Effect on SCE in human chromosomes in vitro of low-level pulsed magnetic field

We analyzed sister chromatid exchanges (SCE) frequencies as an indicator of DNA damage induced in human lymphocytes in vitro by a low-level pulsed electromagnetic field. We studied the effect of low-level pulsed electromagnetic fields on human chromosomes with the cytogenetic assay of sister chromatid exchange (SCE) analysis. After the human peripheral lymphocyte cultures were exposed in vitro to the electromagnetic field at different intensities, no significant differences were observed when comparing with the control group as to the number of SCE.     

A method for detecting sister chromatid exchanges using prematurely condensed chromosomes and immunogold-silver staining

We have developed a method for detecting sister chromatid exchangesusing premature chromosome condensation and immunogold labellingfollowed by silver enhancement. Cells were grown in bromodeoxyuridinebefore sister chromatid exchanges were generated with mitomycinC. Calyculin A was then used to condense the chromosomes prematurely.Finally, incorporated bromodeoxyuridine was detected using agold-conjugated antibody followed by silver enhancement. Exchangedchromatids could easily be seen in the resulting chromosomes.This simple method allows sister chromatid exchanges to be detectedwith great sensitivity and resolution. ¹To whom correspondence should be addressed     

Scanning near field optical/atomic force microscopy of bromodeoxyuridine-incorporated human chromosomes

The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCI with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.     

Sister chromatid exchange and cell cycle patterns of normal human bone marrow cells after in vitro exposure to cytostatic drugs

The effect of different cytostatic chemotherapeutic drugs on the frequency of sister chromatid exchanges and growth kinetics of the bone marrow cells in normal adults was studied by means of an in vitro bromodeoxyuridine chromosome labeling method. Cytosine arabinoside, harringtonine, methotrexate, vincristine, and 6-mercaptopurine were tested. Three of these agents induced significantly high frequency of sister chromatid exchange (p less than 0.01 or p less than 0.05). The sister chromatid exchange frequency induced by cytosine arabinoside and that induced by 6-mercaptopurine were higher than those of the control group. The increased frequency of sister chromatid exchange predominated in C and A group chromosomes. At the concentration adopted by the authors, the growth kinetics were little influenced by these drugs.     

Mapping of amplified c-myb oncogene, sister chromatid exchanges, and karyotypic analysis of the COLO 205 colon carcinoma cell line

We have studied molecular and chromosomal details of cytogenetic status in a human tumor cell line COLO 205 that shows a stable, approximately tenfold amplification of the c-myb oncogene. The amplified copies of c-myb reside in two marker chromosomes that may have evolved from chromosome #6 by complex chromosomal rearrangements. No homogeneously staining regions can be discerned at the site of c-myb amplification. We suggest that c-myb was amplified in situ in a chromosomal segment (6q22-24) that became a part of the marker chromosome, possibly through isochromosome formation followed by duplication, and without the extrachromosomal intermediate form of double minute chromosomes. There is an enhanced frequency of sister chromatid exchanges at the site of amplified c-myb. These results are discussed in the context of models for gene amplification and oncogene activation.     

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.     

Studies on sister chromatid exchanges in patients with Hodgkin's disease

Sister chromatid exchange frequencies were analyzed in bone marrow cells and in stimulated peripheral blood lymphocytes from 20 patients with Hodgkin's disease. Sister chromatid exchange studies were also carried out in the bone marrow of eight and peripheral blood of 11 normal subjects separately. Among the patients, ten were newly diagnosed and ten patients were survivors who had received therapy 30-64 months prior to studies. The mean sister chromatid exchange frequencies in bone marrow and peripheral blood of untreated patients were 3.93 +/- 0.7/cell and 4.09 +/- 0.91/cell, respectively, which were not significantly different from those of normal subjects (3.22 +/- 0.7/cell and 5.16 +/- 1.3/cell in bone marrow and peripheral blood, respectively). The mean sister chromatid exchange frequencies in bone marrow and peripheral blood in five of the untreated patients 3-4 months after initial therapy and in a treated (30-64 months after therapy) group were close to the sister chromatid exchange values of the untreated group. Analysis of the distribution of exchanges per chromosome or chromosome groups showed a nonrandom distribution of exchanges in chromosomes #1, #2, and #3, and in E-, F-, and G-group chromosomes in the normal controls and in Hodgkin's disease patients.     

Cytogenetic properties of Chinese hamster V79-E cells: G-banding, C-banding, nucleolar organizer regions, and sister chromatid exchanges.

A line of the Chinese hamster V79 strain, denominated as V79-E, is cytogenetically characterized. It has a modal chromosome number of 22. Chromosome morphology, G- and C-banding reveal strong differences from the normal complement of the Chinese hamster presumably caused by rearrangements of chromosome segments during the past 20 years of in vitro culture. 4 chromosomes possess terminal nucleolar organizer regions. Spontaneous sister chromatid exchanges occur with a frequency of 0.38 sister chromatid exchanges per chromosome.     

Chromosomal alterations and sister chromatid exchanges induced by zirconium oxychloride in human lymphocytes in vitro with relation to age of donors.

Aqueous solutions of zirconium oxychloride were added to human peripheral blood lymphocyte culture. Cultures were set up from healthy donors of both sexes belonging to age groups of 0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 years. Airdried Giemsa schedule was followed for preparation of chromosomal aberrations and micronuclei count. Flourescence plus Giemsa staining techniques were applied for the study of sister chromatid exchange. The endpoints screened were chromosome and chromatid breaks, dicentrics and rearrangements. The frequencies of chromosomal aberrations and sister chromatid exchanges induced were compared between the samples of different age groups. The frequency of CA could not be related to age of the donor. However, the frequency of SCE increased with increase in age of female donor.     

Relationship between the duration and number of cell cycles after mutagenic exposure and the incidence of sister chromatid exchanges

The incidence of sister chromatid exchanges during the second and third mitoses in Jungar hamster bone marrow cells and human lymphocytes is assessed at different times after mutagenic exposure to thiophosphamide. In the control, the mean number of sister chromatid exchanges in bone marrow cells did not change during three consecutive cycles irrespective of the rate of cell proliferation. Thiophosphamide exposure resulted in replicative or nonreplicative repair of injuries inducing sister chromatid exchanges. About 50–70% of injuries are eliminated during a replicative (mitotic) cycle. Nonreplicative repair is intensive in proliferating bone marrow cells and weakly expressed in blood lymphocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 190–192, February, 1998     

Induction of chromosome aberrations and specific locus mutation but not sister chromatid exchanges in Chinese hamster ovary cells by neocarzinostatin

The induction of chromosome aberrations, sister chromatid exchanges (SCE), cytotoxicity, and 6-thioguanine-resistant mutation by neocarzinostatin (NCS) in Chinese hamster ovary cells was analyzed. It was observed that within the same concentration range of 0.01-0.1 mu/ml NCS, the drug induced a significant increase in all analyzed end-points except in SCE frequencies. There was no increase in SCE frequencies even when the cells were treated at the G1/S border in the first cell cycle and when aberrations were observed in the same cell showing a second cycle differential staining pattern. Our study indicates that the cellular damage induced by NCS leads to expression in chromosome aberrations, cytotoxicity, and mutagenicity but not in sister chromatid exchanges.     

Effects of arachidonic acid and inhibitors of arachidonic acid metabolism on phagocyte-induced sister chromatid exchanges

Stimulated human phagocytes produce toxic oxygen radicals which induce sister chromatid exchanges in cultured mammalian cells. Oxidative damage to membranes initiates lipid peroxidation chain reactions and stimulation of the arachidonic acid cascade. The products of these reactions may mediate the genetic toxicity of oxygen radicals. Arachidonic acid significantly augmented the number of sister chromatid exchanges in target cells exposed to stimulated phagocytes. This genetic damage was abrogated in radical-treated cells preincubated with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (nordihydroguaiaretic acid) or both (piroxicam) pathways.     

Evidence for unequal crossing-over as the mechanism for amplification of some homogeneously staining regions

The mechanism of DNA amplification in homogeneously staining regions (HSR) was studied in the human melanoma cell line MeWo. Three karyotypically distinguishable cell types within this cell line contain HSR on four different chromosomes, but all HSR contain the same amplified sequences derived from the short arm of chromosome #15. We examined metaphases of MeWo cells from different passages for changes in the length and location of the HSR. In addition, we examined the replication patterns of the HSR sequences and the organization of repeated sequences within these structures. We found that variation in the lengths of the HSR was due to fewer or more copies of a unit that consisted of a later-replicating, distamycin A/DAPI-positive block and active nucleolar organizing regions (NOR). Lateral asymmetry studies suggested that the satellite DNA sequences that are present within the HSR are organized in large inverted repeats. This organization would account for the pairing in both orientations with exchanges resulting in the types of derivative chromosomes observed. The frequency of sister chromatid exchanges (SCE) within the HSR was increased over other chromosomal regions and four examples of unequal SCE within the HSR, with prominent looping out of the longer chromatids, were seen. These results support a model of unequal SCE, rather than saltatory replication for the amplification of DNA sequences in these HSR.     

Genotoxicity of two fecal steroids in murine colonic epithelium assessed by the sister chromatid exchange technique

Two components of human feces are known to induce nuclear anomaliesin mice when applied intrarectally, but to be nonmutagenic inSalmonella. We have tested these two compounds for their abilityto induce sister chromatid exchanges in the colonic epitheliumof mice, the same tissue in which they induce nuclear anomalieswhen administered by the same route. One, 4-cholesten-3-one,induced sister chromatid exchanges whereas the other, 5-alpha-cholestan-3-onedid not, even at the maximum feasible dose. The results suggestthat 4-cholesten-3-one is more likely to be a significant factorin human colon cancer than the 5-alpha analog. ²To whom correspondence should be addressed     

Genetic recombination in human melanoma and astrocytoma cell lines involves oncogenes and growth factor genes

We describe here the chromosomal distribution of sister chromatid exchanges (SCEs) in four human tumor cell lines (two melanomas and two astrocytomas), and have mapped the sister chromatid recombination (SCR) sites. A higher incidence of SCR sites than expected on the basis of chromosome length occurred in chromosomes 2, 4, 5 and 15 in both the RPMI 5966 and MEL57 melanoma cell lines, and in chromosomes 1, 5, 13 and 15 of the IJKt and GUVW astrocytoma cell lines. A majority of the recombination sites occurred close to chromosomal fragile sites. A third of these occurred at the same bands as fragile sites. The recombination sites involved the N-ras and the epidermal growth factor gene in the melanomas. In the astrocytomas, the N-ras, Rb and c-mos genes appeared to be involved in the recombination events. The β2-microglobulin gene was involved in both astrocytomas and one melanoma. The erb132 was involved in SCR only in the RPMI melanoma.     

Genotoxicity of tetrodotoxin from puffer fish tested in root meristem cells of Allium cepa L.

Tetrodotoxin (TTX) extracted and purified from puffer fish Arothronnigropunctatus was tested for genotoxicity employing the rootmeristem cells of Allium cepa as the assay system. The genotoxicityendpoints investigated were mitotic index (MI), meta-anaphaseswith spindle aberrations, interphases with micronuclei (MNC)and sister chromatid exchanges (SCE) in metaphase chromosomes.The results demonstrated that TTX inhibited mitosis at concentrationsof     

Hamster origin of metaphases with multiple chromosome rearrangements in first cleavage human-hamster embryos

Laboratories using the human sperm-hamster egg fertilization system to analyse sperm chromosomes obtain, sporadically, metaphases with multiple aberrations. Due to the high number of aberrations, these metaphases cannot be fully karyotyped. In some of them, one or several human chromosomes can be identified, guaranteeing the human origin of the whole metaphase. However, in others, none of the chromosomes can be recognized as human. This latter type of grossly rearranged metaphases is characterized by complex chromatid exchanges, multifragmented chromosomes and pulverized chromosome material. Using fluorescent in- situ hybridization techniques (FISH) with either human or hamster genomic DNA probes, we examined the origin of this second type of metaphase with multiple chromatid exchanges and fragmented chromosomes. Our study demonstrates that all of them hybridize with hamster genomic DNA probes and not with human DNA, proving their hamster origin. Since some of these metaphases seem to be diploid, we suggest that they may arise from hamster eggs that have failed to complete meiosis and have not extruded the second polar body.      

Fertilization and early embryology: Testing the mutagenic potential of polyvinylpyrrolidone and methyl cellulose by sister chromatid exchange analysis prior to use in intracytoplasmic sperm injection procedures

The treatment of infertility due to severe oligoasthenoteratozoospermiahas been revolutionized by the introduction of the techniqueof intracytoplasmic sperm injection. However, techniques whichinvolve injection into the oocyte of polyvinylpyrrolidone solutionas a vehicle for the selected spermatozoon have caused concernsince the possible harmful effects of polyvinylpyrrolidone havenot been fully investigated. This study was performed to investigatethe potential mutagenic effect of polyvinylpyrrolidone on culturedhuman somatic cells, at the concentration used for intracytoplasmicsperm injection, in addition to a possible alternative vehicle,methyl cellulose, using the technique of sister chromatid exchangeanalysis. The results showed no increase in the basal frequencyof sister chromatid exchanges with polyvinylpyrrolidone (median5.0, 95% interval 5.00–6.00) or with methyl cellulose(median 6.0, 95% interval 4.22–6.00) in comparison withthe negative control (saline: median 6.0, 95% interval 5.00–7.00),and in contrast to the positive control (mitomycin C: median25.0, 95% interval 22.23–28.77). This finding suggeststhat polyvinylpyrrolidone and methyl cellulose do not causeDNA lesions resulting in sister chromatid exchanges, and providesreassuring evidence concerning their use in sperm injectionprocedures.     

Modified method of differential staining of sister chromatids

A modified method of obtaining differential staining of sister chromatids is described. It is simple, quick, and highly reproducible, but at the same time is cheap and readily accessible, for the reagents used are widely available. When 5-bromodeoxyuridine was added to a Chinese hamster cell culture 24 h before fixation the proportion of metaphases with differential staining of chromatids was 95–98%, but if the substance was added 28 h before fixation to a culture of human lymphocytes the proportion varied between 75 and 90% depending on the individual. The mean number of sister chromatid exchanges in human lymphocytes was found to be independent of the fixation time.Laboratory of Mutagenesis, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR B. A. Lapin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 242–243, February, 1978.     

Cytogenetic effects of permethrin in cultured human lymphocytes.

The pyrethroid insecticide permethrin was tested for its ability to induce sister chromatid exchanges (SCE), micronuclei (MN) and structural chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Permethrin was tested in the range of 5-500 micrograms/ml in the absence and in the presence of a rat liver activation system (S9 mix). Small elevations in the SCE frequencies were found and even though statistically significant may have no biological meaning, the more so since there was no dose-effect relationship. Permethrin induced both MN and CA when it was evaluated in the absence of a metabolic activation system. Nevertheless, it cannot be said that S9 mix suppressed the activity in itself. The effect of permethrin seemed to be time of exposure dependent. Permethrin could be characterized as a S-phase independent agent with greater potential for inducing chromosomal damage than sister chromatid exchanges.