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Studies on basic and applied aspects of radiation-induced chromosomal aberrations in human cells


Chromosomal aberration is considered to be one of the important biological effects arising as a consequence of exposure to ionizing radiation to man. In the case of radiation accidents, chromosome aberration frequencies in peripheral blood lymphocytes are used to estimate absorbed radiation dose. Chromosomal aberrations in germ cells and somatic cells are associated with congenital malformations in newborns and neoplasms respectively. Thus, understanding of the mechanisms of induction of DNA lesions and formation of chromosomal aberrations is very important. .SP 1 Though the radiation induced DNA double strand break is considered to be the probable DNA lesion leading to chromosomal aberration, factors such as dose rate, chromatin configuration at the time of irradiation, the DNA repair mechanisms that operate and the influence of cell cycle on the type and frequency of aberrations are not well understood. In addition, the influence of low doses and dose rates on the induction of chromosomal aberrations and other biological effects needs further investigations. The basis for the so called adaptive response following low doses of radiation needs to be understood. This project, involving 15 European laboratories aims at elucidating the relative role of the above factors in the yield of radiation induced chromosomal aberrations in mammalian cells.
Research was carried out to determine the frequencies of chromosomal translocations induced by different doses of X-rays in human peripheral blood lymphocytes using a chromosme painting technique. Comparison was made with the frequencies of dicentrics scored by conventional staining procedures.

Human lymphocytes were irradiated with different doses of X-rays (150 kV, 6 mA) and first division metaphases were either stained with Giemsa for scoring dicentrics or painted with chromosome specific probes. For the latter, specific deoxyribonucleic (DNA) libraries for chromosomes number 1, 3, X, 2, 4 and 8 were biotinylated by nick translation and hybridized in situ to chromosome preparations. The hydridized chromosomes were recognized by avidin fluorescein isothiocyanate (FITC) under a fluorescent microscope. Dicentric frequencies as well as translocation frequencies were determined. For calculating the frequencies of translocations for the whole genome, the frequencies obtained for the 6 chromosomes studied were multiplied by a factor of 2.5 this factor being based on the DNA content of these chromosomes. The frequencies of translocations were found to be about 3 times more than the dicentric frequencies. This increased frequency of translocations in comparison to the values reported in literature (usig G banding technique) appears to be due to the higher resolving power of the painting technique in detecting small translocations.

The G2 radiosensitivity of skin fibroblasts derived from cancer patients with various types of malignancy as well as xeroderma pigmentosum, ataxia telangiectasia (both homoand heterozygotes) were studied. In these experiments, the frequencies of chromatid type of aberrations induced by 0.75 Gy of X-rays given in G2 stage of the cell cycle were determined. The results obtained so far indicate that the cells derived from all cancer patients or cancer prone individuals do not respond with increased frequencies of aberrations in comparison to controls. This technique has not given reproducible results.

Chromatid type aberrations are a widely used end point in radiobiological and environmental toxicology studies.
However, their quantification presents problems since the sensitivity to production varies with cycle transit so that no cell system provides a unique yield for a given dose. This is exacerbated by the fact that observed frequencies are markedly influenced by cell kinetic perturbations.

A method was developed which allows identificaton at the time of metaphase scoring, of where (in interphase) the cell was at the time of treatment. Applied to an adaptive response protocol, one can assign the location of a cell at the time of primer (adapting) dose and at the time of challenge dose. Aberrations can then be expressed per classified cell and the composition of any cell mixture that is used for comparison monitored, or controlled.

If bromodeoxyuridine (BradU) (5 to 10 ug/ml) is added to an actively dividing asynchronous culture, cells which are in S-phase being to incorporate bromouracil into newly synthesized chromatin. Thus, cells from S arriving in metaphase contain 2 kinds of chromatin; unsubtituted TT chromatin, made early before the BrdU arrived, and substituted TB chromatin, made after. These 2 types are readily distinguishable.
Both methods have been combined. BrdU is added immediately after the primer is given and the early bands produced identify the position of a cell at that time. Immediately after the challenge dose, replacement with BrdU free medium takes place and the cell position at this time can now be assigned by the pattern of late replicating bands.

This method was tested on a typical adaptive response protocol in stimulated human lymphocytes, using 250 kV X-rays for both primer and challenge.
Using detailed cell classification, it was found that:
the primer did not introduce any measurable mitoticperturbation into the population when given alone, nor did it produce any extra perturbation when given prior to challenge;
at 6 h , the observed adaptive frequency reduction was confined to cells in pre-S at the time of primer;
when cells at defined stages for either primer or challenge dose were summed for all sample times, all evidence for any adaptive depression in frequency disappeared.

The method provides a large amount of information and offers several different way of analysing the data.

Lymphocytes exposed to low doses of radiation first followed by a challenging higher dose in G2 have been found to have lower yield of aberrations in comparison to the expected frequency. This so called adaptive response is found to vary between individuals, and to show cross adaptativity with various types of clastogenic lesions induced by subsequent exposure to chemical mutagens. The research looked at the causes of this variability and the characteristics of cross adaptivity.

Experiments were carried out using cultures of blood from donors which, in previous experiments, had or had not displayed an adaptive repsonse (AR+, AR- ).
The experiments consisted first of exposing cultured human lymphocytes to adaptating treatments and subsequently challenging the cells with high doses of X-ray.
Overall the results indicate that the variability of the adaptive response (AR) found in various donors is not linked to their genetic constitution but depends on some transient physiological parameters. 2 donors, who were AR + in previous experiments, displayed no AR. On the other hand, 2 AR-donors displayed a clear cut AR.

Furthermore, during the experiments, cases were observed in which, under standard culture conditons, the donor displayed no AR, and yet an AR could be evidenced by modifying the culture conditions. Therefore, evidence seems to point to the great importance for AR of the metabolic state of cells during conditioning.

Futher experiments show that deoxyribonucleic acid (DNA) damage induced by methylating agents is able to induce an adpative response that makes the cells resistant to induction of aberrations by subsequent exposure to alkylating agents such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or to X-rays. On the other hand, irrespective of the agent used to induce the adaptive response, cells challenged with methyl methane sulphonate (MMS) show a syngergistic increase in the number of chromatid breaks, indicating that the induced repair system cannot affect certain types of lesions.

Peroxides yield transient radical species that can damage deoxyribonucleic acid (DNA). Such oxygen species are also generated by ionizing radiation. Experiments were carried out using human lymphocytes from different donors in order to analyze the posssible protective effect of pretreatments with low doses of hydrogen peroxide on chromosome damage induced by a challenge treatment with either the true radiomimetic compound Bleomycin which, like X-rays, induces double strand breaks in DNA, or acute doses of X-rays given 24 h after the conditioning treatment with hydrogen peroxide.

The effect of pretreatment with hydrogen peroxide on the yield of chromosomal aberrations induced by Bleomycin was monitored. The total frequencies of chromatid and isochromatid breaks were observed after conditioning treatment with 3 doses of hydrogen peroxide, challenge treatment with 0.015 units/ml BLM until fixation, and consecutive conditioning and challenge treatments in 2 donors. No reduction in the yield of chromosomal aberrations induced by BLM was observed when the lymphocytes were previously conditioned with hydrogen peroxide (lack of adaptive response). Instead, the number of breaks observed after the combined treatments (hydrogen peroxide + BLM) was significantly higher than expected. This synergism is in contrast with previous results on an adaptive response found when analyzing the frequency of chromosomal aberrations in lymphocytes conditioned with hydrogen peroxide and irradiated with X-rays later on. These contrasting results point to the existence of different cell processes dealing with the repair or damage induced by BLM or X-rays after conditioning with hydrogen peroxide.

The frequency of micronuclei was measured in cells conditioned with hydrogen peroxide before irradiation with an acute dose of X-rays.
Human lymphocytes were first given a pulse with different doses of hydrogen peroxide and, 24 h later challenged with a dose of 3.0 Gy of X-rays. Immediately afte r X-ray exposure, 6 230 g/ml cytochalasin B (CYB) was added and the cells harvested 20 h later. Both donors showed a reduction in the frequency of micronuclei observed after the combined treatment (hydrogen peroxide + X-rays).

It has been that when Chinese hamster ovary (CHO) cells are incubated at lowered temperatures (33 C or 29 C) for short periods (transient hypothermia) the G2 phase is lengthened, up to more than 2-fold, so extending the range of time over which repair of rejoining of chromatid breaks can be obvserved. This allows the establishment of more accurate kinetics of chromatid break rejoining than was previously possible. Using this system it was shown that the rate of rejoining of chromatid breaks in CHO cells was similar at 37 C, 33 C and 29 C. It was also found that the rate of rejoining of chromatid breaks in xrs 5 cells was similar to that for its wild type (WT) parental CHO K1 cell line. These results were obtained when equiclastogenic doses of X-rays were employed to damage cells. These experiments have been repeated using the same X-ray dose in both strains. Results show that a similar rate of disappearane of chromatid breaks occurs in both lines (xrs and CHO) following X-irradiaiton. These results seem paradoxical since the overall rate of rejoining of deoxyribonucleic acid (DNA) double strand breaks (dsb) has been shown to be severely reduced in xrs 5 when compared to the WT CHO line. The results indicate that G2 xrs 5 cells are proficient in the repair of DNA dsb. The effect is still underinvestigation and further measurements of repair of dsb, following X-rays, are being made in these cell lines using the neutral filter elution technique.

Protein extracts have been prepared from both CHO and xrs 5 cells and are being assayed in porated cells treated with either X-rays or bleomycin. The date only small changes have been observed in chromosome damage in extract treated versus nontreated samples using the micronucleus assay of Fennech and Moreley (1985) involving cytokinesis block induced by cytochalsin B. This method has proved a valuable method for screening extracts. As a control, T4 ligase was used to treat electroporated and Pvu II treated cells. Experiment s suggest that it leads to little or no change in the frequencies of X-ray induced micronuclei, indicating that although T4 ligase can join restriction endonuclease cuts in cellular DNA, dsb induced by X-rays are not strongly subject to 3' hydroxyl-5' phosphoryl end ligation.

The adaptive response of cells in vivo was examined using whole body exposure to caesium-137 Cs-gamma-rays inhibited temporarily the enzyme thymidine kinase (TdR-K) with a minimum of about 4 h after exposure and full recovery within some 6 h thereafter. This effect was dose dependent up to about 0.01 Gy and it was then dose independent up to at least 1 Gy. Also a diminution in the number of hemopoietic stem cells (CFU-S-7d) in murine bone marrow was seen to be expressed maximally after 2 to 4 h.

After a first exposure to more than 0.1 Gy the reaction of the cells was altered in that TdR-K became temporarily resistant of a repeated exposure with the same dose. The number of hemopoietic stem cells seeding to the spleens of conditioned recipient mice, on the other hand, exhibited an additive effect in terms of colony formation when repeatedly exposed to the same low dose.

Further research was carried out into the reaction of thymidine kinease after repeated irradiation and it was found that after repeated exposures to 0.01 or 0.1 Gy, the reaction of TdR-K was altered, depending upon the radiation free time intervals between the radiation exposure events.

Invesigations concerning the regulation of thymidine kinase activity showed that cells react to low doses of radiation, and that the relation between radiation dose and observed effects is not constant. This suggests cellular adaptation mechanisms arising from a reaction of the cellular radical detoxification system following a short lived radiation insult. The reaction of TdR-K to a change in glutathione concentration in the cell would explain the observed experimental findings after repeated radiation.

The effect of low dose radiation on hemopoietic stem cells was also studied.
The reduction of the CFU-S count by 30% after a single dose of 0.1 Gy seemed to indicate that cell death is not the main underlying mechanism. It is suspected that the low doses produce a change in some stem cells which impels the m either to differentiate or to go into cycle (which by itself reduces the seeding to the spleens) or to reduce seeding capability in a more general way, perhaps through a change in membrane receptors which are responsible for the colonization of the transplanted stem cells in the spleen.

In all, in a synoptic approach to interpreting the response pattern of murine bone marrow, a change in cell cycle and thus in the availability of cells for seeding appears to be the dominant cause for the reduction of CFU-S, rather than the damage in receptor function of transplanted cells.

The thyroid hormone molecule (triiodothyronine T3) labelled with iodine-125 was investigated as a potential carrier of radioactive iodine isotopes to cell nucleus. The 2 cell lines used were Chinese hamster ovary (CHO) and GC, originating from tissues having low and high number of T3-receptors.

The uptake of iodine-125-T3 was found to be cell specific. After incubation in the same external iodine-125-T3 activity concentration, the uptake to GC cells was 20 times higher than to CHO cells. About 75% of the iodine-125 activity in GC cells was localized to the nuclei while no preferential nuclear accumulation was observed in CHO cells.

In GC cells the frequency of micronuclei was positively correlated to the nuclear iodine-125-T3 activity concentration and to the incubation time. The frequency of micronuclei in iodine-125-T3 incubated CHO cells was not significantly different from the controls independent of the incubation time. Using the same cell lines a comparative study was performed with iododeoxyuridine-125. In both cell lines the uptake of iododeoxyuridine-125 was linearly increased when the cells we incubated in medium containing a range of iododeoxyuridine-125 concentrations. However, linear dose effect curves (micronuclei frequency) were found for iododeoxyuridine-125 concentration below 30 (GC) and 75 (CHO) decays per cell. Above these doses the number of micronuclei was almost constant. The 2 times higher sensitivity in GC cells may be due to the longer doubling time for GC cells.
The efficiency of iododeoxyuridine-125 to iododeoxyuridine-125 to induce micronuclei was about 5 to 10 times higher than iodine-125 in GC cells.

Adaptable cell lines are being studied for the purpose of selecting a suitable test material for studies of the mechanism(s) of adaptation or induction of mutation prone conditions. Within the frame of this work the statement of Mendiola-Cruz and Morales-Ramirez (Radiation Res 118, 1989, 131) that mercaptoethylamine (MEA) completely abolishes sister chromatid exchange (SCE) induced in vivo in the mouse was subject to experimental verification (in view of the hypothesis that SCE requires induction of a recombinaton function of significance to dose rate effects). It was found impossible to induce SCE by X-rays, even in the animal strain used by the authors. Mathematical models for adaptation and for error prone repair with thresholds (or quasi thresholds in consequence) have been developed and applied to published or previous data.

Since the inference that MEA acts by inhibition of transcription is indirect, experiments for verification of the hypothesis at the level of induction of messenger ribonucleic acid (mRNA) synthesis have been started.

Human lymphocytes were irradiated with different doses (5, 11 and 15 cGy) of fission neutrons (0.4 MeV average energy) in vitro and posttreated with hydroxyurea (HU) arabinfuranosilcytosin (ara C) and caffeine and the frequency of chromosomal aberrations was evaluated.

Standard whole blood cell cultures were used. The cell cultures were irradiated at 70 to 72 h after stimulation and posttreated with the inhibitors for 3 h before fixation.

The distribution of chromosomal aberrations among the cells depicted a Poisson distribution. The effects of 3 h posttreatment with caffeine, ara C and HU on the frequency of chromatid aberrations induced in human lymphocytes by different doses of fission neutrons were monitored.

The data suggest that also for the repair of double strand breaks (DSB) both caffeine and HU interfere with some steps of their repair processes occurring in G2 phase. In the case of ara C further data have to be collected and protocol has to be modified in order to follow the kinetic of the repair processes.
Different laboratories will be involved in different aspects of the research, depending on their expertise.

Radiation induced DNA lesions and chromosomal aberrations
G0/G1 human lymphocytes and G1 mammalian fibroblasts (Leiden, Stockholm NIRP):
human blood samples or isolated lymphocytes will be irradiated with XArays or fast neutrons (1 MeV) and the frequencies and distribution of aberrations will be studied (University of Leiden).
G2 human/mammalian fibroblasts (St Andrews, Harwell):
elucidation of the molecular mechanism of conversion of primary radiation damage (DNA DSBs) into G2 chromatid deletions and exchanges will be studied (St Andrews); this will include a study of the kinetics of formation of chromatid deletions and exchanges in the G2 stage of cell lines of different radiosensitivities under extended G2 conditions (lowered temperature).

Adaptive response for radiation induced chromosomal aberrations (Rome, Sevilla, Harwell, Julich)
An initial small dose of radiation (adaptive dose) to human lymphocytes in G1 has been found to reduce the frequencies of aberrations induced by a subsequent high dose of radiation (challenging dose) in G2. (University of Rome). This phenomenon has been termed the adaptive response(AR).

Studies on the effects of low doses and dose rates (Stockholm 2, Leiden, Uppsala)
Experiments are planned to study the mechanisms by which dose rates can influence the risk with the purpose of ultimately reducing the uncertainty of risk estimates at low doses and dose rates (Stockholm 2).

G2 sensitivity to high LET (Viterbo)
The objectives of this project are:
to irradiate blood in vitro with high LET irradiation to examine the influence of different classes of DNA repair inhibitors on the induction of chromosomal aberrations in the G2 phase of the cell cycle;
to analyze the data for interindividual variation; effect of the different classes of DNA repair inhibitors on the yield of chromosomal aberrations.

Studies using the HPRT locus as a tester gene
The estimate of the carcinogenic risk associated with exposure to low doses of ionizing radiations is generally based on the extrapolation of the results of exposures at high doses, but it largely depends on various hypotheses concerning the form of the dose response curve. In this project we want to set up the HPRT clonal assay to study in vivo induced human mutants. Because this method allows selection of living mutant cells, characterization of mutation should be possible.

Aberrations in G0 and G2 normal and tumour human cells
In the first part of this project, premature chromosome condensation (PCC) will be used to measure directly induction and repair of chromatid damage in normal and tumour cells in an attempt to elucidate the mechanisms underlying the phenomenon of G2 radiosensitivity fluctuations. In the second part, experiments complementary to those carried out in the previous funding period (BI6-E-206-GR) will be conducted for the development of a biological dosimeter for the early assessment of radiation injury and the establishment of absorbed dose estimates in accidental overexposures. For this purpose, chromosomal aberrations scored in C-banded lymphocyte PCCs will be used.

Sensitivity of lymphocytes from babies and juveniles
Lymphocytes taken from newly born babies and 12 month old juveniles will be irradiated with 0.5 and 1.0 Gy of gamma radiation after stimulation in the G2 phase of the cell cycle. Chromatid aberrations arising in the cells will be scored and the sensitivity of the children to radiation will be evaluated as a function of age. Comparisons will also be made with sensitivity of healthy adults and measurements will be made of the chromosome aberrations induced in the lymphocytes when irradiated prior to stimulation in the G0 phase of the cell cycle

Funding Scheme

CSC - Cost-sharing contracts


72,Rapenburg 70
2311 EZ Leiden

Participants (13)

Direcção-Geral do Ambiente - Ministério do Ambiente e Recursos Naturais
10,Estrada Nacional
2685 Sacavem
Wilhelm Johnen Strasse
52425 Juelich
Instituto Português de Oncologia de Francisco Gentil
Rua Professor Lima Bastos
1093 Lisboa
Medical Research Council (MRC)
United Kingdom
20 Park Crescent
W1N 4AL London
Terma Patriarchou Grigoriou
15310 Athens
Universitetsvaegen 10
10691 Stockholm

171 16 Solna
10,Arrheniusplan 2C
750 07 Uppsala
C/ San Fernando 4
41004 Sevilla
Piazzale Aldo Moro 5
00185 Roma
United Kingdom
College Gate, 66 North Street
KY16 9AH St Andrews
Via S. Camillo De Lellis
01100 Viterbo
Université de Liège
27,Boulevard Du Rectorat
4000 Liège