Human Lymphocyte Chromosomes Research Articles

The depth-dependence of the biological effectiveness of 60Co gamma rays in a large absorber determined by dicentric chromosomes in human lymphocytes

Radiobiological evidence is shown concerning a significant depth-dependence of the maximum relative biological effectiveness at limiting low doses (RBE(M)) of (60)Co gamma rays in a cubic polymethylmethacrylate (PMMA) phantom of 30 cm edge length. Using the dose-response curve for the dicentric data in human lymphocytes obtained in the present experiment at a depth of 20 cm, together with the comprehensive and consistent data set determined earlier at smaller depths of the PMMA phantom, there is an increase in the RBE(M) value by a factor of 2.18 +/- 1.25 at a depth of 20 cm relative to 1 cm in the phantom. All the dicentric data are based on identical exposure durations and irradiation temperatures as well as identical culture and evaluation conditions, with blood from the same donor.

Effect of dithioerythritol on human lymphocyte chromosome length.

The reversible formation of SS-bridges between chromosomal proteins has been suggested to be involved in the chromatin and chromosome contraction process. This study investigated the effect of simultaneous in vitro exposure of human lymphoid cells to dithioerythritol (DTE) and colchicine for 4 or 20 h on metaphase chromosome length. The results show that DTE exposure induced small changes towards longer average chromosome length. The difference was statistically significant when samples drawn from cultures exposed to the highest concentration of DTE (10-3M) were compared to those drawn from cultures exposed only to colchicine. Chromosomal contraction induced by 3 h exposure to colchicine could be partially reversed by hypotonic treatment with solutions of DTE. The results corroborate other studies suggesting involvement of SS-bridge formation in chromosome contraction.

A New Cytogenetic Screening Methodology to Evaluate Individual Susceptibility to Radiation Sensitivity

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 hybridization (FISH) enables to measure stable translocations for biodosimetry and address old or long-term exposures. In addition, premature chromosome condensation (PCC), 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, amifostine, or heat treatment during G2 to M-phase transition, reconfirmed the importance of G2-chekpoint in determining radiosensitivity and of the cdk1/cyclin-B activity in the conversion of DNA damage into chromatid breaks. G2-chromosomal radiosensitivity offers therefore a basis for the identification or testing of key genetic targets for modulation of radiation effects, and a new screening method to detect intrinsic radiosensitivity is presented.

Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis.

Non-random distribution of high density chromatin detected at opposite ends of T-banded human metaphase chromosomes

Previous research using microdensitometric scanning and computer graphic image analysis showed that T-banded segments of human metaphase chromosomes usually exhibit an asymmetrical distribution of high density (HD) chromatin between sister chromatids. Here, we employed the same methods to analyze HD chromatin distribution at opposite ends of T-banded human lymphocyte chromosomes. This study revealed that in most chromosomes with an asymmetrical distribution of HD chromatin at both ends, the highest densities of each arm were located in opposite chromatids. The frequency of this configuration was 0.792 per chromosome, indicating that the highest chromatin densities of the terminal segments of T-banded human chromosomes were non-randomly distributed at opposite chromosome arms. The possible relationship of this observation to the mode of replication of the terminal chromosome region is briefly discussed.

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.

The effect of the β-emitting yttrium-90 citrate on the dose–response of dicentric chromosomes in human lymphocytes: a basis for biological dosimetry after radiosynoviorthesis

The production of dicentric chromosomes in human lymphocytes by beta-particles of yttrium-90 (Y-90) was studied in vitro to provide a basis of biological dosimetry after radiosynoviorthesis (RSO) of persistent synovitis by intra-articular administration of yttrium-90 citrate colloid. Since the injected colloid may leak into the lymphatic drainage exposing other parts of the body to radiation, the measurement of biological damage induced by beta-particles of Y-90 is important for the assessment of radiation risk to the patients. A linear dose-response relationship (alpha = 0.0229 +/- 0.0028 dicentric chromosomes per cell per gray) was found over the dose range of 0.2176-2.176 Gy. The absorbed doses were calculated for exposure of blood samples to Y-90 activities from 40 to 400 kBq using both Monte Carlo simulation and an analytical model. The maximum low-dose RBE, the RBE(M) which is equivalent to the ratio of the alpha coefficients of the dose-response curves, is well in line with published results obtained earlier for irradiation of blood of the same donor with heavily filtered 220 kV X-rays (3.35 mm copper), but half of the RBE(M) relative to weakly filtered 220 kV X-rays. Therefore, it can be concluded that for estimating an absorbed dose during RSO by the technique of biological dosimetry, in vitro and in vivo data for the same radiation quality are necessary.

Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites

Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interface of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G(2) but not in G(1) phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G(2) phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle.

RBE of quasi-monoenergetic 60 MeV neutron radiation for induction of dicentric chromosomes in human lymphocytes

The production of dicentric chromosomes in human lymphocytes by high-energy neutron radiation was studied using a quasi-monoenergetic 60 MeV neutron beam. The average yield coefficient [see text] of the linear dose-response relationship for dicentric chromosomes was measured to be (0.146+/-0.016) Gy-1. This confirms our earlier observations that above 400 keV, the yield of dicentric chromosomes decreases with increasing neutron energy. Using the linear-quadratic dose-response relationship for dicentric chromosomes established in blood of the same donor for 60Co gamma-rays as a reference radiation, an average maximum low-dose RBE (RBEM) of 14+/-4 for 60 MeV quasi-monoenergetic neutrons with a dose-weighted average energy [see text] of 41.0 MeV is obtained. A correction procedure was applied, to account for the low-energy continuum of the quasi-monoenergetic spectral neutron distribution, and the yield coefficient alpha for 60 MeV neutrons was determined from the measured average yield coefficient [see text]. For alpha, a value of (0.115+/-0.026) Gy-1 was obtained corresponding to an RBEM of 11+/-4. The present experiments extend earlier investigations with monoenergetic neutrons to higher energies.

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.

Induction of Chromosomal Aberrations and Sister Chromatid Exchanges by Cyproterone Acetate in Human Lymphocytes

The genotoxicity study of cyproterone acetate used as antiandrogen was carried out on human lymphocyte chromosomes using chromosomal aberrations and sister chromatid exchanges as parameters. The study was carried out in the presence as well as absence of metabolic activation (S9 mix). The effect of cyproterone acetate was studied as 5, 10, 20, 30 µM and was found to be genotoxic at 20 and 30 µM both in the presence as well as absence of metabolic activation system. The results suggest a genotoxic and cytotoxic effect of cyproterone acetate in human peripheral blood cultures in vitro.

Dicentric chromosomes in monolayers of human lymphocytes produced by monochromatized synchrotron radiation with photon energies from 1.83 keV to 17.4 keV.

A systematic approach was used to examine the induction of dicentric chromosomes in human lymphocytes irradiated in vitro with monochromatized synchrotron radiation produced with photon energies in the range from 1.83 keV to 17.4 keV. To avoid potential confounding factors that could influence the outcome of the experiments, only blood from one individual was used. Since for the irradiation experiments with these low photon energies the local dose variations would become unacceptable, monolayers of lymphocytes attached within 3 h PHA stimulation have been used. The culture conditions ensured that the chromosome analysis could be performed exclusively in metaphases of the first cell cycle in vitro. There is a strong indication for a systematic change of the alpha coefficient of the linear quadratic dose-response relationship from 1.83 keV (alpha=1.26+/-0.28) with increasing energy up to 6.9 keV (alpha=8.24+/-0.41) and a decrease with further increase of energy up to 17.4 keV (alpha=3.83+/-1.72). A tendency for a systematic change of the beta coefficient seems to be present at energies of 6.9 keV (beta=8.04+/-0.40) and 4.8 keV (beta=9.48+/-1.57) as well as at energies of 3.10 keV (beta=2.99+/-0.51) and 1.83 keV (beta=0.40+/-0.25). These results agree in essence with the previously published large data set from Sasaki's laboratory.

Brush-like fibers on the human chromosome periphery.

A brush-like border apparently composed of fibers protruding from metaphase chromosomes of human lymphocytes was observed for the first time using transmission (TEM) and scanning electron microscopy (SEM). On the basis of size and sensitivity to colcemid, the fibers may be related to microtubules and spindle organization. The brush-like fibers were observed when chemically fixed metaphase chromosome spreads were placed on glass slides and maintained under "wet" conditions (not allowed to air dry after fixation for conventional cytogenetic protocols) until postfixation protocols for TEM and SEM were performed. The purpose of this study was to establish the occurrence of the brush-like fibers and to determine the effects of colcemid on these fibers.

Measuring the mitotic index in chemically-treated human lymphocyte cultures by flow cytometry

In the human lymphocyte chromosome aberration assay, the mitotic index (MI) is the standard cytotoxic parameter for determining which test concentrations will be evaluated for chromosome aberrations. Assessment of the MI is performed microscopically by determining the frequency of mitotic cells in a population of 1000 cells. With the commercial availability of antibodies to the mitosis-specific marker, phosphorylated-histone H3 at serine 10, automating the assessment of the MI using flow cytometry is now possible [Cytometry 32 (1998) 71]. Our laboratory has utilized and validated this technology to measure the mitotic index of chemically-treated human lymphocyte cultures. Comparisons between the microscopic and flow MI frequencies from 24 h treatments with mitomycin-C, aphidicolin, eugenol, etoposide, hydroxyrurea, potassium cyanide, staurosporine, ethyl alcohol, noscapine and colcemid ® are presented. Our results show that the mitosis specific H3-P marker is excellent for measuring the MI frequency in human lymphocyte cultures treated up to toxic concentrations. In addition, this study demonstrates that automation of analysis by flow cytometry is an excellent alternative to the microscopic method of analysis producing less variability than the microscopic scoring and a more complete dose response curve.

Effect of Oxymetholone on SCE frequency in human lymphocyte chromosomes in vitro

Genotoxicity evaluation of a commonly used synthetic androgen, Oxymetholone, was carried out in human peripheral blood lymphocytes in vitro. Sister chromatid exchange (SCE) was used as genetic end point. The concentrations of the drug were determined after observing its effects on the mitotic index. A wide range of concentrations, i.e., 25, 50, and 100 micro g/ml of the drug, were used to determine the genotoxic effects in the absence as well as in the presence of rat liver microsomal activation system (S9 mix). The drug did not induce any significant increase in the SCE frequency at any of the concentrations either in the presence or in the absence of S9 mix. The maximum value of SCE was observed in the absence of S9 mix at 100 microg/ml concentration (i.e., 1.38+/-0.080/cell), which was not significant statistically. Moreover, the drug was not effective in increasing the SCE frequency even in the presence of S9 mix. The maximum value of SCE (i.e., 1.78+/-0.103/cell) was observed at 50 microg/ml of concentration in the presence of S9 mix. A dose relationship was also not observed. It was concluded that Oxymetholone does not affect the genetic material in human lymphocytes at a wide range of concentrations in the SCE assay.

Additive action of vitamins C and E against hydrocortisone-induced genotoxicity in human lymphocyte chromosomes.

In earlier reports, hydrocortisone administration to human lymphocytes in culture was shown to cause chromosomal aberrations and increased sister chromatid exchanges. With a view to study the ameliorative action of some antioxidants against this effect, vitamins C and E were used separately and in combination along with hydrocortisone treatment, at different dosage and for different durations, on human lymphocyte cultures. The levels of chromosomal aberrations and sister chromatid exchanges were lowered, suggesting a protective role of vitamins against genotoxic damage. Administration of vitamins C and E combined appeared to be more effective in preventing chromosomal damage than separate administration, demonstrating the additive action of these vitamins against steroid-induced genotoxicity.

Relative Biological Effectiveness of 144 keV Neutrons in Producing Dicentric Chromosomes in Human Lymphocytes Compared with60Co Gamma Rays under Head-to-Head Conditions

The RBE for neutrons was assessed in a head-to-head experiment in which cultures of lymphocytes from the same male donor were irradiated simultaneously with 144 keV neutrons and with 60Co gamma rays as the reference radiation and evaluated using matched time, culture conditions, and the end point of chromosomal aberrations to avoid potential confounding factors that would influence the outcome of the experiment. In addition, the irradiation time was held constant at 2 h for the high-dose groups for both radiation types, which resulted in rather low dose rates. For the induction of dicentric chromosomes, the exposure to the 144 keV neutrons was found to be almost equally as effective (yield coefficient alpha(dic) = 0.786 +/- 0.066 dicentrics per cell per gray) as that found previously for irradiation with monoenergetic neutrons at 565 keV (alpha(dic) = 0.813 +/- 0.052 dicentrics per cell per gray) under comparable exposure and culture conditions (Radiat. Res. 154, 307-312, 2000). However, the values of the maximum low-dose RBE (RBE(m)) relative to 60Co gamma rays that were determined in the present and previous studies show an insignificant but conspicuous difference: 57.0 +/- 18.8 and 76.0 +/- 29.5, respectively. This difference is mainly due to the difference in the alpha(dic) value of the 60Co gamma rays, the reference radiation, which was 0.0138 +/- 0.0044 Gy(-1) in the present study and 0.0107 +/- 0.0041 Gy(-1) in the previous study. In the present experiment, irradiations with 144 keV neutrons and 60Co gamma rays were both performed at 21 degrees C, while in the earlier experiment irradiations with 565 keV neutrons were performed at 21 degrees C and the corresponding reference irradiation with gamma rays was performed at 37 degrees C. However, the temperature difference between 21 degrees C and 37 degrees C has a minor influence on the yield of chromosomal alterations and hence RBE values. The large cubic PMMA phantom that was used for the gamma irradiations in the present study results in a larger dose contribution from Compton-scattered photons compared to the mini-phantom used in the earlier experiments. The contribution of these scattered photons may explain the large value of alpha(dic) for gamma irradiation in the present study. These results indicate that the yield coefficient alpha(dic) for 144 keV neutrons is similar to the one for 565 keV neutrons, and that modification of the alpha(dic) value of the low-LET reference radiation, due to changes in the experimental conditions, can influence the RBE(m). Consequently, alpha(dic) values cannot be shared between cytogenetic laboratories for the purpose of assessment of RBM(m) without verification of the comparability of the experimental conditions.

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures

Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 μg/ml) had a limited antimutagen effect on cisplatin (0.5 μg/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment.

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4 Gy of cobalt-60 γ-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 μM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly ( P<0.05–0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 μM. Therefore, fresh blood samples were incubated with/without 17.5 μM Ot/Vc for 30 min and then exposed to 0.5–4 Gy of γ-radiation. Radiation increased the MN frequency linearly ( r 2=0.99) with dose. Pre-treatment with Ot or Vc significantly ( P<0.01–0.001) reduced the MN counts to 51–67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.

Evaluation of genotoxic potential of synthetic progestins-norethindrone and norgestrel in human lymphocytes in vitro

The genotoxicity study of two widely used contraceptive synthetic progestins, i.e. norgestrel and norethindrone was carried out on human lymphocyte chromosomes using chromosomal aberrations (CA), sister chromatid exchanges (SCE) and cell growth kinetics as parameters. The study was carried out both in the presence as well as in the absence of metabolic activation (S 9 mix). The lymphocytes were exposed to three different concentrations of the drugs (20, 40 and 75 μg/ml for norethindrone and 10, 25 and 50 μg/ml for norgestrel) for three different durations (24, 48 and 72 h). The drug norethindrone was found to be non-genotoxic at any concentration and at any exposure duration either in the presence or in the absence of S 9 mix. But another drug norgestrel was found to affect the genetic material. It induces CA, SCE at significant level, and inhibits lymphocyte proliferation at 25 and 50 μg/ml of concentrations only. In the presence of S 9 mix the values obtained for CA, SCE and mitotic index (MI) were more significant. A time and dose relationship was also observed. It was concluded that norgestrel itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.