Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective or radiosensitizing effects as elucidated by the PCC and G2-assay.

Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140µM curcumin and 2.2 to 220µM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity testing, which simulates conditions similar to those of the highly radiosensitive lymphocytes of AT patients. The results demonstrate for the first time the cell-cycle-dependent action of these polyphenols. When non-cycling cells are irradiated, the radioprotective properties of curcumin and trans-resveratrol are more prominent. However, when cycling cells are irradiated during G2-phase, the radiosensitizing features of these compounds are more pronounced. This observation offers a new biological basis for the mechanisms underlying the action of these polyphenols in cancer radiotherapy.

Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes.

Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8-24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

Lack of association between GSTT1 polymorphism and endogenous or benzo[a]pyrene-induced sister chromatid exchanges as analyzed in metaphase or G2-phase lymphocytes.

The objectives of the present work are (1) to verify whether genetic polymorphism in the detoxification gene GSTT1 influences the endogenous sensitivity in terms of sister chromatid exchanges (SCEs)/cell in healthy donors and (2) to test whether in vitro exposure to B[a]P in terms of SCEs/cell can be associated with polymorphism of GSTT1 gene. The presence or absence of the homozygous deletion in GSTT1 gene was determined in peripheral blood cells using multiplex-PCR. For SCEs quantitation, the cytogenetic method used thus far is based on the analysis in metaphase chromosomes. Consequently, G2-arrested cells are not included in the analysis. To overcome this shortcoming of the conventional method, we applied here SCE analysis in G2-phase prematurely condensed chromosomes (G2-PCCs) induced by calyculin-A, using a modified fluorescence-plus-Giemsa staining protocol. Compared to metaphase, a statistically significant increase in the yield of SCEs was notified in the G2-phase analysis after 48 h exposure of peripheral blood lymphocytes to 0.01-1 mM B[a]P, in both GSTT1-positive and -null donors. Therefore, the analysis of SCEs in the G2-phase using calyculin-A induced PCC methodology was shown to be more sensitive compared to the analysis at the metaphase level. Nevertheless, the results obtained do not show an association between the GSTT1 polymorphism with increased endogenous and/or B[a]P-induced SCE-frequencies in peripheral blood lymphocyte chromosomes in vitro. These results highlight not only the effect of B[a]P on cell cycle kinetics but also they demonstrate that conventional cytogenetic analysis at metaphase underestimates the cytogenetic effects of chemicals that delay cell cycle progression in G2-phase.

Investigation of bystander effects in hybrid cells by means of cell fusion and premature chromosome condensation induction.

The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by premature chromosome condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

Cytogenetic methods for biodosimetry and risk individualisation after exposure to ionising radiation.

Measurement of dicentric chromosomes in human lymphocytes has been applied to assess dose received by potentially overexposed people and estimate risk for health effects. Since the dicentrics in exposed people decrease with time, the introduction of fluorescent in situ hybridisation enables to measure stable translocations for biodosimetry and address old or long-term exposures. In addition, premature chromosome condensation, which enables analysis in interphase, offers several advantages for biodosimetry. However, dose and risk estimates derived using cytogenetics and adequate calibration curves are based on the assumption that all individuals respond equally to radiation. Since increased radiosensitivity has been associated with cancer proneness, there is particular interest for risk assessment at the individual level. Towards this end, the efficiency of dynamics that govern DNA repair and apoptosis, as well as the conserved cellular processes that have evolved to facilitate DNA damage recognition using signal transduction pathways to activate cell cycle arrest and preserve genomic integrity, are being investigated. Recent work in cancer cytogenetics and on the modulation of radiation effects at the chromosome level using changes in gene expression associated with proteins or factors such as caffeine or amifostine treatment during G(2) to M-phase transition, reconfirmed the importance of G(2) chekpoint in determining radiosensitivity and of the cdk1/cyclin-B activity in the conversion of DNA damage into chromatid breaks. G(2)-chromosomal radiosensitivity may offer, therefore, a basis for the identification or testing of key genetic targets for modulation of radiation effects and the establishment of a screening method to detect intrinsic radiosensitivity.

SCE analysis in G2 lymphocyte prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action.

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a "possible human carcinogen". The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used.

Radioprotective effect of amifostine on cells from cancer prone patients and healthy individuals studied by the G2 and PCC assays.

PURPOSE: To investigate whether amifostine is effective at reducing the yield of chromatid breaks when present during G(2)-phase irradiation of human normal cells and cells from cancer prone patients, as well as to study the mechanisms underlying the radioprotective effect of amifostine. MATERIALS AND METHODS: G(2) chromosomal radiosensitivity in the presence or absence of amifostine was studied in healthy donors, cancer patients, ataxia-telangietasia (A-T) patients and five human lymphoblastoid cell lines with genes predisposing to cancer. The yield of chromatid breaks following gamma-irradiation in G(2) phase was obtained at the subsequent metaphase using the G(2) assay. For scoring chromatid damage directly in G(2) or G(0) phase, premature chromosome condensation was used. RESULTS: When amifostine was present during irradiation, the mean yield of radiation-induced chromatid breaks as visualized by the G(2) assay was significantly reduced in healthy donors (t-test, p=0.001), in cells from cancer patients (p=0.001) and in cell lines from patients with genes predisposing to cancer (p=0.01) except ATM(-/-) (0.1

A new cytogenetic approach for the evaluation of mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase.

The aim of the present study was to develop and standardize a cytogenetic approach for evaluation of the mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase. Even though cytogenetic end-points such as sister chromatid exchange (SCE) have been extensively used to indirectly assess the DNA-damaging potential of various chemicals, they are based on metaphase chromosome analysis. Cells delayed in G2 phase after chemical exposure are not included in conventional SCE analysis. The yield of SCEs obtained, therefore, can be biased, since predominantly undamaged cells proceed to metaphase without delay. To overcome this shortcoming of conventional SCE analysis, the use of a new cytogenetic approach for genotoxic studies is presented that enables the analysis of SCEs directly in G2 phase using drug-induced premature chromosome condensation in cultured peripheral blood lymphocytes. By means of this method, firstly, the possibility that SCE analysis in metaphase chromosomes underestimates the mutagenic potential of various chemicals was tested. Secondly, whether the genotoxic potential of suspected carcinogens could be evaluated using SCE analysis in G2 phase, even at exposures that arrest cells in G2 phase, was examined. Thirdly, whether an important part of the background variation in SCE frequency among individuals is due to the delay of affected cells in G2 phase, rather than to a true biological variation in the cytogenetic end-point used, was tested. The results showed that a higher SCE frequency was scored in G2 phase than in metaphase. Subsequently, the mutagenic potential of chemicals that temporarily arrest cells in G2 phase could now be evaluated more accurately. In addition, it may be of interest to further examine the involvement of cell cycle kinetics in the baseline SCE variation among individuals since a lesser SCE variability was observed when the analysis was carried out in G2 phase rather than at metaphase.

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.

Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved.

PURPOSE: To test the hypothesis that deficient DNA repair as measured by increased G2 chromosomal radiosensitivity results from up-regulation of cdk1/cyclinB and cell cycle control mechanisms during the G2 to M transition. MATERIALS AND METHODS: A total of 185 cancer patients and 25 normal individuals were tested for G2 chromosomal radiosensitivity. The chromatid breaks were analysed in metaphase using the G2 assay or directly in G0 and G2 phase using premature chromosome condensation (PCC). The activity of cdk1/cyclinB, a key regulator of the G2 to M-phase transition, was measured by histone H1 kinase activity and correlated with the development of chromatid breaks after irradiation of cell lines in vitro. RESULTS: Based on the G2 assay, cancer patients on average showed increased chromosomal radiosensitivity above controls. When the analysis was carried out directly in G0 or G2 lymphocytes using PCC, no differences in the induction of chromosomal damage and its repair were observed between G2 assay-sensitive and G2-normal donors. Using the G2 assay to test G2 radiosensitivity in various cell lines, it was found that the higher the cdk1/cyclinB activity level of the cell line tested, the higher the yield of chromatid breaks scored. Furthermore, when mitotic cells from these cell lines were used for PCC induction in irradiated G2 lymphocytes it was observed that the higher the cdk1/cyclinB activity level of mitotic cells used, the higher was the induced yield of chromatid breaks. CONCLUSION: The cdk1/cyclin-B activity levels during the G2 to M transition impair DNA repair processes and play a major role in the yield of chromatid breaks induced after G2-irradiation. Regulation of cdk1/cyclinB complex activity rather than deficient repair enzymes of DNA damage may underlie the mechanisms of G2 radiosensitivity.

Conversion of DNA damage into chromosome damage in response to cell cycle regulation of chromatin condensation after irradiation.

Cell fusion, premature chromosome condensation (PCC) and conventional cytogenetics were used to test whether the biochemical process of chromatin condensation-decondensation throughout the cell cycle, which depends on cyclin-regulated histone H1 kinase activity, affects the conversion of DNA damage into chromosome damage and determines intrinsic cell cycle-stage radiosensitivity. Results from three sets of experiments are presented. Irradiated G0 human lymphocytes were fused to exponentially growing hamster cells and time allowed for repair, while following the hamster cells in their progress towards mitosis. Severe fragmentation was observed in the induced lymphocyte PCCs when hamster cells entered mitosis 13 h after irradiation, suggesting conversion of DNA damage into non-repairable chromosome damage during G1/S transition. When PCC was used to analyse chromosome damage directly in G0 and G2 phase lymphocytes, the induction of breaks per cell per chromatid per Gy was found to be similar, suggesting that G2 increased radiosensitivity is related to chromatin condensation occurring during G2/M transition and not to an inherent chromatin structure at this phase. When chromatin condensation-decondensation at the G1/S and G2/M transitions was modified after irradiation by using conditioned media or elevated temperature (40 degrees C), a dramatic change in the yield and the type of chromosomal aberrations was observed. All results obtained were consistent with the proposed hypothesis. They may be also helpful in the characterization of a DNA-chromosome damage conversion process which could give a biochemical explanation of the variability in radiosensitivity observed at the various stages of the cell cycle as well as among mutant cells and cells of different origin. The proposed conversion process is cell cycle-regulated and, therefore, subject to up-regulation or down-regulation following mutagen exposure and genetic alterations.