Induced Chromatid Aberrations Research Articles

Cytotoxic and genotoxic potential of Bulgarian Rosa alba L. essential oil – in vitro model study

ABSTRACTRosa alba L., also known as the white oil-bearing rose, has been cultivated in relatively small areas of Europe – predominantly in the Rose Valley of Bulgaria. An increasing number of studies in vitro and in vivo, including clinical studies, have demonstrated the therapeutic efficacy of rose oils in recent years. However, little is known about the cytotoxic and genotoxic potential of the phytocomplex oil extract derived from Rosa alba L. The aim of the present work was to investigate the cytotoxicity and clastogenic effects of Rosa alba L. essential oil and its main constituent – geraniol, as well as citral A, associated with allergies and contact dermatitis. We used classical cytogenetic methods and comet assay. 1-Methyl-3-nitro-1-nitrosoguanidine was used as a standard mutagen. The data showed that R. alba L. essential oil (in concentrations up to 1000 μg/mL) did not exhibit a statistically significant cytotoxic effect. The essential oil did not significantly increase the level of mitotic disturbances (micronuclei and aneuploidy effects) and had no significant effect on the induction of chromatid aberrations, compared to the untreated control sample. Only geraniol and citral A (in the concentrations used) increased significantly the percentage of migrated DNA in the comet tail, compared to the whole oil extract. This study gives a reason to believe that R. alba L. oil has potential as a supplementary component in some therapeutic strategies. It would be harmless to normal cells and tissues, but with potential for additive or synergistic cytotoxicity against cancer cells.

In vivo versus in vitro individual radiosensitivity analysed in healthy donors and in prostate cancer patients with and without severe side effects after radiotherapy

Background: A high cellular radiosensitivity may be connected with a risk for development of severe side effects after radiotherapy and indicate cancer susceptibility. Hence, a fast and robust in vitro test is desirable to identify radiosensitive individuals.Materials and methods: The study included 25 prostate cancer patients with severe side effects (S) and 25 patients without severe side effects (0) after radiotherapy as well as 23 male healthy age-matched donors. Blood samples were exposed to 0.5 Gy or 1 Gy of γ-rays. The initial level of double-strand breaks (dsb) and repair kinetics measured by phosphorylation of histone H2A (γ-H2AX-assay), apoptosis (Annexin V-assay) and the induction of chromatid aberrations after irradiation in the G2-phase of the cell cycle (G2-assay) were analysed.Results: A significant higher chromatid aberration yield was found in lymphocytes from prostate cancer patients when compared to healthy donors. We found no significant differences between patients S and patients 0.Conclusions: There is no obvious correlation between clinical and cellular radiosensitivity in lymphocytes of prostate cancer patients when all chosen in vitro assays are considered. Although 25% of the patients showed both severe side effects and increased radiation-induced chromosomal sensitivity, predictive value of G2-assay is doubtful.

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H 2O 2 treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H 2O 2, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.

No adaptive response of Chinese hamster ovary cells to low doses of ionizing radiation.

Chinese hamster ovary (CHO) cells that had been exposed to very low doses of ionizing radiation from incorporated tritiated thymidine or X-rays were shown not to be more resistant to the induction of chromatid aberrations by a subsequent higher dose of ionizing radiation than previously untreated cells. The adaptive response to ionizing radiation was absent both in cells challenged with high doses of X-rays (1.25 Gy) and in cells post-treated with thermal neutrons (0.5-1 x 10(12) n/cm2). In agreement with these observations in CHO cells we were unable to demonstrate an adaptive response to X-rays also in human lymphocytes from five different donors.

The Role of the Telomere/ telomerase System in the Bystander Effect

Bystander responses and genomic instability are both non-targeted effects of irradiation. There have been suggestions that the telomerase system is involved in radiation induced genomic instability and therefore may also play a role in bystander mechanisms. The aim of this study was to investigate the possible relationship between the bystander effect and the telomere/ telomerase system as well as the involvement of telomerase in the development of chromosomal damage and its repair in G2 phase. We examined the relationship between several response criteria, including cell survival, chromosomal damage (using micronuclei, breaks and exchanges in prematurely condensed chromosomes, G2 induced chromatid aberrations) and double strand breaks of the DNA (using γ-H2AX and Rad 51) in two cell lines, BJ human foreskin fibroblasts and BJ1-hTERT- a telomerase-immortalised BJ cell line after direct irradiation and bystander exposure. Our findings suggest that telomerase does not play any major role in the bystander effect. However, telomerase appears to be involved in the development of chromosomal damage and its repair in G2 phase and may play a role in the control of DSB repair. The data, taken overall, reinforce the idea that telomerase or other elements of the telomere/telomerase system may defend the chromosomes from breakage, but breaks which occur in cells which do not have telomerase activity are more easily repaired.

Wavelength dependence of cellular responses in human melanocytes and melanoma cells following exposure to ultraviolet radiation

Purpose: To examine the wavelength dependence of cellular responses in human melanocytes and human melanoma cells exposed to ultraviolet radiation (UVR).Materials and methods: Primary human melanocytes and G361 human melanoma cells were exposed to ultraviolet-C (UVC), ultraviolet-B (UVB), or ultraviolet-A (UVA) radiation. Dose-response relationships for clonal cell survival were assessed, and flow cytometry was used to monitor cell cycle distributions for up to one week post-irradiation. Chromosomal aberrations were scored in exposed and unexposed melanoma cells.Results: G361 melanoma cells were more sensitive than melanocytes to killing by UVB and UVC radiation. This difference in sensitivity between cell types was much less marked following UVA irradiation. The melanoma cells showed a sustained, dose-dependent G2/M block following exposure with all wavelengths; in addition, transit through S phase was slowed following UVA irradiation. There was no apparent block to G1 cells entering S phase at any wavelength. Melanocytes, on the other hand, showed a marked G1 arrest, particularly following UVA irradiation. Cytogenetic results showed a dose-dependent increase in chromatid-type aberrations, mostly gaps, breaks and exchanges, in exposed melanoma cells.Conclusion: These results show that G361 malignant melanoma cells have lost the ability to regulate the cell cycle at the G1/S checkpoint and are more sensitive than melanocytes to cell killing by UVC and UVB but not UVA radiation. Similarly, exposure of these melanoma cells to UVC and UVB, and to a much lesser extent UVA, induced chromatid aberrations. UVA nevertheless induced strong cell cycle delays in both cell types, indicating that UVA exposure can significantly affect genome metabolism.

Early and delayed effects of carboplatin on the blood system.

Carboplatin injected intravenously in a maximum permissible dose to Wistar rats inhibited erythro- and granulocytopoiesis in the bone marrow, caused hyporegenerative anemia, leukopenia, and thrombocytopenia in the peripheral blood, led to hypoplasia of the thymus and spleen, and produced moderate apoptosis-inducing effects. These effects were observed within the first month after treatment. In mice carboplatin induced chromatid aberrations in the bone marrow and increased the count of erythrocytes with micronuclei. Moderate hypoplasia of erythro- and granulocytopoiesis and accelerated involution of the thymus were observed 3 and 6 months after cytostatic treatment. Our results indicate that carboplatin possesses higher myelotoxic activity compared to cisplatin.

Increased sensitivity to chromatid aberration induction by bleomycin and neocarzinostatin results from alterations in a DNA damage response pathway

DNA damage response pathways coordinate the cellular response to DNA damage. To investigate the roles of tumor suppressor genes in these pathways, human lymphoblastoid cells (wild-type, p53−/−, ATM−/−) were treated for 1 h with 0–3 μg/ml of the radiomimetic compound bleomycin (BLM), and cells treated in G 2 were analyzed for chromatid aberrations. BLM-induced aberration frequencies were significantly increased, to the greatest extent in the ATM−/− cells and, to a lesser extent, in the p53−/− cells compared to wild-type cells. These observations are consistent with p53 and ATM acting in a damage response pathway activated by DNA strand breaks. The consequences of disrupting this pathway were further investigated by studies using wortmannin, a PI-3 kinase and DNA repair inhibitor. Wortmannin significantly increased the BLM-induced aberration frequencies in all but the ATM−/− cells, elevating the sensitivity of p53−/− cells to ATM−/− levels and that of wild-type cells to intermediate levels. These differential sensitivities suggest that the ATM phenotype is the result of dual cellular defects, one involving p53 and the other a wortmannin-sensitive component. Similar studies in Brca1+/− and Brca2+/− human lymphoblasts showed no increased sensitization to BLM in the absence of inhibitor, and differential sensitization by wortmannin. To determine if there was any substrate specificity for p53- and ATM-mediated DNA damage responses, chromatid aberrations were assessed in wild-type, p53−/−, and ATM−/− cells exposed to 0–0.4 μg/ml neocarzinostatin (NCS) for 1 h. In contrast to results with BLM, the p53−/− cells exhibited a low sensitivity to NCS-induced aberrations, similar to wild-type, while ATM−/− cells remained highly sensitive. This suggests that the response to BLM- and NCS-induced lesions involves different mechanisms.

XPD polymorphisms: effects on DNA repair proficiency.

XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Involvement of G 2-dependent DNA double-strand break repair in the formation of ultraviolet light B-induced chromosomal aberrations

Wortmannin, an inhibitor of DNA double-strand break (DSB) repair added 19 h before harvest enhanced the incidence of ultraviolet light B (UVB)-induced chromatid aberrations in Chinese hamster V79 cells. Posttreatment with wortmannin for last 3 h of culture also enhanced the yield of breakage-type chromatid aberrations and suppressed the yield of exchange-type chromatid aberrations almost completely. Thus, the inhibition of DSB repair in the G 2 phase stimulated the breakage-type aberration formation, while suppressing the exchange-type aberration formation. We propose the model of UVB-induced chromatid-type aberration formation which might be fully related to G 2-dependent DSB repair pathway.

The relationship between p53 status, DNA repair and chromatid aberration induction in G2 mouse embryo fibroblast cells treated with bleomycin.

The involvement of p53 in the formation of chromosome aberrations was assessed by analyzing bleomycin-induced, chromatid-type aberrations in G2 phase fibroblasts derived from embryos from wild-type and p53 knock-out mice. Cells that were p53+/- or p53-/- were more sensitive to the induction of aberrations than the p53+/+ cells, particularly at concentrations of 7.5 and 10.0 microg/ml. The p53-deficient cells also showed an overdispersed distribution of bleomycin-induced chromatid aberrations, a greater amount of overall genomic instability and a possible loss of a cell death pathway. These data are interpreted as indicating a role for p53 in DNA repair in the G2 phase, with a loss of p53 leading to an increased frequency of deletions (incomplete repair) and interchanges (misrepair). The specific role remains to be elucidated. The mitotic index decreased with increasing bleomycin concentration to a similar extent in all three cell lines, indicating that the loss of a G2 checkpoint in p53-/- and p53+/- cells was not an explanation for the increased sensitivities in these cells compared with the p53+/+.

Comparative study of radiation-induced G2 phase delay and chromatid damage in families with ataxia-telangiectasia

Two assay systems, radiation-induced chromosome aberrations and flow cytometry, were compared for the detection of ataxia-telangiectasia (A-T) heterozygotes. In three A-T families, the frequencies of chromatid aberrations in phytohemagglutinin-stimulated blood lymphocytes after 1 Gy of gamma-irradiation were twofold higher in A-T homozygotes than in obligate A-T heterozygotes, which were in turn approximately twofold higher than in normal control cells. Other consanguineous relatives of A-T patients had intermediate levels of induced chromatid aberrations, suggesting that they were carriers of the gene. Matched Epstein-Barr virus—transformed lymphoblastoid cell lines from A-T homozygotes showed a greater radiation-induced accumulation in the G2 phase of the cell cycle than did control cells. In family B, both obligate heterozygotes had increased G2 delay, as did the one heterozygote available for family C, and two of the grandparents in that family were in the high range for G2 delay. Neither parent in family A had high G2 phase delay after irradiation although the induced chromatid aberrations were in the heterozygote valve range. These results show a good concordance between the two assay systems for A-T heterozygotes, with the chromatid aberrations somewhat more consistent.

Chromatid damage induced by 238Pu alpha-particles in G2 and S phase Chinese hamster V79 cells.

The comparative induction of chromatid aberrations by 238Pu alpha-particles, or by 250 kVp X-rays was investigated in V79 Chinese hamster cells. Metaphases were sampled at hourly intervals postirradiation up to 8 h and BrdU/FPG staining methods were used to distinguish G2, S and G1 phase cells. Two experiments were performed. In the first, an alpha-particle dose of 0.41 Gy was compared with an X-ray dose of 1.5 Gy used in a previously published study. In the second, an X-ray dose of 1.2 Gy was used in parallel with 0.41 Gy of alpha-particles to produce a similar overall frequency of interchanges, and allow comparative ratios to be derived for other aberration types. At these isoexchange doses, alpha-particles produce relatively less gaps and breaks, particularly in late G2, and significantly more isochromatid deletions. A very high proportion of the isochromatid deletions were incomplete after alpha-particles compared with X-rays, but no difference in incompleteness was found for interchanges. With X-rays, about 6% of interchanges are complex intra-interchange forms. At similar exchange frequencies this increases to 26.7% for alpha-irradiation, suggesting increased multiple lesion interaction. Differences in dose distribution between alpha-particles and X-rays are discussed and mitotic delay is examined after separation of the analysed cells into damaged and undamaged classes.

Combined application of MH and TEM for conditioning treatment simultaneously triggers two protective functions against induction of chromatid aberrations in Vicia faba root tip meristem cells

Conditioning pre-exposure of Vicia faba root tip meristem cells to triethylenemelamine (TEM) does not trigger an adaptive response to maleic hydrazide (MH) and vice versa. Since TEM conditioning treatment can induce protective effects (as evident from the yield of metaphases with chromatid aberrations) against TEM challenging treatment and MH conditioning can trigger an adaptive response to MH challenging treatment, two different protective functions are apparently triggered in dependence on the agent used for conditioning pre-exposure. When a mixture of TEM and MH is being used for conditioning treatment, adaptive responses to both TEM and MH can simultaneously be induced and significantly reduce the yield of metaphases with chromatid aberrations observed after challenge treatment with TEM or MH.

Chromosomal changes during the acquisition of resistance to adriamycin in tumorigenic human synovial cells.

Chromosome analysis of tumorigenic human synovial cells at different stages of acquisition of resistance to doxorubicin was undertaken. Exposure of the sensitive cells to adriamycin for 2h induced chromatid aberrations, while exposure to the drug for 6h did not show any mitosis. However, drug resistant cells continued to show high frequency of mitosis even in presence of the drug. High frequency of chromatid exchanges were seen during the process of the development of drug resistance by exposure to increased drug concentration. Double minutes, a characteristic feature of resistant cells appeared only when the drug resistance was established in the cells, which was confirmed by drug sensitivity assay.

Further evidence for a high position-specific effect in the action of chemical mutagens on the chromosomes of barley

Further convincing evidence for the decisive role of chromosome constitution in the processes underlying the specific induction of structural mutations by chemical mutagens is described. The most important conclusion to be drawn from the results obtained in experiments with maleic hydrazide (MH) and ethyl methanesulphonate (EMS) is that segments 39 and 47, situated next to the secondary constrictions of standard chromosomes 6 and 7, when tandemly combined by reciprocal translocation in chromosome 7(6) of reconstructed karyotype T-21, behave in a rather similar way. This is independent of the nature of the chemical mutagens applied, as far as the distribution pattern of induced chromatid aberrations is concerned. The phenomenon may be characterized as follows: (1) the segments in question in the new position appeared to be the most pronounced aberration "hot spots", the expressivity in segment 39 always being higher; (2) the pronounced "hot spot" character of these segments proved to be due mainly to their preferential involvement in intercalary deletions and duplication-deletions; (3) the specific constitution of chromosome 7(6) resulted in the majority of cases in a marked increase of the region involved in the aberration types mentioned above in segment 39. This is one of the very few examples of true position effect in the expression of chemically induced structural mutations at the chromosome level.

Effect of pH shifts on radiosensitivity of human lymphocytes irradiated in the G2 stage

Experiments were carried out using human lymphocytes in order to test the effect of pH shifts on radiosensitivity of cells irradiated in the G2 stage. In our culture conditions constant variations in medium pH over the range of 7.4–6.8 were observed as a function of incubation time after PHA stimulation; in addition the pH of the medium was adjusted in parallel cultures over the range of 5.9–8.2. Then we exposed the cultures with different pH to a treatment of X-rays delivered 2 h before fixation.The pH of the medium was found to greatly affect the yield of induced chromatid aberrations.

Involvement of phytochelatins in NiCl 2-triggered protection against induction of chromatid aberrations by TEM and MH in Vicia faba root tip meristems?

Conditioning treatment of Vicia faba root tip meristem cells with NiCl 2 prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) triggered protective functions against both these clastogens, i.e., resulted in a significantly reduced yield of metaphases with chromatid aberrations. Protection was prevented by pretreatment with buthionine sulfoximine (BSI), an inhibitor of the synthesis of plant phytochelatins (PCs), indicating that the NiCl 2-triggered PC synthesis may be involved in the protective functions induced by NiCl 2 conditioning treatment. BSI (instead of NiCl 2) conditioning treatment triggered protection against MH but not against TEM.

Analysis of Chromatid Damage in G2Phase Induced by Heavy Ions and X-rays

The induction of chromatid aberrations was examined in Golden hamster embryo (GHE) cells 2 h after exposure to X-rays or heavy ions. Compared with X-rays the frequencies of gaps and deletions were almost one-half in the case of He ions, and much lower in cells irradiated with N ions. The induction of exchanges by He ions and N ions was also less than that by X-rays. The relative number of gaps, deletions and exchanges were 0.4, 0.5 and 0.4 for He ions, and 0.2, 0.2 and 0.4 for N ions. The mitotic indices were almost the same in X-irradiated and heavy-ion-exposed cells. Chromatid aberrations induced by heavy ions tended to persist longer than those induced by X-rays during successive culture passages after exposure. The present results suggest a difference in the quality of chromatid damage induced by X-rays and heavy ions.

Cytogenetic investigations in a family with ataxia telangiectasia.

Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normal control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7-11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.