Spontaneous unusual expression of frequency of chromosome aberrations and common fragile in human lymphocytes of colorectal cancer patients induced by Aphidicolin

Chromosomal fragile sites are distributed all over the human genome. Aphidicolin mediated expression frequency of common fragile sites and other chromosomal changes were evaluated in prometaphase/metaphase chromosomes obtained from peripheral blood lymphocytes of colorectal cancer patients. The present study reveals first time high incidence i.e. 6 % of aphidicolin induced chromosome breaks / gaps designated as "common fragile sites" in cell population of clinically diagnosed patients of colorectal cancer patients in Nepalese population. These chromosomal changes including structural and numerical were compare to clinically healthy normal individual of same sex / age groups, act as controls for statistical analysis. The frequency of chromosomal aberration in cancer patients were significantly higher (p<0.001) when compare to normal individuals. The increased genetics instability probably either due to nutritional factor i.e. lack of folic acid component in diet--an essential component required for DNA synthesis or unknown environmental factor for such genetic disorder. The present study indicates aphidicolin high frequency of induced chromosome aberrations and "common fragile sites" because of late replication of DNA in mitosis in colorectal cancer patients suggesting these sites could be used as suitable marker for determining genetic predisposition in cancer patients.     

Adriamycin-induced chromosome aberrations in human fibroblasts.

Adriamycin (AM) induced chromosome lesions and cell division delay in human foreskin fibroblasts. Cells treated with 0.01, 0.03, and 0.05 mug AM/ml culture medium for 1 hour and evaluated 5-12 hours post treatment exhibited a wide spectrum of cytogenetic injuries, ranging from moderately damaged metaphases with predominantly simple chromatid-type lesions to heavily damaged metaphases with chromosome stickiness and fragmentation. In moderately damaged metaphases that could be scored for specific types of aberrations, we observed a paucity of chromatid exchanges and chromosome-type lesions even in cultures having a very high frequency of breakages. Further, the distribution of breaks among chromosomes within groups A-G appeared to be random, which suggested that the drug does not show breakage specificity in human fibroblasts; The number of heavily damaged metaphases increased with an increase in concentration of AM and with longer periods of recovery.     

The in vitro induction of sister chromatid exchanges and chromosome aberrations in human lymphocytes by styrene derivatives

Three structural analogues of styrene, vinyltoluene (40% para-,60% meta -isomers), 4-methoxy-trans-ß-chlorostyreneand trans-ß-nitrostyrene, were tested for their potentialto induce chromosome aberrations and sister chromatid exchange(SCE) in phytohaemag-glutinin-stimulated human lymphocytes culturedfor 48 h (aberration analysis) or 72 h (SCE analysis). The treatmentswere carried out 24 h (aberrations) or 48 h (SCEs) before harvest.The toxicity of vinyltoluene to cultured lymphocytes was similarto that of styrene while 4-methoxy-ß-chlorostyrenewas about 10 times and ß-nitrostyrene about 100 timesmore toxic than styrene. A dose-dependent increase of SCEs andchromosome aberrations was observed in cells treated with vinyltoluene(0.33–4.00 mM) or with 4-methoxy-ß-chlorostyrene(0.5–1.00 mM). No effect could be seen in cultures treatedwith ß-nitrostyrene (0.004–0.044 mM). The resultssuggest that, like styrene, vinyltoluene and 4-methoxy-ß-chlorostyrenealso are converted in vitro to reactive metabolites, presumablyepoxides.     

Effect of heterocyclic amines and beef extract on chromosome aberrations and sister chromatid exchanges in cultured human lymphocytes

Two of the major bacterial mutagens formed in heated meat products,2-amino-3-methylimidazo[4, 5-f]quinoline and 2-amino-3, 8-dimethylimidazo[4,5-f quinoxaline or the basic fraction of beef extract induceda low frequency of sister chromatid exchanges in human lymphocytecultures in the presence of metabolic activation. Structuralchromosome aberrations were not induced at comparable high concentrationsin human lymphocytes with intact repair system, suggesting thatrepair or induction of point mutations are involved in the DNA-damagingeffect of heterocyclic amines rather than structural chromosomeaberrations. Accordingly it may be concluded that mammaliancells with both intact repair and enzyme systems are more relevantthan bacterial systems for evaluating the carcinogenic potentialof heterocyclic amines.     

碳离子辐照诱导人外周血淋巴细胞染色体畸变的时间和剂量效应

目的：研究碳离子辐照诱导淋巴细胞染色体畸变的时间和剂量效应。方法：以加速的碳离子为辐射源,吸收剂量分别为2、4Gy的碳离子辐照人外周血淋巴细胞后,分别培养48、72、84h后收集细胞,用姊妹染色单体区别染色法分析淋巴细胞第一次分裂中期染色体畸变,以研究畸变的时间效应;吸收剂量为0、0.5、1、2、3、4Gy的碳离子辐照人外周血淋巴细胞后,培养48h,用常规染色体技术研究碳离子辐照诱导淋巴细胞染色体畸变的剂量效应。结果：辐照后分别培养48、72和84h得到的双着丝粒和着丝粒环畸变（＂双＋环＂畸变）频率没有明显差异;在0.5～4Gy剂量范围内,＂双＋环＂畸变的量效关系符合线性关系：Y=0.0005＋0.689D。结论：在本实验辐照条件下,碳离子辐射诱导淋巴细胞染色体畸变不存在时间效应,可以用48h的培养时间来研究碳离子的生物学效应,并发现染色体＂双＋环＂畸变随剂量的增加而呈线性增加。     

Association between TP53 gene ARG72PRO polymorphism and chromosome aberrations in human cancers

It is well known that the TP53 gene considerably influences on DNA repair processes. Polymorphisms in the TP53 gene, particularly the well‐known Arg72Pro in codon 72 of exon 4 (Ex4+119 G>C; rs1042522), can modify the functionality of the p53 protein and activation of DNA repair. Actually, polymorphic variants Arg and Pro were found to have different properties of regulation of TP53‐dependent DNA repair target genes, that can effect various levels of chromosome aberrations in cancer patients with these genotypes. Here, we studied frequency of chromatid breaks (CB), chromosome‐type aberrations (CTA) and aberrant cells (AC) in cancer patients (n = 102) with various Arg72Pro genotypes. It was shown that the Arg variant of TP53 gene is associated with high frequency of AC and chromatid breaks. That is Arg/Arg carriers have more different chromosome aberrations in comparison to individuals with Arg/Pro and Pro/Pro genotypes (P < 0.05). Conversely, the lowest level of AC and chromatid breaks were detected in cancer patients with the Pro/Pro genotype. A completely unexpected result was that women with Arg/Arg genotype had the most high frequency of CB and AC in comparison to Arg/Pro and Pro/Pro women carriers (P < 0.001). In the group of male patients we did not show any differences in chromosome aberrations between carriers of Arg72Pro genotypes. In conclusion, the TP53 gene Arg72Pro polymorphism appreciably influence on occurrence of chromosome aberrations in cancer. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

Antipain-mediated suppression of X-ray-induced chromosomal aberrations in human lymphocytes

The protease inhibitor antipain is known to modulate the numberof chromosomal aberrations induced by the S-phase-dependentalkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Experimentshave now been carried out to see if antipain might also affectthe yield of aberrations induced by X-rays, which are S-independentand thus produce chromosomal aberrations by a different mechanism.The results show that human lymphocytes exposed to 0.4 or 1.5Gy of X-rays at 48 h of culture and fixed at 3, 6, 8, 10 or12 h thereafter contain 27–52% fewer chromatid breaksif the cells are also treated with antipain before irradiation.Because previous studies postulated that antipain could affectthe induction of chromosomal aberrations by suppressing freeradical reactions within cells, we also tested whether antipainaffects X-ray-induced aberrations when present only during thetime of irradiation, as is the case for free radical scavengers,such as L-cysteine. The results indicate that, in contrast toL-cysteine, antipain can suppress the induction of X-ray-inducedaberrations even when administered as late as 2 h after irradiation,suggesting that the effects of antipain on aberrations are notattributable to its interference with short-lived radicals withinthe cells. Although the exact mechanism whereby antipain decreasesthe yield of chromosome aberrations induced by the S-independentagent X-rays is unknown, these data indicate that the formationof chromosome aberrations by S-independent agents too can involvean antipain-sensitive process.     

Sister chromatid exchanges and chromosome aberrations in lymphocytes of nurses handling antineoplastic drugs.

Chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) were investigated in peripheral lymphocytes of 15 nurses and nurse's aides handling cytostatic agents in hospital oncology units. Significantly increased frequencies were noted for both CA and SCE rates when the exposed individuals were compared with 15 nurses working in other hospital units and to a control sample matched by sex and age. This points to the need for emphasizing protective measures in the handling of anti-neoplastic agents.     

Chromosome aberrations in the peripheral lymphocytes induced by brachytherapy and external cobalt teletherapy

In the present study, the induction efficiencies of chromosome aberrations were analyzed in patients receiving various modalities of interstitial radiotherapy with small radiation sources in the oral cavity area and compared with those in patients treated with external telecobalt irradiation in the thoracic region. Further, as a local nonstochastic effect, the acute mucosal reaction was investigated. The mucosal reaction in the area treated by brachytherapy reached a maximum at two to three weeks after the implantation. The frequencies of dicentrics plus rings of peripheral lymphocytes, on the other hand, revealed rapid increases and approached plateau levels as early as two days after the implantation. The whole-body effects, evaluated on the basis of the chromosome aberration frequencies of peripheral lymphocytes in the patients who underwent brachytherapy, were compared with the effects observed in those patients treated with external radiotherapy or bleomycin injection and discussed with regard to their effectiveness in control of the tumor and side effects. The radiation doses used in the patients who received brachytherapy or a single external irradiation were found to exert the same effects on the chromosome aberration induction. However, even in the case of brachytherapy in which whole-body side effects were believed to be trivial, the peripheral lymphocyte count was temporarily reduced to one half or less of the preirradiation level. An equivalent whole-body dose of 50 cGy was obtained from the frequencies of dicentrics and rings.     

Mitochondrial genotypes and radiation-induced micronucleus formation in human osteosarcoma cells in vitro

We investigated whether or not the mitochondrial genotypes affect radiation-induced micronucleus (MN) formation. For that purpose, the rho+, KT1 and rho0 human osteosarcoma cell lines were used, which carry the wild-type mitochondrial DNA (mtDNA), the tRNALys mutant mtDNA and no mtDNA, respectively. Despite no significant difference in the clonogenic radiosensitivity, the rho+, KT1 and rho0 cells exhibited high, intermediate and low radiosensitivities, respectively, to the MN induction in cytokinesis-blocked binucleated cells. Such differential MN inductions were correlated with high, intermediate and low levels of cellular ATP in the rho+, KT1 and rho0 cells, respectively, but not exactly with ROS production. Antimycin A that inhibits the respiratory complex III reduced the rate of radiation-induced MN induction in the rho+ and KT1, but not rho0 cells. Thus, the functional status of the mtDNA to produce ATP appears to play a significant role for radiation-induced MN.     

Thyroid nodularity and chromosome aberrations among women in areas of high background radiation in China

Thyroid nodularity following continuous low-dose radiation exposure in China was determined in 1,001 women aged 50-65 years who resided in areas of high background radiation (330 mR/yr) their entire lives, and in 1,005 comparison subjects exposed to normal levels of radiation (114 mR/yr). Cumulative doses to the thyroid were estimated to be of the order of 14 cGy and 5 cGy, respectively. Personal interviews and physical examinations were conducted, and measurements were made of serum thyroid hormone levels, urinary iodine concentrations, and chromosome aberrations in circulating lymphocytes. For all nodular disease, the prevalences in the high background and control areas were 9.5% and 9.3%, respectively. For single nodules, the prevalences were 7.4% in the high background area and 6.6% in the control area (prevalence ratio = 1.13; 95% confidence interval = 0.82-1.55). There were no differences found in serum levels of thyroid hormones. Women in the high background region, however, had significantly lower concentrations of urinary iodine and significantly higher frequencies of stable and unstable chromosome aberrations. Increased intake of allium vegetables such as garlic and onions was associated with a decreased risk of nodular disease, which seems consistent with experimental studies suggesting that allium compounds can inhibit tumor growth and proliferation. The prevalence of mild diffuse goiter was higher in the high background radiation region, perhaps related to a low dietary intake of iodine. These data suggest that continuous exposure to low-level radiation throughout life is unlikely to appreciably increase the risk of thyroid cancer. However, such exposure may cause chromosomal damage.     

Lack of a correlation between micronucleus formation and radiosensitivity in established and primary cultures of human tumours.

The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.     

Chromosomal aberrations induced by chemotherapy and radiotherapy in lymphocytes from patients with breast carcinoma

: Stable chromosomal aberrations (SCAs) have been found in circulating lymphocytes from patients treated for breast carcinoma. Therefore, we tried to define their incidence in such patients, to determine an in vitro dose-effect relationship, and to correlate these data with clinical parameters.

: This prospective study included 25 patients who, after surgery, underwent either radiotherapy (RT) alone (n = 15) or RT combined with chemotherapy (n = 10). SCAs were scored using the fluorescent in situ hybridization technique before RT and 4 and 12 months after RT. Dose-effect curves were established by in vitro irradiation of blood samples with 2 and 4 Gy, before and after treatment.

: In all patients, the rate of SCAs increased significantly after external irradiation. No significant decrease in SCAs was observed during the first year after RT. RT and chemotherapy had no effect on the lymphocyte in vitro dose-effect relationship. No relationship was found in the distribution of patients between the yield of SCAs scored after external irradiation and after in vitro irradiation. SCAs after RT or in vitro irradiation did not correlate with family history of breast carcinoma or acute toxicity of treatment. More significantly, the yield of SCA after external irradiation was strongly related to the irradiation of the internal mammary chain and the supraclavicular lymph node area, suggesting that the volume of irradiated blood vessels was an essential parameter in determining the rate of SCAs.

: A high and stable yield of SCAs persisted at least 1 year after external irradiation. The nature of the volume irradiated containing large blood vessels was the major determinant of the observed biologic dose.     

Acute cytogenetic effects of antineoplastic drugs on peripheral blood lymphocytes in cancer patients chromosome aberrations and micronuclei

AIMS AND BACKGROUND: The aim of the present study was to evaluate the individual sensitivity of cancer patients to different antineoplastic drugs administered in standard protocols by assessing their acute cytogenetic effects on peripheral blood lymphocytes. METHODS AND STUDY DESIGN: In 12 patients undergoing cancer chemotherapy, acute cytogenetic effects on peripheral blood lymphocytes were evaluated by analysis of structural chromosome aberrations and micronuclei. All patients were given antineoplastic drugs, mainly as polychemotherapy. The frequencies of both cytogenetic biomarkers determined after the first chemotherapy cycle were compared with their pre-treatment (baseline) values. RESULTS: All chemotherapy protocols employed induced clear cytogenetic effects in both tests studied. The results obtained indicate interindividual variations between cytogenetic damage in peripheral blood lymphocytes among cancer patients. Statistically significant increases in the total number of structural chromosome aberrations and micronuclei in lymphocytes analyzed after chemotherapy compared to pre-therapy samples were observed in almost all patients studied. The highest level of chromosome damage as well as the highest incidence of micronuclei was observed following administration of the ACOP protocol (adriamycin, cyclophosphamide and vincristine). The proportions of signal-positive and signal-negative micronuclei were evaluated using DAPI staining, while silver staining revealed Ag-NOR+ and Ag-NOR- micronuclei. In some patients the incidence of signal-positive and Ag-NOR+ micronuclei after treatment was increased, indicating a more pronounced susceptibility of particular chromosomes to damage caused by antineoplastic drugs. CONCLUSIONS: With regard to the results obtained we may conclude that both parameters used in the present study on peripheral lymphocytes are sensitive biomarkers and can be successfully employed for biomonitoring of acute cytogenetic effects induced by antineoplastic drugs in standard clinical protocols for cancer treatment.     

The micronucleus assay in human lymphocytes: screening for inter-individual variability and application to biomonitoring

Micronuclei levels were assessed in cytokinesis-blocked lymphocytesof 200 male and female healthy donors not occupationally exposedto genotoxic risks and of 33 male industrial painters handlinggenotoxic substances. Frequency of micronucleated cells was9.87 ±3.1 per 1000 in the control population and wasshown to have a large inter-individual variability. The studyof factors contributing to this variability showed that onlysmoking could affect micronucleated cell rate, inducing an increaseof 25%, whereas age and sex had no effect. Among the industrialpainters, frequency of micronucleated cells averaged 18.30 ±7.39 per 1000: the difference between the two populations studiedwas shown to be statistically significant by the Mann-Whitneyrank sum test (one-sided U test) and indicated that exposedpainters need preventive measures.     

Numerical chromosome aberrations in fibrothecoma.

Cytogenetic analysis on a 7-day-old culture of a fibrothecoma showed only numerical chromosome abnormalities: 57, XX, +4, +5, +6, +10, +12, +12, +14, +17, +18, +19, +20. The finding of an extra copy of chromosome 12 in mesenchymal tumors, mostly benign and originating from the female genital tract, may possibly point towards their common embryonic origin.     

人外周血淋巴细胞高分辨染色体制备技术的研究

目的： 建立一种具有分裂指数高和染色体分散好等优点的550~850条带纹高分辨染色体的制备方法。方法：取10例健康人外周血为样本制备淋巴细胞高分辨染色体。固定5-氟尿嘧啶核苷、尿嘧啶核苷、胸腺嘧啶核苷、溴化乙锭、秋水仙胺的剂量,进行5个因素3个水平的正交设计15种实验方案。5个因素分别为培养时间、胸腺嘧啶核苷、溴化乙锭、秋水仙胺作用时间以及低渗时间。3个水平分别为培养时间64、72、80 h;胸腺嘧啶核苷作用时间16、17、18 h;溴化乙锭作用时间3、4、5 h;秋水仙胺作用时间10、15、20 min以及低渗时间30、40、50 min。每例样本同时采用15种方案进行实验,比较各方案带纹在550条以上时染色体分裂指数和分裂相分散的情况。结果：在15种方案中培养时间以及秋水仙胺作用时间对分裂指数有显著影响 (P＜0. 01),其中培养72 h后加5-氟尿嘧啶核苷和尿嘧啶核苷进行同步化和秋水仙胺作用15 min高分辨染色体分裂指数最大。37 ℃低渗40 min高分辨染色体分散效果最佳。结论：本实验方案的高分辨染色体制备方法,具有分裂指数高和染色体分散好等优点,具有较好的推广价值。     

Relationship between anaphase aberrations and carcinogenicity in the trout embryo microinjection assay

An increased incidence of anaphase aberrations was observed in the tissues of rainbow trout embryos (Salmo gairdneri) within 96 hours of exposure to mitomycin [(MM) CAS: 50-07-7; 50 and 100 ng/embryo] and aflatoxin B1 [(AFB1) CAS: 1162-65-8; 13 and 25 ng/embryo] with the use of the trout embryo microinjection assay. High numbers of anaphase aberrations in embryos exposed to MM were associated with low mitotic indices, cell pyknosis, and high embryo mortalities. When the anaphase aberration data for embryos exposed to AFB1 were compared to previously reported carcinogenesis data from the microinjection assay, the incidence of anaphase aberrations in embryos did not show the same quantitative variations as the incidence of hepatic carcinomas in adult trout. While anaphase aberrations may be an indicator of the clastogenic effects of chemicals on fish embryos, it is unlikely that these aberrations can be used as an early indicator of a carcinogenic response in the trout embryo assay.     

Relationship between cell ploidy and glucocorticoid induced death in human lymphoid cell lines

We have found a relationship between sensitivity to glucocorticoid induced cell death (at 10 microM glucocorticoid) and ploidy in the human lymphoid cell line CCRF/CEM-C7. Most sensitive clones are diploid, whilst resistant clones and the resistant parent line CCRF/CEM are tetraploid. Diploid sensitive clones have a tendency to become aneuploid within a few months of isolation, with alterations in their kinetic responses to glucocorticoids. This is followed by a doubling in DNA content which results in reversion to the tetraploid glucocorticoid resistant state of the parent line CCRF/CEM. A few sensitive clones have been found to be tetraploid but with different kinetic responses to glucocorticoids as compared to diploid clones. The principal difference being an extended lag period (48-72 h) prior to lethal response. The relationship between ploidy and glucocorticoid sensitivity does not appear to extend to other human lymphoid cell lines.