Single Chromatid Breaks Research Articles

Cytotoxic Evaluation of <i>Eurycoma longifolia</i> Jack Root Extract on Chromosome Aberrations in Human Lymphocytes <i>In vitro</i>

This study aimed to investigate chromosomal aberrations of Eurycoma longifolia Jack (EL) root extract in human lymphocytes in vitro. Human whole blood was cultured in medium solution that treated with distilled water, 20% DMSO, extract of EL roots at the concentration of 2.5, 5, 10, 20, 40, 80 µg/mL (extracted with distilled water and ethanol), and nontreated (blank: only culture medium and whole blood). All experiments were cultured for 72 hours in the 37°C incubator. The effects of EL roots extract on cytotoxicity were compared with the control groups including the blank, distilled water, and 20% DMSO. This study found that EL root extract significantly decreased metaphase cell number and increased chromosome aberrations dose dependent manner (p<0.01). The 7 types of chromosome aberration that were observed consisted of dicentric chromosome, single chromatid breaks, isochromatid break, isochromatid gap, single chromatid gap, fragmentation, and deletion. The dicentric chromosome was the most common chromosomal aberrations type that was treated with EL root extract both distilled water and ethanol. Moreover, the ethanolic extract of EL root was more effective to stimulate chromosome aberrations compared to the water extract of EL root (the deletion and fragmentation were not found in the water extract of EL root). This study demonstrated that the phytochemicals of EL root extract had cytotoxicity effect (decreased metaphase cells and increase cells death) and genotoxic effect (increased chromosomal aberrations. The use of EL root crude extract with distilled water is therefore safer for cells. However, when EL is used at high levels, it may lead to the inhibition of cell division process and cause side effects (toxicity). EL extracts consist of various phytochemicals with different properties and dosages, thus more studies should be conducted on the effect of those substances on cytotoxicity, especially their effects on genotoxicity humans.

Analysis of chromosome abnormalities in the Asian swamp eel (Monopterus albus) affected by arsenic contamination near a gold mine area

ABSTRACT The paper reports a study of chromosome abnormalities (CA/s) in the Monopterus albus near a gold mine area compared to a non-affected area. The arsenic (As) concentrations were determined in the water, sediment and M. albus muscles. The average concentrations of As in the water and sediment samples near the gold mine were higher than allowable by Thailand’s water quality standards. The average concentrations of As in the M. albus samples were lower than the standards. The diploid chromosome number of M. albus in both areas was 2n = 24. The percentage of CA of M. albus in the gold mine area was higher than in the non-affected area. Seven types of CA were single chromatid gap (SCG), fragmentation (F), sister chromatid gap, single chromatid breaks, deletion, sister chromatid fragmentation and centric fusion. In the affected area, the most common CAs in the samples were SCG and F. The specific CA in M. albus samples was SCG on chromosome pair 1. It can be concluded that As contamination caused structural CA, but did not affect the diploid chromosome number in the Asian swamp eel from the gold mine area. The accumulation of As in M. albus has potential effects on human health in consumers of M. albus from this contaminated area.

Cytotoxic evaluation of rice field frogs (Fejervarya limnocharis) from gold mine area with arsenic contamination

The objectives of this study were to investigate chromosomal aberrations in rice field frogs (Fejervarya limnocharis) from gold mine area compared to those from non-affected area. Three frogs were collected from each site, and chromosomal aberrations were studied in their bone marrow. Arsenic (As) was also measured in water and sediment from both areas. The average As concentrations in samples from the gold mine and the non-affected areas were 0.03 ± 0.003 mg/L and not detected in water and 351.59 ± 5.73 and 1.37 ± 1.07 mg/kg in sediment, respectively. The values in the gold mine were still higher than that allowed by the water and soil quality standards; however, the As concentrations in the samples from the non-affected area were acceptable. The average concentrations of As in the frog samples from the gold mine and the non-affected areas were 0.35 ± 0.33 and 0.07 ± 0.01 mg/kg, respectively, which were both lower than the standard of As contamination in food. The diploid chromosome number of frogs in both areas was 2n = 26, and the percentage of chromosomal aberrations in rice field frogs in the gold mine area was higher than in the non-affected area. There were seven types of chromosomal aberrations observed: single chromatid gap, isochromatid gap, single chromatid breaks, deletion, fragmentation, centric fragmentation and polyploidy. The most common chromosomal aberration in the samples from the affected area was the single chromatid gap. The difference in the percentages of chromosomal aberrations in rice field frogs from each area was statistically significant (p < 0.05).

High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.

The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

Chromosomal Aberration of Snakehead Fish (Channa striata) in Affected Reservoir by Leachate with Lead and Mercury Contamination

The objectives of this study are to investigate chromosomal aberrations of snakehead fish in a leachate-affected reservoir located 100 meters from a municipal landfill which compared to non-affected reservoir. Three snakehead fish were collected and chromosomal aberrations were studied using kidney tissue. Lead and mercury were measured in water, sediment and snakehead fish from the affected reservoir at three sampling sites. The results showed that the average concentrations of lead and mercury in water were 0.0012±0.0003 and 0.0053±0.0036 mg/L, respectively. The average concentrations of lead and mercury in sediment were 3.3650±2.1930 and 0.0890±0.0272 mg/kg, respectively. These values did not exceed the standard for water and soil quality except for the mercury in the water, which was higher than acceptable. Lead was not found in snakehead fish from either reservoir. The average concentrations of mercury in snakehead fish from both reservoirs were 0.1330±0.0792 and 0.1180±0.0350 mg/kg, respectively, which were higher than the standard for mercury contamination in food. This study showed that the accumulation of mercury in snakehead fish was higher than in sediment because it accumulates in organisms through the consumption hierarchy. The diploid chromosome number of snakehead fish in both reservoirs was 2n = 42 and the percentage of chromosomal breakages of snakehead fish in the affected reservoir was higher than the non-affected reservoir. There were four types of chromosomal breakages: single chromatid gap, isochromatid gap, single chromatid breaks and isochromatid breaks. The difference in percentage of chromosomal breakages in snakehead fish from both reservoirs was statistically significant (p<0.05).

Application of mFISH for the analysis of chemically-induced chromosomal aberrations: a model for the formation of triradial chromosomes

Using a human lymphoblastoid cell line WTK-1, we applied multicolor fluorescence in situ hybridization (mFISH) technique to analyze mitomycin C (MMC)-induced chromatid exchanges, focusing especially on the triradial chromosomes. It was found that the triradial chromosomes were formed with a specific rearrangement, “recipient and donor” relationship. The exchange sites of the recipient chromosomes were on single chromatid breaks and distributed randomly throughout the interstitial, pericentromeric, and terminal regions. In counterpart, donor chromosomes exchanged on isochromatid breaks of their telomeric and/or subtelomeric regions with the single chromatid breaks of recipient chromosomes. More than 80% of the scored triradial chromosomes were formed with such rearrangements, and few acentric chromosome fragments derived from the donor chromosomes could be detected in the metaphases observed. We therefore suggest that biological mechanisms of breakages between the recipient and donor chromosomes are different: the former due to direct DNA-damage by MMC, but the latter due to indirect DNA-damage depending on telomeric specific structure/function.

Chromosomal aberrations in native small mammals (peromyscus leucopus) at a petrochemical waste disposal site: II. Cryptic and inherited aberrations detected by G-band analysis

Wild rodents (Peromyscus leucopus) collected from a hazardous waste site polluted with petrochemicals and known to have significantly increased frequencies of somatic chromosome aberrations in a standard metaphase chromosome aberration (SMCA) assay were examined by G-banding, a differential staining technique. Several classes of aberrations not detectable with SMCA were identified, including Robertsonian fusions, pericentric and paracentric inversions, and interstitial deletions. Four classes of these more cryptic aberrations were significantly higher in animals from the hazardous waste site compared to animals from reference sites. Several animals also carried apparently inherited aberrations in chromosomes 5, 10, and 21, suggesting that aberrations induced in a parent may be transmitted to offspring. When all classes of aberrations were converted to numbers of lesions per cell, animals from the hazardous waste site did not show elevated frequencies of lesions or aberrant cells, compared to animals from reference sites, unless inherited aberrations were removed from the data set. Then animals collected from the hazardous waste site showed a significantly increased number of aberrations per cell. Two chromosomes (5 and 21) sustained more aberrations than the other members of the complement, suggesting that some chromosomes may be more susceptible to induced damage than others. The method of G-banding itself may obscure much of the damage attributable to single chromatid breaks in SMCA but reveals types of damage not detectable in SMCA, indicating that the application of any single genetic assay technique may seriously underestimate actual levels of induced damage in exposed organisms.

Chromosome aberrations in workers at a tannery in Iraq

Blood samples were collected from 17 healthy chromium-exposed workers at a tanning plant near Baghdad city and 13 controls matched for age, period of service and social background. For each individual more than 100 lymphocyte metaphases were examined. The results showed no significant differences in the per cell frequencies of chromatid and isochromatid gaps, single chromatid breaks, various chromosome-type aberrations and all aberrations combined. However, smoking workers exhibited statistically higher frequency of chromosome-type aberrations than non-smoking workers and smoking controls.

Sister-chromatid exchanges and chromosomal aberrations in lymphocytes of male albino rats treated with an alkylating agent, apholate (NSC 26, 812; ENT 26, 316)

A lymphocyte culture system was used to study the frequency of chromosomal aberrations and SCEs at various time intervals after an international injection of apholate (0.5 mg/kg b.w.) in male albino rats. Treatment with apholate increased the frequency of chromosomal aberrations significantly ( P<0.001). The majority of chromosomal aberrations were single chromatid breaks, although other aberrations were also present. The distribution of chromatid breaks was non-random and the large chromosomes were affected to a greater extent. SCEs frequency in lymphocytes of apholate-treated rats was more than 5-fold higher in comparison to controls. The highest number of chromosomal aberrations and SCEs/rat was recorded at 48 h post-treatment, after which there was a gradual decline in both SCE and chromosomal aberration frequency. The results indicate that apholate is a mutagenic compound and it may also be carcinogenic.

Restitution of chromatid and isochromatid breaks induced in the G2 phase by actinomycin D.

Although actinomycin D (AMD) appears to cause chromatid breakage, it has been suggested that it merely weakens the chromosomal proteins, causing breaks to occur during the stress of fixation. To determine if this was true, we examined lymphocytes treated with AMD (3.5 micrograms/ml) for 90 min prior to harvesting for evidence of DNA repair. Besides single chromatid breaks, we observed a high frequency of chromatid exchange figures (translocations, inversions, rings) and closed isochromatid breaks, indicating that a type of DNA repair leading to chromosome aberration had occurred prior to cell fixation. Therefore, at least some of the breaks observed after AMD treatment in G2 represented true DNA damage and were not merely artifacts of fixation.

Chromatid breaks induced in Chinese hamster cells by low doses (12?100 rad) of? ?-mesons

Monolayer cultures of the fibroblast-like Chinese hamster cell-line 19/1 were irradiated in the G2-phase of the cell cycle by pi--mesons (6 rad/min peak-pion dose rate). Frequencies of induced single- and isochromatid breaks, acentric fragments and interchanges were compared with data obtained from 140 kV X-rays. The RBE-values were for the pion dose peak between 0.8--1.2 and for the pion dose plateau 0.5--0.9. Whereas for single chromatid breaks there was no significant difference between X-rays and peak pions for identical physical doses, the isochromatid breaks alone showed a significantly higher frequency for 100 rad peak pions.

The effects of increasing dosages of X‐radiation on the chromosomes of the central mudminnow, Umbra limi (Kirtland) (Salmoniformes: Umbridae)

Fish subjected to 350 R, 660 R and 990 R of X‐radiation showed chromosomal aberrations such as chromatid breaks and gaps, and chromatid exchanges between several chromosomes. The frequency of aberrations/metaphase increased with radiation dosage. Likewise, the percentage of aberrant cells increased with increased irradiation. The countable metaphases fish was lower for higher doses of radiation. At lower doses single chromatid breaks accounted for most of the aberrations whereas complex aberrations involving the breakage and exchange of fragments between several chromosomes were more frequent in fish subjected to 990 R. Gill tissue yielded three times as many countable metaphases as did spleen tissue.

Effect of two chemosterilants apholate and hempa on the bone-marrow chromosomes of mice.

Mice were injected with 0.1% solution of apholate and hempa at the rate of 1.5 ml per 100 gm body weight and their bone-marrow cells were fixed at different intervals between 15 min and 144 hr. Specimens injected with distilled water served as controls.Only three aberrations were found in 4,100 metaphases in the control series. In the hempa-treated series, out of 4,100 metaphases examined one chromatid break, one subchromatid break, two gaps and one exchange were found at 24 hr only.In the apholate-treated series aberrations were absent only at 15 min and 144 hr post injection. At other times the aberrations were single chromatid breaks, subchromatid breaks, aniso- and isochromatid breaks, constrictions, gaps and lesions and exchanges. The aberration peak of 28.2% was reached at 24 hr, and fell to 0.3% at 120 hr. The breaks were non-randomly distributed between and within chromosome groups. Groups I and II and distal region of all affected groups were most susceptible.The mode of action of apholate and hempa in the induction of chromatid aberrations in mice, their localization and non-random distribution are discussed. The role of the aziridine groups in inducing aberrations has been considered. It is suggested that the breaks were due to some direct action and that the chromatids of mice contain some weaker regions.

In vitro cytogenetic investigation of calcium cyclamate, cyclohexylamine and triflupromazine

Cytogenetic effects after varied times of exposure to calcium cyclamate, cyclohexylamine (CHA) or triflupromazine HCl (TFP) were determined in a cell line derived from the kidney of the rat kangaroo ( Potorous tridactylis apicalis). A preliminary study, using aflatoxin and mitomycin C, which are known to produce abnormalities in cell systems, established the suitability of this cell line for the detection of chromosomal aberrations. In the present study, mitomycin C produced frequent chromatid gaps and breaks in the arms of the chromosomes. Aberrations of the chromosome-type as well as exchanges were noted with aflatoxin. The X-chromosome was extremely sensitive to these agents. CHA produced single chromatid breaks and TFP produced single chromatid breaks and exchange figures. The X-chromosome was not exceptionally sensitive to either of these agents. Calcium cyclamate at concentrations up to 200 μg/ml produced no chromosomal aberrations.

Cytogenetic studies in rats of cyclohexylamine, a metabolite of cyclamate.

Cyclohexylamine, the major known metabolite of cyclamate, was tested in vivo for possible cytogenetic effects. In rats injected with this metabolite, there was a direct relation between dose concentration and percentage of spermatogonial and bone marrow cells showing chromosomal breaks. Single chromatid breaks predominated with infrequent exchange figures.

Chromosome studies on bone marrow from Chinese hamsters treated with benzodiazepine tranquillizers and cyclophosphamide

Chinese hamsters were treated for two weeks with high doses of chlordiazepoxide hydrochloride, diazepam, and medazepam. Chromosome preparations of bone marrow were made 5–8 h after the last drug application. Single chromatid breaks were found in 0.27% of the cells, an incidence not differing from control values. Cyclophosphamide was used as a known chromosome-breaking control substance in order to test the sensitivity and peculiarities of the bone marrow system. Doses of less than 16 mg/kg produced little chromosome damage whereas after doses of 32 mg/kg the incidence of chromosome aberrations was easily quantitated. The time interval between the last drug application and preparing the bone marrow proved to be of importance: after 10 applications of 64 mg/kg cyclophosphamide in a 14-day course of treatment, the percentage of metaphases with damaged chromosomes dropped from 96% at 6 h after the last medication to 16% at 24 h, 2–5% at 3 days, and to virtually zero after 6 days.