Effect of follicle-stimulating hormone (FSH) on radiation-induced chromosomal aberrations in mouse germ cells and Chinese hamster cells in vitro

Earlier observations on the induction by X-rays of reciprocal translocations in stem-cell spermatogonia of the rhesus monkey have established a correlation between the level of follicle-stimulating hormone (FSH) in blood at the moment of irradiation and the final recovery of translocations (van Buul, 1980). In the present study, FSH treatment of mice did not induce chromosomal aberrations in bone-marrow cells or stem-cell spermatogonia, nor did it change the radiosensitivity of stem-cell spermatogonia for the induction of chromosomal translocations. Experiments in vitro with Chinese hamster ovary cells (CHO), however, showed a clear radiosensitizing effect of FSH on the induction of structural chromosomal aberrations.     

Induction of chromosomal aberrations in cultured Chinese hamster cells in a superoxide-generating system

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.     

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.     

Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

Trenimon-induced SCEs and structural chromosomal aberrations in early- and late-dividing lymphocytes

In cultures of human peripheral lymphocytes the frequencies of Trenimon-induced SCEs in second post-treatment metaphases and of structural chromosomal aberrations in first, second and third post-treatment metaphases were clearly lower at late as compared with early fixation times. These results, which are discussed, indicate that T cells (early dividing) are more sensitive with respect to the induction of SCEs and structural chromosomal aberrations by Trenimon as compared with B cells (late dividing).     

Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.     

Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside

Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA-strand breaks by ara C.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Molecular mechanisms involved in the production of chromosomal aberrations

Chinese hamster ovary cells (CHO cells) and mouse fibroblasts (PG 19) were permeabilized with inactivated Sendai virus, treated with different types of restriction endonucleases (Eco RV, Pvu II, Bam HI, Sma I, Asu III, Nun II), and studied for the occurrence of chromosomal aberrations at different times following treatment. The pattern of chromosomal aberrations observed was similar to that induced by ionizing radiations. Restriction endonucleases that induce blunt double-strand breaks (Eco RV, Pvu II) were more efficient in inducing chromosomal aberrations than those that induce breaks with cohesive ends (Bam HI, Nun II, Asu III). Ring types were very frequent among the aberrations induced by restriction enzymes. Cytosine arabinoside, an inhibitor of DNA repair, was found to increase the frequencies of aberrations induced by restriction enzymes, indicating its effect on ligation of double-strand breaks. The relevance of these results to the understanding of the mechanisms of chromosomal aberration formation following treatment with ionizing radiations is discussed.     

Anti-clastogenic ingredients in Cassia nomame extract

The suppressing effect of the hot-water extract of Cassia nomame (Sieb.) HONDA was studied on the frequency of chromosomal aberrations in Chinese hamster ovary K1 (CHO-K1) cells. CHO-K1 cells were pretreated with 2.5 microM Mitomycin C (MMC) for 1 h and incubated with or without the extract in medium for 10-24 h. The frequency of chromosome aberrations in observed 100 metaphase cells was significantly lower with the extract than that without the extract. Moreover, the suppressing effect of the four fractions collected by high performance liquid chromatography (HPLC) was also examined on the same procedure. The frequency of cells with chromosome aberrations in cells cultured with each collected fraction was lower than in those without the extract. The suppressing effect of the collected fractions on chromosomal aberrations, however, was less than that of the total extract. This result suggests that the ingredients which have the suppressing effect of chromosomal aberrations are also contained in the other fraction of the extract.     

Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells

Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations.

In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor.

The significance of these results is discussed.     

Genome-wide examination of chromosomal aberrations in neuroblastoma SH-SY5Y cells by array-based comparative genomic hybridization

Most neuroblastoma cells have chromosomal aberrations such as gains, losses, amplifications and deletions of DNA. Conventional approaches like fluorescence in situ hybridization (FISH) or metaphase comparative genomic hybridization (CGH) can detect chromosomal aberrations, but their resolution is low. In this study we used array-based comparative genomic hybridization to identify the chromosomal aberrations in human neuroblastoma SH-SY5Y cells. The DNA microarray consisting of 4000 bacterial artificial chromosome (BAC) clones was able to detect chromosomal regions with aberrations. The SH-SY5Y cells showed chromosomal gains in 1q12 approximately q44 (Chr1:142188905-246084832), 7 (over the whole chromosome), 2p25.3 approximately p16.3 (Chr2:18179-47899074), and 17q 21.32 approximately q25.3 (Chr17:42153031-78607159), while chromosomal losses detected were the distal deletion of 1p36.33 (Chr1:552910-563807), 14q21.1 approximately q21.3 (Chr14:37666271- 47282550), and 22q13.1 approximately q13.2 (Chr22:36885764-4190 7123). Except for the gain in 17q21 and the loss in 1p36, the other regions of gain or loss in SH-SY5Y cells were newly identified.     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

Reduced expression of the BRCA1 gene and increased chromosomal instability in MCF-7 cell line.

Using clonal cell cultures, a significant increase in chromosomal aberrations (aneuplolidy, dicentrics and chromatid breaks) were observed in MCF-7 cells compared with HeLa. BRCA1 expression was lower in MCF-7 cells than in HeLa cells. Since BRCA1 is known to play a role in the maintenance of chromosomal integrity, the increase in chromosomal aberrations in MCF-7 clones suggests that downregulation of BRCA1 expression could be one of the possible mechanisms for increased chromosomal instability in this cell line.     

Chromosome constitution and its bearing on the chromosomal radiosensitivity in man

Blood samples obtained from patients with various types of inborn chromosome abnormalities were exposed to γ-rays and the relationship between the chromosome constitution and chromosomal radiosensitivity of lymphocytes was studied by analysing types and frequencies of radiation-induced chromosome aberrations. The results showed that the chromosomal radiosensitivity was consistently higher in the cells which were trisomic for the whole or a part of a chromosome than in the cells with normal karyotype, but it was not significantly influenced by the monosomic conditions, reciprocal translocation and inversion. Age of the subjects also affected the chromosomal radiosensitivity, which was elevated in the neonates.

The analysis of chromosome aberrations showed that the high frequency of radiation-induced chromosome aberrations was due to the increased production of exchange aberrations and that the level of deletions was not affected either by factors of the chromosome constitution or of the age of the subject. A hypothesis to explain the increased chromosomal radiosensitivity of the trisomic cells was given in line with the effects of altered enzyme activity on the production of exchange aberrations.

The parallelism between the increased chromosomal radiosensitivity in the trisomic cells and the susceptibility of the affected persons to neoplasia allowed us to recognize that the trisomic cells are particularly cancer-prone and that the illegitimate repair of chromosome damage, which is intrinsic to the trisomic cells, may play an important role in the development of cancer.     

Clastogenicity of carbazole in mouse bone marrow cells in vivo

The protective effect of Nigella sativa seed extract and its main constituents thymoquinone (TQ) was studied on mouse cells infected with schistosomiasis. Bone marrow cells in the in vivo experiments and spleen cells in the in vitro one were used to evaluate the potentially protective effect of these natural compounds on the induction of chromosomal aberrations. Karyotyping of the mice cells illustrated that the main abnormalities were gaps, fragments and deletions especially in chromosomes 2, 6 and some in chromosomes 13 and 14. Both N. sativa extract and TQ were considered as protective agents against the chromosomal aberrations induced as a result of schistosomiasis.     

DNA topoisomerase II inhibition and gene amplification in V79/B7 cells

Topoisomerase II inhibitors such as etoposide (VP16) are able to stabilize the enzyme—DNA complex by trapping the topoisomerase on DNA without affecting its strand-break activity. To test if this inhibition resulting in chromosomal breakage via double-strand breaks could underlie gene amplification, we performed VP16 treatments followed by selection for PALA resistance in V79/B7 Chinese hamster cells. We found that VP16 induced PALA-resistant cells very efficiently, and in a dose-dependent manner. On the other hand VP16 in combination with 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase involved in DNA repair, reduced the frequency of PALA-resistant cells. Cytogenetic analysis revealed a higher number of chromosomal aberrations in VP16-treated cells than in cells treated with VP16 plus 3AB. These results suggest a correlation between frequency of chromosomal aberrations and frequency of PALA-resistant cells, and are consistent with models which consider chromosomal breakage as an important step in initiating gene amplification.     

Induction of chromosomal aberrations in CHO cells by restriction endonucleases: effects of blunt- and cohesive-ended double-strand breaks in cells treated by 'pellet' methods

In recent reports it has been suggested that restriction endonucleases (RE) producing cohesive-ended double-strand breaks (dsb), are of comparable effectiveness to those producing blunt-ended dsb in causing chromosomal aberrations (CA) in mammalian cells. In several of these reports, trypsinized cells or suspension cultures were treated as cell 'pellets' in small volumes containing RE and storage buffers. In this study we have examined this by comparing 2 'pellet' methods in which trypsinized Chinese hamster cells were treated with RE in small volumes, after cells were centrifuged to a pellet. In the first method, cells were treated with RE in storage buffer as previously reported (e.g. Obe et al., 1985). In the second method, cells were treated as pellets with Sendai virus and purified RE. For both methods we show that the frequency of chromosomal aberrations was higher in cells treated with RE causing blunt-ended dsb than those causing cohesive-ended dsb. The first method however was found to lead to substantial loss in cell viability. The results strengthen the conclusion drawn from our earlier work, using treatment of attached V79 or CHO-K1 cells with Sendai virus, that cohesive-ended dsb are less effective than blunt-ended dsb in causing chromosomal aberrations.