and MN in barley root cells with different effective concentrations and with different trends as treatment concentrations increased. At high concentrations of 0.5–30.0 mM, SO 2 hydrates inhibited the mitotic activity and the growth of barley roots by cell cycle delay and cell death, but at 0.1 mM, the chemicals slightly stimulated mitotic activity and root growth. These remarkable effects in causing DNA damage and consequent chromosome damage suggest that SO 2 is genotoxic agent and its genotoxicity may influence the mitotic activity and plant growth under SO 2 stress.

Fanconi Anemia Protein FANCD2 Promotes Immunoglobulin Gene Conversion and DNA Repair through a Mechanism Related to Homologous Recombination

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

Genotoxicity and mutagenicity of melanoidins isolated from a roasted glucose–glycine model in human lymphocyte cultures, intestinal Caco-2 cells and in the Salmonella typhimurium strains TA98 and TA102 applying the AMES test

Melanoidins are formed during household cooking procedures and are part of our daily diet, but data on their toxicological potential are still scarce. Therefore, the mutagenic, cytotoxic and genotoxic activity of the water soluble total fraction (sol A), the water soluble high molecular weight fraction (HMW; Molecular weight > 12,400 Da) and the remaining water soluble low molecular weight fraction (LMW) isolated from a glucose–glycine model system roasted at 125 °C was comprehensively studied in human lymphocytes (genetic end point: sister chromatid exchange (SCE)), Caco-2 cells (SCE, cell viability, cell proliferation) and in the Salmonella typhimurium strains TA98 and TA102 (Ames test). Tests were performed in a dose- and time-dependent manner. The results indicate a significant increase in SCE formation in human lymphocytes after the exposure to 0.05% and 0.1% of the melanoidin fractions. In Caco-2 cells, only the exposure to LMW increased the SCE formation as a matter of concentration. Cell's proliferation and viability decreased significantly after exposure to melanoidins. In the AMES test, melanoidins did not show a mutagenic potential, neither using the TA98 nor the TA102 strain. These results show that melanoidins isolated from the glucose–glycine mixture exhibited modest but significant genotoxic effects in human lymphocytes and, in particular the LMW, in Caco-2 cells, but they induce neither in low nor in very high concentrations mutagenicity in bacteria strains.