SCE Frequency Research Articles

Analysis for Sister Chromatid Exchange.

Sister chromatid exchange (SCE) is the exchange event of genetic material between two identical sister chromatid. Elevation of SCE frequency is considered as a result of replication stress from genetic defects, ROS stress, and genomic damages. SCE staining needs extra processes compared to regular Giemsa staining. Usually two rounds of cell cycle progress are required to observe SCE under microscope. SCE can be visualized with the fluorescence plus Giemsa (FPG) staining method or fluorescence stainingmethods with immunocytochemistry to BrdU or Click reaction to EdU whichprovide more clear images of SCE. This chapter will provide the detailed method for the SCE staining andmeasurement for the traditional FPG staining, BrdU monoclonal antibody staining method, and newly developed EdU Click reaction staining method.

Association between lead exposure and DNA damage (genotoxicity): systematic review and meta-analysis

Studies suggest that chronic lead (Pb) exposure may induce deoxyribonucleic acid (DNA) damage. However, there is no synthesised evidence in this regard. We systematically reviewed existing literature and synthesised evidence on the association between chronic Pb exposure and markers of genotoxicity. Observational studies reporting biomarkers of DNA damage among occupationally Pb-exposed and unexposed controls were systematically searched from PubMed, Scopus and Embase databases from inception to January 2022. The markers included were micronucleus frequency (MN), chromosomal aberrations, comet assay, and 8-hydroxy-deoxyguanosine. During the execution of thisreview, we followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Mean differences in the biological markers of DNA damage between Pb-exposed and control groups were pooled using the random-effects model. The heterogeneity was assessed using the Cochran-Q test and I2 statistic. The review included forty-five studies comparing markers of DNA damage between Pb-exposed and unexposed. The primary studies utilised buccal and/or peripheral leukocytes for evaluating the DNA damage. The pooled quantitative results revealed significantly higher DNA damage characterised by increased levels of MN and SCE frequency, chromosomal aberrations, and oxidative DNA damage (comet assay and 8-OHdG) among Pb-exposed than the unexposed. However, studies included in the review exhibited high levels of heterogeneity among the studies. Chronic Pb exposure is associated with DNA damage. However, high-quality, multicentred studies are required to strengthen present observations and further understand the Pb's role in inducing DNA damage. CRD42022286810.

Studies on Cytotoxic and Genotoxic potential of Ethanolic extract of Lawsonia inermis leaves

Lawsonia inermis is widely known as henna and belongs to Lythraceae family. It contains various phytochemica constituents in every part of plant and shows various pharmacological properties. Secondary metabolites are acts as antioxidants and these are very high in this plant species. In this research studies, ethanolic extract of Lawsonia inermis was performed in the blood sample culture for cytotoxicity and genotoxicity effects. The both of the effects of plant extracts were evaluated through measurement of mitotic index (MI), sister chromatid exchanges (SCE) and cell proliferation kinetics (CPK). The blood sample culture was treated with control and various concentrations of extracts (500μg/ml and 1000μg/ml). The mean of MI, SCE and CPK for control were found to be 4.51, 5.19 and 0.52 respectively where as for 500μg/ml of Lawsonia inermis extracts are 4.33, 4.87 and 0.50 respectively and for 1000 μg/ml extracts are 4.18, 4.68 and 0.48 respectively. From the results, we can conclude that the MI and frequency of SCES of ethanolic plant extracts are remarkably similar to that of control which indicates that the Lawsonia inermis plant extracts have no substantial cytotoxicity and genotoxicity effects in cultured blood lymphocytes.

Genotoxicity of Two Nanoparticles: Titanium Dioxide and Zinc Oxide

Nanoparticles (NPs) and nanoparticle-based materials have been increasingly used in various consumer and medical applications. However, investigations have disclosed that some NPs may generate toxic effects in different cell types and organisms. In this study, the cytotoxic and genotoxic potential of titanium dioxide (TiO2) and zinc oxide (ZnO) NPs were examined by using four genotoxicity tests, chromosome aberrations-CAs, sister chromatid exchange-SCE, micronucleus-MN, and comet, in human lymphocytes in vitro. The results showed that both NPs significantly increased the frequency of aberrant cells, CA/Cell, SCE, and DNA damage, and decreased mitotic index in some treatments. These results demonstrated that TiO2 and ZnO NPs induce genotoxic effects. Therefore, more detailed in vitro and in vivo experiments should be conducted for the safe usage of both NPs.

Evaluation of baseline frequency of sister chromatid exchanges in an Italian population according to age, sex, smoking habits, and gene polymorphisms.

Increased SCEs frequencies in human lymphocytes are an indicator of spontaneous chromosome instability and could be influenced by different exogenous and endogenous factors. In this study, we evaluated the influence of age, sex, smoking habits, and genetic polymorphisms on the background levels of SCEs in peripheral blood lymphocytes. Two hundred-thirty healthy Italian subjects were recruited. Data about age, sex and smoking habits were recorded. Subjects were also genotyped for GSTT1, GSTM1, GSTP1 A/G, CYP1A1 Ile/Val, CYP2C19 G/A, ERCC2/XPD Lys751Gln, XRCC1 Arg194ATrp, XRCC1 Arg399Gln, and XRCC1Arg208His gene polymorphisms. The frequency of SCEs/cell was 5.15±1.87, with females showing a significantly higher SCEs value with respect to males (5.36±2.10 and 4.82±1.39, respectively). Smokers showed significantly increased levels of SCEs with respect to nonsmokers (5.93±1.75 and 4.70±1.79, respectively) whereas no differences were observed between heavy and light smokers. Age correlated with the RI value (P=.01) but not with the SCEs frequency (P=07), although the 31-40 age group showed a significantly lower SCEs frequency with respect to the other age groups. A significant association was also found between GSTP2C19-AA, GSTT1-null, GSTM1-null, ERCC2/XPD Gln751Gln, and XRCC1 His208His genotypes, and higher frequencies of SCEs. We describe the association between some phase I, phase II, and DNA-repair gene polymorphisms with increased SCEs frequencies, reinforcing the importance of genetic analysis in biomonitoring studies. Sex and age were found to be important endogenous factors that affect the level of genomic damage and the replicative capacity of cells, respectively.

Association of GSTT1 null, XPD 751 CC and XPC 939 CC genotypes with increased levels of genomic damage among hospital pathologists

Context: Hospital workers are at risk for genotoxic damage following occupationally exposure to xenobiotics. Pathologists are exposed to chemicals during their use in health care environments, particularly throughout inhalation of airborne agents, absorption through skin or contact with the patient’s body fluids.Objective: We evaluated the level of genomic damage in a sample of 61 hospital pathologists (occupationally exposed to antineoplastic drugs and sterilizing agents) and 60 control subjects.Materials and methods: Lymphocytes were analyzed by SCEs and CAs assays and genotyped for GSTT1, GSTM1, CYP1A1 Ile/Val, XPD (A751C) and XPC (A939C) gene polymorphisms.Results: Pathologists showed significantly higher frequencies of SCEs and CAs with respect to control subjects. GSTT1 null genotype was found to be associated with higher SCEs and CAs frequencies, whereas XPD 751 CC and XPC 939 CC genotypes only with a higher level of SCEs.Discussion and conclusions: The SCEs and CAs results are consistent with other published data, placing hospital workers as a category at risk for genotoxic damage caused by chronic exposure to xenobiotics. The higher levels of cytogenetic damage observed among GSTT1 null, XPD 751 and XPC 939 CC homozygote subjects confirm the importance of the genetic polymorphisms analysis associated to genotoxicological studies.

Early detection of lung cancer potential among Egyptian wood workers.

Wood dust is known to be a human carcinogen, with a considerable risk of lung cancer. The increased cancer risk is likely induced through its genotoxic effects resulting from oxidative DNA damage. This study aimed at assessing the genotoxicity of wood dust and demonstrating the role of sputum PCR as a screening tool for early prediction of lung cancer among wood workers. The study was carried out in the carpentry section of a modernized factory involved with the manufacture of wooden furniture in Greater Cairo, Egypt. Environmental assessment of respirable wood dust concentrations was done. Frequency of chromosomal aberrations (CA%) and sister chromatid exchanges (SCE%) in peripheral blood lymphocytes (PBL) was assessed and comet assays were performed in samples from among the study population (n=86). Levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes were measured. The polymerase chain reaction (PCR) was used to study hypermethylation of p16 and ̸or O6-methylguanine-DNA methyltransferase (MGMT) gene promoters in sputum DNA. The concentrations of respirable wood dust exceeded the Egyptian and international permissible limits with highest levels generated by sawing operations. Laboratory investigations revealed statistically significantly higher frequencies of CA and SCE as well as increased comet tail length associated with significant decrement in the levels of SOD and GPx among exposed group. A statistically significant elevation in the extent of hypermethylation was detected for the p16 and MGMT gene promoters in the sputum DNA of studied wood workers. The study results support the conclusion that prolonged unprotected occupational exposure to wood dust is associated with possible genotoxicity and oxidative stress that might raise the risk for carcinogenesis including lung cancer.

Premature chromatid separation and altered proliferation of human leukocytes treated with vanadium (III) oxide

Vanadium is a widely distributed metal in the Earth’s surface and is released into the environment by either natural or anthropogenic causes. Vanadium (III) oxide (V2O3) is present in the environment, and many organisms are exposed to this compound; however, its effects at the cellular and genetic levels are still unknown. Therefore, in this study, the ability of V2O3 to induce chromosomal damage and impair cell proliferation was tested on human leukocytes in vitro. The cultures cells were treated for 48 h with different concentrations 2, 4, 8 or 16 μg/mL of V2O3, and we use the sister chromatid exchange’s (SCE) test and the viability assay to evaluate the effects.In the results, no change was observed in either the viability or the frequency of SCE; however, a significant increase was observed in the incidence of premature chromatid separation (PCS), and a decrease was observed in both the mitotic index (MI) and the replication index (RI). Therefore, it can be suggested that V2O3 induces a genotoxic effect at the centromere level, indicating that it is a cause of aneuploidy that is capable of altering cell cycle progression.

Genotoxic effects of the carbamate insecticide Pirimor-50® in Vicia faba root tip meristems and human lymphocyte culture after direct application and treatment with its metabolic extracts.

The aim of the study was to evaluate genotoxic effects of Pirimor-50®, a pirimicarb-based formulation (50 % active ingredient), in human lymphocyte cultures and Vicia faba root meristems. Furthermore, the objective was to examine a combined influence of insecticide treatment with mammalian microsomal S9 and vegetal S10 metabolic fractions or S10 mix metabolic transformation extracts (after Vicia faba primary roots treatment with Pirimor-50®). We used sister chromatid exchange assay-SCE and measured cell cycle progression and proliferation (proportion of M1-M3 metaphases and replication index ratio-RI). Two processes were used for plant promutagen activation: in vivo activation-Pirimor-50® was applied for 4 h to the plant and then S10 mix was added to lymphocytes; and, in vitro activation-lymphocytes were treated with Pirimor-50® and S10 or S9 for 2 h. Direct treatment induced significantly higher SCE frequencies in meristems at 0.01 mg mL-1. In lymphocytes, significantly higher SCE was at 1 mg mL-1 with decrease in RI and M1-M3 metaphase proportions at 0.5 mg mL-1 and cell division stop at 2.5 mg mL1. S10 mix lymphocyte treatment showed significantly elevated SCE values at 2-2.5 mg mL-1, with cell death at 3 mg mL-1. Lymphocyte treatment with Pirimor-50® together with S9 or S10 showed slightly elevated SCE frequency but had a significant influence on RI decrease, with lowest values in S9 treatment. Since no data are available on the genotoxicity of Pirimor-50®, this study is one of the first to evaluate and compare its direct effect in two bioassays, animal and vegetal, and also the effect of plant and animal metabolism on its genotoxic potential.

Genotoxic monitoring of nurses handling cytotoxic drugs

Objective:Several biomarkers may be used to detect harmful exposure and individual susceptibility to cancer. Monitoring of biomarkers related to exposure may have a significant effect on early detection of cell transformation, thereby aiding the primary prevention of various chronic and malignant diseases. Nurses who handle cytotoxic drugs are exposed to carcinogenic agents, which have the potential to interrupt the cell cycle and to induce chromosomal aberrations. The presence of high chromosomal aberrations indicates the need for intervention even when exposure to these carcinogens is low.Methods:Nationally representative samples of 552 nurses were investigated by a follow-up monitoring system. The measured biomarkers were clinical laboratory routine tests, completed with genotoxicological (chromosome aberrations [CAs] and sister chromatid exchanges [SCEs]) and immunotoxicological monitoring (ratio of lymphocyte subpopulations and lymphocyte activation markers) measured on peripheral blood lymphocytes. Results were compared to the data of 140 healthy, age-matched controls.Results:In nurses exposed to cytostatics, we observed a significantly increased frequency of CAs and SCEs compared with those in the controls. Cytostatic drug exposure also manifested itself in an increased frequency of helper T lymphocytes. Genotoxicological and immunotoxicological changes, as well as negative health effects (i.e., iron deficiency, anemia, and thyroid diseases), increased among cytostatic exposed subjects.Conclusions:These results raised concerns about the protection of nursing staff from chemical carcinogens in the working environment.

SiO2 Nanoparticule-induced size-dependent genotoxicity – an in vitro study using sister chromatid exchange, micronucleus and comet assay

Fine particles with a characteristic size smaller than 100 nm (i.e. nanoparticlesspread out in nowadays life. Silicon or Si, is one of the most abundant chemical elements found on the Earth. Its oxide forms, such as silicate (SiO4) and silicon dioxide, also known as silica (SiO2), are the main constituents of sand and quartz contributing to 90% of the Earth's crust. In this work, three genotoxicity systems “sister chromatid exchange, cytokinesis block micronucleus test and single cell gel electrophoresis (comet) assay” were employed to provide further insight into the cytotoxic and mutagenic/genotoxic potential of SiO2 nanoparticules (particle size 6 nm, 20 nm, 50 nm) in cultured peripheral blood lymphocytes as in vitro. It was observed that there is a significant decrease in Mitotic index (MI), Cytokinesis block proliferation index (CBPI), proliferation index (PRI) values expressed as Cell Kinetic parameters compared with negative control (p < 0.05). There is a statistically significant difference between negative control culture and culture exposed to SiO2 (6 nm, 20 nm, 50 nm) (p < 0.01, p < 0.01, p < 0.05, respectively). It is found that SiO2 nanoparticles at different size (6, 20, 50 nm) progressively increased the SCE frequency and DNA damage on the basis the AU values compared with negative control (p < 0.05). Results showed that the genotoxic/mutagenic and cytotoxic effects of SiO2 nanoparticules is dependent to particule size.

Clastogenic and Mutagenic Effect Induced by Some Fast Foods

The present work aims at disclosing the capability of some fast foods ininducing clastogenic effect assessed by analysis of chromosomes of Allium cepa and Viciafaba genome, and mutagenic effect assessed by in vivo induction of sister chromatidexchanges in mice genome. To achieve such a purpose Corn chips; Potato chips Burger andmice were locally purshased and used. The present investigation showed that the tested fastfoods were proven to be clastogenic agents, since they were capable to interfere with spindlefibers, and C-metaphases were observed, giving a strong evidence that the water-solublefraction of potato and corn chips as well as of Burger contained contaminants capable tointeract with chromosomes arid spindle fibers as well. However they have mutagenic activityas sister chromatid exchanges revealed obtained since positive frequencies of SCEs wereobserved.

Crossbreeding Effect on Genome Stability in Pig (&lt;I&gt;Sus scrofa scrofa&lt;/I&gt;)

Aneuploid cell percentages and frequencies of CAs and SCEs were investigated in 10 Calabrian pigs, 10 LW pigs and 19 Calabrian x LW crossbred pigs, in order to compare genome stability between an autochthonous pig breed and a highly selected one and to verify if genome stability of their progeny, as other phenotypic traits, are influenced by heterosis. The mean number of cells per animal with structural aberrations, excluding gaps, was 6.20 +/- 2.39, 4.90 +/- 2.02 and 4.52 +/- 3.34 in Calabrian, LW and crossbred pigs, respectively, while the mean number of total CAs without gaps was 0.14 +/- 0.38, 0.11 +/- 0.35 and 0.11 +/- 0.35, respectively. The mean number of SCEs was 7.30 +/- 3.24 in Calabrian pigs, 6.45 +/- 2.74 in LW pigs and 6.28 +/- 2.90 in the crossbred ones. Percentages of cells with aneuploidy were 7.30, 10.10 and 10.79 in Calabrian, LW and crossbred pigs, respectively. In particular, the Calabrian breed showed higher values compared to LW in each test, however, there were statistically significant differences only in the mean number of SCEs per cell (P<0.01). In addition, there is a positive effect of crossbreeding on baseline levels of genome stability in the crossbred group that shows in all tests, excluding gaps, mean values of cellular or chromosome damage similar to the LW group.

Antigenotoxic Effect of Curcumin and Carvacrol against Parathion Induced DNA Damage in Cultured Human Peripheral Blood Lymphocytes and Its Relation to GSTM1 and GSTT1 Polymorphism

In recent years, the use of organophosphorus pesticides has been extensively increased and these compounds signify a major class of agricultural pesticides today. We studied antigenotoxic potential of curcumin and carvacrol against the parathion induced DNA damage in cultured peripheral blood lymphocytes using sister chromatid exchanges as a biomarker of genotoxicity. Heparinised fresh blood from healthy individuals was treated with 2.5 μg/mL concentration of parathion in presence of curcumin and carvacrol in order to observe the antigenotoxic potential of both curcumin and carvacrol. Significant reduction (P < 0.05) was observed in the frequencies of SCEs in presence of 10 μg/mL and 15 μg/mL concentrations of curcumin as compared to parathion exposed sample. Similarly carvacrol had significant (P < 0.05) antigenotoxic effect at the concentrations of 2.5 μg/mL and 5.0 μg/mL against the parathion. We also studied the effect of GSTT1 and GSTM1 on genotoxicity of parathion and antigenotoxic potential of curcumin and carvacrol. We did not observe any significant effect (P > 0.05) of GSTT1 and GSTM1 polymorphism on genotoxicity of parathion and antigenotoxic potential of curcumin and carvacrol.

The Effect of Environmental Factors on Sister Chromatid Exchange Incidence in Domestic Horse (Equus caballus) Chromosomes

The SCE test is often used as a sensitive and reliable technique in the biomonitoring of genotoxicity of mutagenic and carcinogenic agents. This study analysed the frequency of sister chromatid exchange in domestic horse chromosomes depending on the habitat and age of the analysed horses. The chromosome preparations were obtained from an in vitro culture of peripheral blood lymphocytes stained using the FPG technique. Both the habitat and the age significantly influence SCE frequency. A higher SCE incidence was observed in horses that lived in a large urban agglomeration than in those from the country. Also, a higher SCE incidence was identified in the group of horses above 6 years of age in comparison with the younger ones. Additionally, the frequency of SCEs in the first, second and third chromosomes and the X sex chromosome were analysed in detail. More exposed to the effect of environmental pollutants, the horses from the urban environment developed more double and triple SCEs in comparison with the village horses. The urban horses also developed quadruple SCEs, in addition to the less frequent exchanges.

The ameliorative effect of Cetraria islandica against diabetes-induced genetic and oxidative damage in human blood

Context: The aqueous extracts of Cetraria islandica (L.) Ach. (Parmeliaceae) is traditionally used in many countries against a number of conditions, including inflammatory conditions.Objective: The present study aimed to assess, for the first time, the effectiveness of C. islandica in cultured primary blood cells of Type 1 diabetes subjects.Materials and methods: Diabetic and control blood samples were treated with or without aqueous lichen extract (5 and 10 μg mL−1) for 48 h. The activity of antioxidant enzymes in erythrocytes and also malondialdehyde levels in plasma were determined to evaluate the oxidative status. DNA damages were analyzed by SCE, MN and comet assays in cultured human lymphocytes. Additionally, proliferation index (PI) was evaluated in peripheral blood lymphocytes.Results: There were significant increases in observed total DNA damage (comet assay) (240.2%) and SCE (168.8%), but not in MN frequencies of cultures with diabetes as compared (p > 0.05) to controls. Whereas, the significant reductions of total DNA damage (69.2 and 65.3%) and SCE frequencies (17.7 and 12.3%) were determined when the 5 and 10 mg mL−1 lichen extract was added to the cell culture medium, respectively. However, lichen extract did not completely inhibit the induction of SCEs in lymphocytes of patients with diabetes. C. islandica extract was also useful on PI rates.Discussion: In conclusion, the antioxidant role of C. islandica in alleviating diabetes-induced genomic instability and for increasing cell viability was firstly indicated in the present study.

Sister Chromatid Exchange in Polish White Improved Goats (Capra hircus)

The study was aimed at evaluating the frequency of spontaneous sister chromatid exchange in Polish White Improved goats (Capra hircus). The mean number of SCEs/cell was 2.73 +/- 1.84. The effect of sex and age on SCE incidence was also investigated. No statistically significant differences in the number of SCEs/cell were observed between the males and females. On the other hand, age was found to significantly influence SCE frequency. A lower SCE frequency was observed in younger goats. A positive correlation between chromosome length and SCE number was identified. The longer the chromosome, the more exchanges occurred. The highest number of SCEs was observed in the interstitial region, the lowest in the distal area.

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)11Selenium and vitamins A, C and E, selenium ACE. against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight22mg/kg body weight (mg/kg b.wt.). alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCE's)33Sister chromatid exchanges (SCE's). in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCE's, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Genotoxic Effect of Dye Effluents in Chromosomes of Indigenous Goats (Capra hircus)

The genotoxic effect of dye effluents was assessed in indigenous goats inhabiting textile industry areas encompassing dyeing and bleaching units by means of mitotic drive, mitotic index, chromosome aberrations and the mean SCEs frequency. Peripheral blood leucocyte cultures were set up with culture medium and pokeweed mitogen. The cultures were supplemented without bromodeoxyuridine (BrdU) for chromosome aberrations and with BrdU for the mean SCEs frequency for 2 cell cycles. Air-dried slides were stained with Giemsa for chromosome aberrations and the fluorescent plus Giemsa (FPG) technique was adopted for the display of sister chromatid differentiation and the estimation of the mean SCEs frequency. No gross structural and numerical chromosome abnormalities were noticed in all animals screened based on G-banding and karyotyping. The mitotic drive for the control and exposed populations was 37.58±1.29 and 26.13±0.78%, respectively. The mitotic drive in exposed population was significantly (p<0.01) lower when compared to control animals. The mitotic index for the control and exposed populations was 12.49±0.42 and 11.49±0.43%, respectively. Though there was a decrease in mitotic index in the exposed population, it was not statistically significant. The frequency of SCEs did not follow the Poisson distribution in both the control and exposed populations of goats. The pooled mean±S.E (range) of SCEs frequency for the control and exposed populations of goats were 4.83±0.16 (1–11) and 12.98±0.16 (8–19), respectively. There was significant (p<0.01) increase in the mean SCEs frequency of goats (exposed) reared in industrial areas. The number of SCEs in chromosome 1 was significantly (p<0.05) higher than that expected from relative chromosomal length.

Homologous Recombination Repair of DNA Strand Breaks Induced by the Nucleoside Analogue CNDAG.

AbstractAbstract 1189We hypothesize that the novel deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl) guanine] may share the common action mechanism with its cytosine congener CNDAC [2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine], a prodrug of which, sapacitabine, is undergoing clinical trials in myeloid leukemias. CNDAC induces single strand breaks following incorporation into DNA. Subsequent processing or DNA replication across the unrepaired nicks would generate double strand breaks (DSBs) [1]. Because cytosine and guanine nucleoside congeners have remarkably different clinical activities, e.g., cytarabine (acute myelogenous leukemia) and nelarabine (T-cell malignancies), it will be useful to pursue investigations to fully characterize the metabolism and actions of CNDAG. This study was aimed at defining cellular response and damage repair mechanisms for two CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each prodrug is a substrate for adenosine deaminase (ADA), the action of which generates CNDAG. First, growth inhibition by both CNDAG prodrugs was dependent upon both concentration and time of exposure; the proliferation of T-cell malignant lines (CCRF-CEM and Jurkat) was suppressed more to B-cell lines (Raji and IM-9). This may be attributed to relatively low activity of deoxycytidine kinase in the latter cell lines. Second, p53 knocked-out and parental HCT116 cells were equally sensitive to CNDAG 6-NH2 in a clonogenic assay, indicating that cytotoxicity of CNDAG is independent of p53 status. Third, similar to CNDAC, CNDAG prodrugs activated repair proteins in multiple DNA damage response pathways, as revealed by immunoblotting. 24-hr incubation of CCRF-CEM cells with 50 microM either prodrug increased the phosphorylation of Ser-1981 on ATM, Ser-345 on Chk1, Thr-68 on Chk2, Ser-966 on SMC1, Ser-343 on Nbs1 and g-H2AX. In contrast, there was no increase in phosphorylation of two other sensor kinases, DNA-PKcs (Ser-2056) which participates in repair of double strand breaks by non-homologous end-joining, and ATR (Ser-428) which senses stalled DNA replication forks. Fourth, we investigated the role of components of homologous recombination (HR) in CNDAG-induced DNA damage repair. The clonogenic survival of human fibroblasts deficient in ATM or those transfected with an empty vector were approximately 20- to 30-fold more sensitive to CNDAG prodrugs than cells complemented with full-length ATM cDNA. Chinese hamster cells deficient in Rad51D or either of the two Rad51-interacting proteins, Xrcc3 and Brca2, conferred greater than 30-fold sensitivity to CNDAG prodrugs relative to respective wild type lines. Similar sensitization was also observed with CNDAC. In contrast, cells lacking HR function were not more sensitive to ara-C or ara-G compared to their parental and complemented cells, indicating HR is a unique repair mechanism for 2'-C-cyano-2'-deoxy-nucleoside analogues. Finally, a cytogenetic approach was used to analyze sister chromatid exchange (SCE, a hallmark for HR) formation in metaphase cells exposed to 2 microM CNDAG 6-NH2. The frequencies of SCEs in AA8 cells incubated with CNDAG for two cell cycles (mean 14.2 per metaphase) were 2-fold of those exposed for one cell cycle (mean 7.4 per metaphase, n>20, p<0.001), the latter greater than control (mean 6 per metaphase, p<0.05). Together these results demonstrate that DNA damage caused by CNDAG activates ATM-dependent signaling pathways and is repaired through homologous recombination. Thus, this is a class effect caused by 2'-C-cyano-2'-deoxy-nucleoside analogues. Our study suggests that despite relatively less potency, CNDAG might have distinct clinical activity from that of CNDAC. [1] Liu X, et al. Blood, Blood. 2010 May 17, Epub ahead of print, PMID: 20479284. [Display omitted] Disclosures:No relevant conflicts of interest to declare.