Chromatid Lesions Research Articles

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.

The cytotoxicity and genotoxicity of hexavalent chromium in medaka ( Oryzias latipes) cells

Chromium is an increasing health concern for aquatic environments, however, the mechanism of chromium toxicity in aquatic species is yet unknown. We used a medaka ( Oryzias latipes) fin cell line to investigate the cytotoxicity and genotoxicity of sodium chromate, a soluble form of hexavalent chromium. We used a clonogenic cytotoxicity assay to measure sodium chromate cytotoxicity, gamma-H2A.X immunofluoresence to measure DNA double-strand breaks, and chromosome damage to measure clastogenicity. We found that sodium chromate is cytotoxic to medaka fin cells, with toxicity increasing in a concentration-dependent manner. Treatments of 0.5, 1, 5, 10, 25, 50 and 100 μM sodium chromate caused 100, 103.5, 87.8, 77.5, 40.9, 15 and 2.7% survival, respectively, relative to the control. We visualized DNA double-strand breaks in medaka cells through the formation of gamma-H2A.X foci. Breaks could be detected at concentrations as low as 1 μM. We also found that sodium chromate induces chromosomal aberrations, causing chromatid lesions and exchanges that increase with concentration. Treatments of 0, 1, 5, 10 and 25 μM sodium chromate damaged 10.3, 17, 32.3, 43 and 51.6% of metaphases and induced 13, 23, 44, 69 and 118 total aberrations in 100 metaphases, respectively. These data show that hexavalent chromium is both cytotoxic and genotoxic to fish cells. Our results set the context for future work in the medaka cell culture model and provide important tools for investigating mechanisms of toxicity in aquatic organisms.

XRCC1 protects cells from chromate-induced chromosome damage, but does not affect cytotoxicity

Hexavalent chromium Cr(VI) is a well known human carcinogen. This genotoxic metal induces DNA strand breaks and chromosome damage. However, the relationship between these lesions is uncertain. Our study focused on examining the role of XRCC1 in sodium chromate-induced cytotoxicity and chromosomal aberrations in Chinese Hamster Ovary (CHO) cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant) and H9T3 (EM9 complemented with human XRCC1 gene). Results show that concentration-dependent decreases in relative survival are similar in all three cell lines, indicating that XRCC1 is not crucial for protecting cells from sodium chromate-induced cytotoxicity. Similarly the frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total number of Cr(VI)-induced chromosome aberrations was exacerbated by XRCC1 deficiency and the spectrum of chromosome damage changed dramatically. Specifically, chromatid and isochromatid lesions were the most prominent aberrations induced in the cell lines and XRCC1 was essential to reduce the formation of chromatid lesions. In addition, XRCC1 deficiency caused a dramatic increase in the number of chromatid exchanges indicating that it is involved in protection from Cr(VI)-induced chromosome instability.

XRCC1 Protects against Particulate Chromate–Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells

Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

XRCC1 Protects against Particulate Chromate–Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells

Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions, but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

Mutagen sensitivity in humans: A comparison between two nomenclature systems for recording chromatid breaks

Currently, there are two systems in use for recording chromatid breaks, either spontaneously occurring or induced by mutagens: The International System for Human Cytogenetic Nomenclature (ISCN) and the Chatham Barrs Inn Conference (CBIC) recommendation. The former system considers that a chromatid break is recognized only when the chromatid fragment is displaced to the other side of its sister chromatid, while all others, regardless of the distance between the two broken ends, are called chromatid gaps. The CBIC system recognizes a chromatid break when the intervening achromatic segment is longer than or equal to the diameter of the chromatid, whether the fragment is displaced or not. Minor lesions are called chromatid gaps. We conducted experiments using bleomycin treatment of human cells (primary cultures or lymphoblastoid cell lines) and read the chromatid lesions both ways. We conclude that the CBIC system appears to have more direct biologic relevance than the ISCN system.

Induction of chromosomal aberrations by the anthracycline antitumor antibiotics N,N-dimethyldaunomycin and aclacinomycin A.

The clastogenic effect of the anthracycline antitumor antibiotics, N,N-dimethyldaunomycin and aclarcinomycin A, was studied in a murine hemopoietic cell line (Friend leukemia cells). A dose-dependent increase in chromatid lesions, i.e., achromatic lesions, chromatid breaks, chromatid deletions and triradial or quandriradial chromosomal exchange fiqures, was found. It appears that the clastogenicity of N,N-dimethyldaunomycin and aclacinomycin A is lower than that of the classic anthracycline, daunomycin, which is also a potent mutagen and carcinogen. The data demonstrate that the capacity of chemicals to induce point mutations and chromosomal aberrations may not necessarily be correlated: aclacinomycin A is devoid of mutagenic activity in bacterial (Salmonella typh.) and mammalian cell (HGPRT) mutagenesis assays, and is non-carcinogenic in rats. Nevertheless, it was now found to possess clastogenic activity.

Differential mutagen susceptibility in cultured lymphocytes of normal individuals and cancer patients

When cultured human lymphocytes were treated with a radiomimetic chemical bleomycin, metaphase chromosomes from different individuals exhibited a wide spectrum of responses in terms of number of chromatid lesions. This variability is interpreted as the result of capability for DNA repair. Among 100 healthy, normal individuals tested, nearly 60% showed a mean breaks per cell rate in the range of 0.20–0.60, whereas, only 12% showed breaks per cell rates above 1.00. On the other hand, among 75 cancer patients, 60% showed breaks per cell rates above 1.00. It is believed that differential responses to a mutagen have genetic implications. Individuals with DNA repair deficiencies may be more susceptible to carcinogens and, therefore, are more susceptible to neoplastic induction.

Chromatid lesions and chromatid core morphology.

A silver-staining technique revealed the core morphology of metaphase chromosomes of irradiated CHO cells with chromatid lesions (breaks, gaps). These cells were photographed before and after silver staining. As a rule, the core was not continuous in chromatid gaps, suggesting that the chromatid is broken in many so-called gaps. Ten cytogeneticists who were asked to classify chromatid gaps and breaks from photographs of chromosome lesions before silver core staining agreed in only 19 of 53 cases.

Vulnerability of specific rat chromosomes to in vitro chemically induced damage

The treatment of rat embryo secondary cultures with DMBA or DMBA-3H for 5, 9, or 24 h resulted in chromosome damage consisting mainly of chromatid type aberrations. There was an increase in the percentage of labelled nuclei and metaphases with increasing length of exposure. In terms of incidence of chromatid lesions, the largest telocentric chromosome (No. 2) was the most susceptible of the autosomes. Banding pattern analysis demonstrated that the region associated with negative band 2q24 of the No. 2 chromosome had the highest number of lesions. An increased accumulation of DMBA-3H label occurred in approximately the same chromatid area of a small fraction of cells exposed for either 5 or 9 h prior to mitosis. The complete loss of DMBA-3H chromosomal labelling after DNAse treatment suggests that the visible grains represent carcinogen-bound DNA. After DMBA and BrdUrd, there was an increase in the number of sister chromatid exchanges compared to controls treated with BrdUrd only; the location of the exchange points on chromosome No. 2 was similar in samples treated with either DMBA and BrdUrd or BrdUrd alone. Additional experiments with thymidine-3H showed that the non-random chromatid lesions on chromosome No. 2 may result from endogenous radiation from the incorporated tritium. These studies demonstrate that a specific chromosome may be affected by diverse agents and that chromatid lesions frequently occur at the site of sister chromatid exchanges.

Continuous induction of chromatid lesions by DNA-intercalating compounds

Continuous induction of chromatid lesions by DNA-intercalating compounds

Cytogenetic study on individuals occupationally exposed to DDT

Workers from three insecticide plants in direct contact with 2,2-bis (β-chlorophenyl)-1,1,1,-trichloroethane (DDT) did not differ significantly in the frequencies of cells with chromosomal aberrations when compared with controls from the same plants but not in direct contact with the drug. The same was true when a group of workers from one plant was compared with a control group from the Instituto Butantan, with no history of occupational exposure to DDT. Yet, when the control group from one of the three plants, which showed high DDT plasmic levels, was added to the group in direct contact with the insecticide, the frequency of cells with chromatid aberrations was significantly higher, suggesting that DDT causes chromatid lesions. A positive correlation was found between DDT levels and times of exposure, but being in direct or indirect contact with DDT was not always correlated with the degree of contamination.

Cytogenetic study on individuals occupationally exposed to DDT

Workers from three insecticide plants in direct contact with 2,2-bis (β-chlorophenyl)-1,1,1,-trichloroethane (DDT) did not differ significantly in the frequencies of cells with chromosomal aberrations when compared with controls from the same plants but not in direct contact with the drug. The same was true when a group of workers from one plant was compared with a control group from the Instituto Butantan, with no history of occupational exposure to DDT. Yet, when the control group from one of the three plants, which showed high DDT plasmic levels, was added to the group in direct contact with the insecticide, the frequency of cells with chromatid aberrations was significantly higher, suggesting that DDT causes chromatid lesions. A positive correlation was found between DDT levels and times of exposure, but being in direct or indirect contact with DDT was not always correlated with the degree of contamination.

Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure to pesticides

Lymphocyte cultures from 42 pesticide applicators and 16 controls were scrutinized for chromosomal aberrations during the midwinter ebb in pesticide spraying operations, and again during the peak summer period of intense spraying activity. Cultures prepared from the exposed individuals during the heavy spraying period showed a marked increase in the frequency of chromatid lesions. This trend was especially noticeable among workers exposed primarily to herbicides. A few chromatid exchange figures were also noted among the exposed group. Also discussed is the need for better methods of estimating the potential mutagenic threat, if any, of such observed clastogenicity.

Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure to pesticides

Lymphocyte cultures from 42 pesticide applicators and 16 controls were scrutinized for chromosomal aberrations during the midwinter ebb in pesticide spraying operations, and again during the peak summer period of intense spraying activity. Cultures prepared from the exposed individuals during the heavy spraying period showed a marked increase in the frequency of chromatid lesions. This trend was especially noticeable among workers exposed primarily to herbicides. A few chromatid exchange figures were also noted among the exposed group. Also discussed is the need for better methods of estimating the potential mutagenic threat, if any, of such observed clastogenicity.

The endometrial karyotypic profiles of women after clomiphene citrate therapy.

SummaryCytogenetic studies were made before and after clomiphene therapy on endometrial tissue obtained from seven patients with anovulation. In a further six subjects, endometrial karyotypic patterns were studied before drug administration. Statistical analysis of a large number of cells revealed that after treatment there was an increase in heteroploidy as well as in chromatid lesions and aberrations. The biological significance of these findings remains uncertain. They may reflect the action of the compound at the cellular level and could thus explain the occurrence of persistent infertility despite the successful induction of ovulation.

Interaction of chromatid lesions induced by N-nitroso-N-methylurea and N-nitroso-N-ethylurea inVicia faba

After treatment of primary roots ofVicia faba with N-nitroso-N-methylurea (NMU) and afterwards with N-nitroso-N-ethylurea (NEU) only an additive effect has been found. When NEU was used for the first treatment and NMU was applied afterwards an interaction of induced lesions was observed in the production of interchanges. These results might be interpreted as follows: NMU induces primary chromatid lesions in a later stage of interphase than NEU, so that these lesions cannot be induced in the same cell, if NMU is applied first. Because of the complexity of events leading to aberrations, a lot of which are unknown, our interpretation should be accepted with due reserve and cannot be more than a working hypothesis for further experimental tests.

THE INDUCTION OF CHROMATID LESIONS BY CYTOSINE ARABINOSIDE IN POST-DNASYNTHETIC HUMAN LEUKOCYTES.

When cultures of human leukocytes are treated with tritiated thymidine and cytosine arabinoside, it is observed that all of the aberrations induced by the cytosine arabinoside are induced in unlabeled (G<sub>2</sub>) cells. Furthermore, very few labeled cells reach metaphase, indicating that the cells in S at the time of treatment are either slowed or stopped in their progression to metaphase. The time from the end of S to metaphase is estimated to be 2 ½ to 3 ½ h in normal cultures.