In vitro Antigenotoxicity of Ulva rigida C. Agardh (Chlorophyceae) Extract against Induction of Chromosome Aberration, Sister Chromatid Exchange and Micronuclei by Mutagenic Agent MMC

Objective To determine the in vitro possible clastogenic and cytotoxic activities of Ulva rigida crude extracts （URE）, and identify their antigenotoxic and protective effects on chemotherapeutic agent mitomycine-C （MMC）. Methods Anti-clastogenic and anti-genotoxic activities of Ulva rigida crude extracts （URE） were studied using chromosome aberration （CA）, sister chromatid exchange （SCE）, and micronuclei （MN） tests in human lymphocytes cultured in vitro. Results The chromosome aberration, sister chromatid exchange or micronuclei tests showed that URE at concentrations of 10, 20, and 40 lag/mL had no clastogenic activity in human lymphocyte cell culture. Three doses of URE significantly decreased the number of chromosomal aberrations and the frequencies of SCE and MN when compared with the culture treated with MMC （P〈0.0001）. Conclusion Although URE itself is not a clastogenic or cytotoxic substance, it possesses strong antigenotoxic, anti-clastogenic, and protective effects on MMC in vitro.

Evaluation of antigenotoxic effects of carotenoids from green algae Chlorococcum humicola using human lymphocytes

Objective:To identify the available phytochemicals and carotenoids in the selected green algae and evaluate the potential genotoxic/antigenotoxic effect using lymphocytes.Methods:Organic,solvent extracts of Chlorococcum humicola(C.humicola)were used for the phytochemical analysis.The available carotenoids were assessed by HPLC,and LC-MS analysis.The genotoxicity was induced by the benzo(a)pyrene in the lymphocyte culture,the genotoxic and antigenotoxic effects of algal carotenoids with and without genotoxic inducer were evaluated by chromosomal aberration(CA),sister chromatid exchange(SCE)and micronucleus assay(MN).Results:The results of the analysis showed that the algae were rich in carotenoids and fatty acids.In the total carotenoids lutein,β-carotene andα-carotene were found to be present in higher concentration.The frequency of CA and SCE increased by benzo(a)pyrene were significantly decreased by the carotenoids(P<0.05 for CA,P<0.001 for SCE).The MN frequencies of the cells were significantly decreased by the treatment with carotenoids when compared with the positive controls(P<0.05).Conclusions:The findings of the present study demonstrate that,the green algae C.humicola is a rich source of bioactive compounds especially carotenoids which effectively fight against environmental genotoxic agents,the carotenoids itself is not a genotoxic substance and should be further considered for its beneficial effects.

Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model

AIM： To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma （EAC）- bearing murine model.METHODS： Male Swiss albino mice were divided into four groups： Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal （ip） inoculations of rapidly proliferating 2 × 10^5 viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract （ALE） of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation （ALE control）; and Group D was the EAC ＋ ALE- treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein （MT） protein, sister-chromatid exchanges （SCEs）, and DNA alterations.RESULTS： Treatment of the EAC-bearing mice with the ALE significantly （P 〈 0.001） reduced viable tumour cell count by 68.34% （228.7 × 10^6 ± 0.53） when compared to EAC control mice （72.4 × 10^6 ± 0.49）, and restored body and organ weights almost to the normal values. ALE administration also increased （P 〈 0.001） mean survival of the hosts from 35 ± 3.46 d in EAC control mice to 83 ± 2.69 d in EAC ＋ ALE-treated mice. Haematological indices also showed marked improvement with administration of ALE in EAC-bearing animals. There was a significant increase in RBC count （P 〈 0.001）, hemoglobin percent （P 〈 0.001）, and haematocrit value （P 〈 0.001） from 4.3 ± 0.12, 6.4 ± 0.93, and 17.63 ± 0.72 respectively in EAC control mice to 7.1 ± 0.13, 12.1 ± 0.77, and 30.23 ± 0.57 respectively in EAC ＋ ALE-treated group, along with concurrent decrement （P 〈 0.001） in WBC count from 18.8 ± 0.54 in EAC control to 8.4 ± 0.71 in EAC ＋ ALE. Furthermore, treatment with ALE substantially improved hepatocellular architecture and no noticeable neoplastic lesions or foci of cellular alteration were observed. Daily administration of the ALE was found to limit liver MT expression, an important marker of cell proliferation with concomitant reduction in MT immunoreactivity （62.25 ± 2.58 vs 86.24 ± 5.69, P 〈 0.01）. ALE was also potentially effective in reducing （P 〈 0.001） the frequency of SCEs from 14.94 ± 2.14 in EAC control to 5.12 ± 1.16 in EAC ＋ ALE-treated group. Finally, in comparison to the EAC control, ALE was able to suppress in vivo DNA damage by abating the generations of＇tailed＇ DNA by 53.59% （98.65 ± 2.31 vs 45.06 ± 1.14, P 〈 0.001）, and DNA single-strand breaks （SSBs） by 38.53% （3.14 ± 0.31 vs 1.93 ± 0.23, P 〈 0.01） in EAC-bearing murine liver.CONCLUSION： Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

Evidence of increased chromosomal instability in infertile males after exposure to mitomycin C and caffeine

Aim： To evaluate the genetic instability of 11 fertile and 25 infertile men. Methods： The methodology of sister chromatid exchanges （SCEs） was applied to cultures of peripheral blood lymphocytes, and the levels of SCEss were analyzed as a quantitative index of genotoxicity, along with the values of the mitotic index （MI） and the proliferation rate index （PRI） as qualitative indices of cytotoxicity and cytostaticity, respectively. The genotoxic and antineoplastic agent, mitomycin C （MMC）, and caffeine （CAF） - both well-known inhibitors of DNA repair mechanism - were used in an attempt to induce chromosomal instability in infertile men, so as to more easily detect the probable underlying damage on DNA. Results： Our experiments illustrated that infertile men, compared with fertile ones, demonstrated a statistically significant DNA instability in peripheral blood lymphocytes after being exposed simultaneously to MMC and CAF. Conclusion： The current study showed vividly that there was genetic instability in infertile men which probably contributes to the development of an impaired reproductive capacity. （Asian JAndro12006 Mar; 8： 199-204）

Sister chromatid exchange and DNA damage in human lymphocytes induced by air-dust in Lanzhou City: involvement in free radicals

The air-dust samples collected from petro-chemical industrial region in the suburb of Lanzhou and from a certain rural region 64 km away from the city were extracted, with a mixed solvent (benzene: hexane: isopropanol=7:2:1) for 8 hours. A strong free radical signal at g= 2.00 of air-dust itself and a hyperfine splitting EPR signal of extract from air-dust have been detected. The sister chromatid exchange frequency (SCE) was increased by extracts of both dusts from the industrial region and from the rural region. If a chemical is able to increase SCE up to twice as high as the control, this chemical is considered to be mutagenic and/or carcinogenic. The double SCE frequency concentration is 23 μg/ml for the dust extract obtained from the industrial region and 47μg/ml for that from the rural region. Extracts were able to damage to DNA template. Results indicated that the mutagenicity and/or carcinogenicity of the extracts obtained from the petro-chemical industrial region were stronger than that of the extracts from the rural region.

Chromosome Alterations of Peripheral Lymphocytes in Patients with Cervical Cancer

Sister chromatid exchanged (SCE) and chromosome aberrations in peripheral lymphocytesof 30 patients with cervical cancer and relative lengths of C-band in 20 patients were studied. Verysignificant increase in SCE, hypodiploidies and relative lengths of C-band of chromosome 9 were ob-served, and significant increases in polyploidies, structural aberrations and relative lengths of C-bandof chromosome 1 were also found in cancer patients as compared with the controls, with all in non-randomized distribution. No differende was noted in alterations between stage Ⅱ and Ⅲ cancers.These findings suggest that chromosomal instability in the cervical cancer patients may represent aningerent trait in the patient and be related to the increased susceptibility of the individual to malignan-cy.

EARLY DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES USING CLONAL ANALYSES

Objective: To study the value of clonal analysis to the early diagnosis of myelodysplastic syndrome (MDS). Methods: Four types of clonal analyses were performed on the bone marrow samples from 50 patients suspected of MDS: (1) Conventional Cytogenetics (CC) for clonal chromosomal abnormalities; (2) BrdU-Sister Chromatid Differentiation (BrdU-SCD) for cell cycle kinetics; (3) Fluorescence in Situ Hybridization (FISH) for trisomy 8; (4) Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for N-ras mutation. Results: The diagnosis of forty-three patients was compatible with the FAB criteria for MDS. The other seven cases didn’t meet the FAB criteria, with only one lineage of dyspoiesis or with no obvious dysplastic changes. Among these seven cases, two were morphologically diagnosed with suspicious refractory anemia, one with sideroblastic anemia, one with leukemoid reaction, one with hypercellular anemia and two with chronic aplastic anemia. Clonal analyses of the 7 patients showed that six cases had clonal karyotype abnormalities, four had prolonged cell cycle patterns, four had trisomy 8 of different proportions and one had mutation of the exon 1 of N-RAS. Thus, they were revaluated as MDS patients. Conclusion: The untypical MDS patients with one lineage dyspoiesis or without obvious dysplastic changes can be diagnosed early by combining multiple clonal analysis techniques such as CC, SCD, FISH and PCR-SSCR.

Genotoxic and Cytotoxic Damage by Cyclophosphamide and Adriamycin as a Response to Treatment in Breast Cancer Patients: Pilot Study

The aim of this study was to evaluate the genotoxicity induced by cyclophosphamide-adriamycin treatment in breast cancer patients through the frequencies of Sister Chromatid Exchange (SCE), Replication Index (RI), Mitotic Index (MI) and Cell Proliferation Index (CPI) and to study the possible association between biomarkers of genotoxicity and the early response to treatment. The frequencies were obtained before and immediately after therapy from 17 patients with breast cancer (p < 0.001). Response to treatment was assessed after two years resulting in 12 patients in a state of remission. MI and CPI had high values after treatment in women with active cancers compared to those in a state of remission, however there were not significant differences. Conclusions: It is possible that MI y CPI biomarkers can serve as indicators for early assessment of treatment with cyclophosphamide-adriamycin. It should be noted that these are preliminary results and further study is necessary.

On the regulation of PP2A and its role in controlling sister chromatid cohesion and microtubule-kinetochore attachment

Protein phosphatase 2A(PP2A)plays a critical multi-faceted role in the regulation of the cell cycle.PP2A is involved in such diverse processes by the formation of structurally distinct families of holoenzymes,which are regulated spatially and temporally by specific regulators.Mitosis requires the correct arrangement of sister chromatids so that replicated chromosomes can bind correctly to microtubules and segregate towards opposite poles.A large number of studies have shown that PP2A is mainly involved in a series of phosphorylation processes during the G2/M phase transition and the termination of M phase.Moreover,PP2A shows a more substantial contribution to sister chromatid cohesion and microtubule-kinetochore attachment.These processes are all crucial for proper cell survival and proliferation and are often deregulated in cancer and other diseases.

Is there a connection between synthetic bone grafts and sisters chromatide exchange?

Background: In oral and maxillofacial surgery, synthetic bone grafts are most widely used as bone substitutes, due to the limited sources of autologous bone. The aim of this study was to examine the influence of three different synthetic bone grafts (Cerasorb, Fortoss and Perioglass) on sisters chromatide exchanges (SCEs) in peripheral lymphocytes. Materials and Methods: Peripheral blood samples taken from 68 patients (45 females and 23 males), who underwent oral surgery procedures, such as apical resection, cyst enucleation or periodontal curretage, were obtained for SCE a day before and two months after the surgeries. A control group included 30 patients, while the study group was made of the patients who underwent bone grafting with Cerasorb? (11 patients), Fortoss? VITAL (10 patients) or Perioglass? (17 patients). Results: Comparing with the results of the study group before and after the treatment, it was concluded that the results were statistically significant (p = 0.001). In the Perioglass? subgroup, a greater statistical significance (p = 0.003) was noted, than that in either the Cerasorb? (p = 0.620) or Fortoss? (p = 0.210) subgroups, in which there was no statistical significance. Conclusions: Although further investigations may be necessary, our results suggest that the synthetic bone grafts might have an influence on SCE in peripheral lymphocytes.

Regulation of sister chromatid cohesion during the mitotic cell cycle

Orderly execution of two critical events during the cell cycle––DNA replication and chromosome segregation––ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister chromatid cohesion. Timely establishment and dissolution of sister chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. In this review, we discuss recent progress in the molecular understanding of sister chromatid cohesion during the mitotic cell cycle.

SWI1 Is Required for Meiotic Chromosome Remodeling Events

The Arabidopsis dsy10 mutant was previously identified as being defective in the synapsis of meiotic chromosomes resulting in male and female sterility. We report here the molecular analysis of the mutation and show that it represents a T-DNA insertion in the third exon of the SWll gene. Four mutations have now been identified in SWI1, several of which exhibit different phenotypes. For example, the swil- 1 and dyad mutations only affect meiosis in megasporocytes, while the swil-2 and dsy10 mutations block both male and female meiosis. Furthermore, as part of a detailed cytological characterization of dsy10 meiocytes, we identified several differences during male meiosis between the swil-2 and dys10 mutants, including variations in the formation of axial elements, the distribution of cohesin proteins and the timing of the premature loss of sister chromatid cohesion. We demonstrate that dsy10 represents a complete loss-of-function mutation, while a truncated form of SWll is expressed during meiosis in swil-2 plants. We further show that dys10 meiocytes exhibit alterations in modified histone patterns, including acetylated histone H3 and dimethylated histone H3-Lysine 4.

Effect of Upregulated DNA Replication and Sister Chromatid Cohesion 1 Expression on Proliferation and Prognosis in Hepatocellular Carcinoma

Background: DNA replication and sister chromatid cohesion 1 (DSCC1) (also called DCC1) is a component of an alternative replication factor C complex that loads proliferating cell nuclear antigen onto DNA during S phase of the cell cycle. It is located at 8q24 and frequently amplified in hepatocellular carcinoma (HCC). However, the role of DSCC1 in the carcinogenesis and progress of HCC has not been fully investigated. Here, we aimed to assert the importance of DSCC1 in the HCC. Methods: In this study, copy number variation data and RNA sequencing data were used to calculate the DNA copy number and mRNA expression of DSCC1 in HCC. Quantitative polymerase chain reaction, Western blotting, and immunohistochemistry analysis were used to determine the mRNA and protein level of DSCC1 in HCC. The Kaplan–Meier analysis and univariate and multivariate Cox regression analysis were used to assess the association of DSCC1 with the overall survival (OS) of HCC patients. Moreover, lentiviral shRNA was used to knockdown DSCC1, and then, colony?forming assay, cell cycle assay, and cell proliferation assay were performed to evaluate the impact of DSCC1 silencing on HCC cell lines. Results: We found that DSCC1 was amplified and highly expressed in HCC tumor tissues than in nontumor tissues. We then found that the overexpression of both mRNA and protein of DSCC1 was linked to the bad prognosis of HCC patients. Astonishingly, the protein level of DSCC1 was an independent prognostic factor for OS (hazard ratio, 1.79; 95% confidence interval, 1.17–2.74; P = 0.007). Furthermore, the clonogenic capacity of DSCC1?amplified HCC cell lines (MHCC?97H, MHCC?97L, and Hep3B) was significantly inhibited by transduction of a lentiviral shRNA that targets DSCC1. We also showed that knockdown of DSCC1 induced G0–G1 cell cycle arrest (increased from 60% to more than 80%) and greatly inhibited the proliferation of HCC cell lines. Conclusion: These results suggest that DSCC1 is a putative HCC driver gene that promotes proliferation and is associated with poor prognosis in HCC.